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PATHOLOGY AND PATHOGENESIS OF NIPAH VIRUS INFECTION IN HUMANS AND ANIMAL MODEL

WONG KUM THONG

THESIS SUBMITTED IN FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF MEDICINE

FACULTY OF MEDICINE UNIVERSITY OF MALAYA

KUALA LUMPUR

DECEMBER 2008

i

ABSTRACT

In 1998, an outbreak of acute encephalitis in Malaysia led to the discovery of a novel

paramyxovirus named Nipah virus (NiV). Subsequently, outbreaks were also reported in

Bangladesh and India. So far, the number of people infected is more than 500 with

mortality between 40-70%. NiV is closely related to Hendra virus (HeV), both belonging to

the new genus, Henipavirus (family Paramyxoviridae). The natural host of henipaviruses is

the pteropid or fruit bat whose range includes Africa, Asia-Oceania and Australia. In the

NiV outbreaks in Malaysia and Singapore, the intermediate host was the pig, while in

Bangladesh and India, direct bat-to-human and human-to-human transmission occurred.

Acute NiV infection may be asymptomatic; symptomatic cases present with fever and

headache or acute encephalitis often associated with coma. Most patients recovered without

serious sequelae but a small percentage developed relapsed/late-onset NiV encephalitis.

HeV infection is also associated with relapsed encephalitis but acute encephalitis has not

been previously reported.

To study the human pathology of NiV infection, 31 cases of acute infections and 3

cases of relapsed/late-onset encephalitis were examined. These findings were compared

with human HeV, and infections in a hamster model (Mesocricetus auratus). Tissues were

examined by histology, immunohistochemistry, electron microscopy, in situ hybridisation

and other molecular techniques.

In acute NiV infection, there were systemic vasculitis and discrete, plaque-like,

parenchymal necrosis and inflammation in most organs, particularly in the central nervous

system (CNS). Vascular endothelial damage, multinucleated syncytia and vasculitis resulted

in thrombosis, vascular occlusion, ischaemia and microinfarction. Viral antigens were

immunolocalised to the vascular wall. Viral inclusions, nucleocapsids, antigens and RNA

ii

were also detected in extravascular parenchymal cells especially in neurons. The

pathogenesis of acute NiV infection appears to be a unique dual mechanism of vasculitis-

induced thrombosis, ischaemia/microinfarction, and direct parenchymal cell infection.

In relapsed/late-onset NiV encephalitis, CNS-limited lesions consisted of mildly

inflamed, vacuolated necrotic lesions merging with confluent areas of more extensive

neuronal necrosis and increasing inflammation. Some lesions had a distinctive concentric or

wave-like morphology. There were neuronal viral inclusions, antigen/RNA and

nucleocapsids but vasculitis was absent throughout. These findings suggest that

relapsed/late-onset encephalitis is a recurrent infection rather than post-infectious

encephalitis, and that reinfection is unlikely to have arisen from extraneural foci.

The case of acute HeV infection that had no clinical encephalitis showed evidence

of systemic vasculitis and parenchymal cell involvement. Virus inclusions and

antigens/RNA were found especially in neurons. Relapsed HeV encephalitis was

characterised by severe meningoencephalitis with neuronal infection, inflammatory cells

and reactive blood vessels. Vasculitis was absent. Hence, human HeV infection appears to

be similar to acute and relapsed NiV encephalitis, respectively.

Hamsters with acute henipavirus infections demonstrated vasculitis, thrombosis and

the rare endothelial syncytia in blood vessels of multiple organs. Viral antigens/RNA and

nucleocapsids were localized in vascular and extravascular tissues in the CNS and other

organs. Infectious virus and/or RNA could be recovered from tissues and urine.

The pathology and pathogenesis of acute henipavirus infection in humans and

hamsters appears to be very similar. Human relapsed/late-onset NiV encephalitis and

relapsed HeV encephalitis are both recurrent infections. Thus, the henipaviruses share

common biological characteristics including the ephrin B2 virus entry receptor, pathology

and pathogenesis.

iii

ABSTRAK

Pada tahun 1998, perebakan ensefalitis akut di Malaysia telah terarah kepada penemuan

sejenis “paramyxovirus” yang dikenali sebagai virus Nipah (NiV). Berikutan ini, perebakan

lain telah dilaporkan di Bangladesh dan India. Sehingga kini, jumlah pesakit yang

dijangkiti melebihi 500 orang dengan kadar kematian di antara 40%- 70%. NiV berkaitan

rapat dengan virus Hendra (HeV), kedua-duanya dikategorikan di bawah genus

Henipavirus (Famili: Paramyxoviridae). Perumah semulajadi “henipavirus” adalah kelawar

“pteropid” ataupun kelawar buah yang boleh ditemui di Afrika, Asia-Oceania dan

Australia. Bagi perebakan NiV di Malaysia dan Singapura perumah perantaraan adalah

khinzir, manakala di Bangladesh dan India, ia melibatkan penularan terus dari kelawar

kepada manusia dan dari manusia kepada manusia. Jangkitan akut NiV boleh menjadi

asimptomatik; manakala kes simptomatik dikaitkan dengan manifestasi demam dan sakit

kepala atau ensefalitis akut yang sering dikaitkan dengan koma. Walaupun majoriti pesakit

telah pulih daripada ensefalitis akut tanpa kesan sekuela yang serius, peratusan kecil pesakit

telah menunjukkan perkembangan ensefalitis “relapsed/late-onset” NiV. Jangkitan HeV

juga dikaitkan dengan ensefalitis “relapsed” tetapi ensefalitis akut tidak pernah dilaporkan.

Bagi mengkaji patologi jangkitan NiV, 31 kes manusia dengan jangkitan akut dan 3

kes dengan ensefalitis “relapsed/late-onset” NiV telah diperiksa. Maklumat yang diperoleh

dibandingkan dengan jangkitan HeV dalam manusia, dan jangkitan virus dalam hamster

(Mesocricetus auratus). Tisu-tisu dikaji dengan teknik histologi, immunohistokimia,

mikroskopi elektron, “in situ hybridization” dan teknik-teknik molekular yang lain.

Dalam jangkitan akut NiV, terdapat vaskulitis sistemik, nekrosis parenkima yang

berupa plak dan inflamasi dalam kebanyakan organ, terutamanya sistem saraf pusat.

Kerosakan endotelium vaskular, syncytia multinukleus dan vaskulitis mengakibatkan

iv

trombosis, penyekatan vaskular, iskemia dan infarksi mikro. Antigen virus dikesan pada

dinding saluran darah. Rangkuman virus, nukleokapsid, antigen dan RNA juga telah

dikesan di sel parenkima ekstravaskular terutamanya di neuron. Patogenesis jangkitan akut

NiV adalah unik dengan mekanisme dwinya iaitu trombosis akibat vaskulitis, iskemia dan

infarksi mikro, dan jangkitan terus sel parenkima.

Di dalam ensefalitis “relapsed/late-onset” NiV, lesi-lesi yang terhad kepada sistem

saraf pusat, terdiri daripada lesi nekrosis vakuol yang berinflamasi ringan bergabung

dengan bahagian konfluen yang mempunyai nekrosis neuronal yang luas dan yang

mempunyai inflamasi yang meningkat. Sebilangan lesi nekrosis vakuol mempunyai

bahagian morfologi khusus yang konsentrik atau seperti gelombang. Terdapat juga

rangkuman virus, antigen/RNA dan nukleokapsid di neuron tetapi vaskulitis tidak langsung

dijumpai. Kesemua penemuan ini mencadangkan bahawa ensefalitis “relapsed/late-onset”

NiV adalah jangkitan virus berulang dan bukannya ensefalitis post-infeksi, dan jangkitan

semula ini kemungkinan besar tidak berasal dari luar sistem saraf pusat.

Kes tunggal jangkitan akut HeV yang tiada tanda ensefalitis klinikal, menunjukkan

vaskulitis sistemik dan penglibatan sel parenkima. Rangkuman virus, antigen/RNA telah

dikesan terutamanya di neuron. Dalam ensefalitis “relapsed” HeV, terdapat

meningoensefalitis yang ketara bercirikan infeksi neuron, dan sel-sel inflamasi dan saluran

darah reaktif. Vaskulitis tidak wujud. Oleh itu, jangkitan HeV pada manusia nampaknya

serupa dengan ensefalitis akut dan “relapsed” NiV.

Hamster yang mengidapi jangkitan akut “henipavirus” memanifestasikan vaskulitis,

trombosis, dan juga syncytia (yang jarang dijumpai) dalam saluran darah pelbagai organ.

Antigen virus/RNA dan nukleokapsid telah dikesan di kedua-dua tisu vaskular dan

ekstravaskular di sistem saraf pusat dan organ lain. Virus berinfeksi dan/atau RNA virus

boleh diperoleh daripada kebanyakan tisu dan air kencing.

v

Patologi dan patogenesis jangkitan akut “henipavirus” di dalam manusia dan

hamster adalah sangat serupa. Kedua-dua ensefalitis “relapsed/late-onset” NiV dan

ensefalitis “relapsed” HeV di manusia adalah jangkitan berulang. Oleh itu, “henipavirus”

berkongsi ciri-ciri biologi yang sama seperti reseptor kemasukan virus ephrin B2, patologi

dan patogenesis.

vi

ACKNOWLEDGEMENT In an effort like this that spanned several years, there are numerous people who have helped

along the way, in one way or another.

I humbly acknowledge with gratitude the great contribution and assistance from

staff of the University of Malaya Medical Centre, Hospitals in the Ministry of Health

(Malaysia), Centers for Disease Control and Prevention (Atlanta, Georgia, USA), Pasteur

Institute and INSERM (France), National Institute of Infectious diseases (Japan), in this

research effort.

For the initial human Nipah virus infection study, the monumental effort to collect

all the materials together resulted in a collaborative effort with my friends and fellow

pathologists Dr Norain Karim and Dr Shalini Kumar from the Ministry of Health,

Malaysia, and Dr SR Zaki, Dr WJ Shieh, C Goldsmith and Dr J Guarner in the CDC,

Atlanta, USA. The University of Malaya generously supported the trip and gave me leave

to join the group in Atlanta. For the unstinting generosity to share study materials and

expertise, and scientific enthusiasm and the spirit of international collaboration that I

experienced, I am most grateful to members of this group. Many other staff from the

pathology labs in Malaysia and the CDC helped with tissue preparation,

immunohistochemistry and electron microscopy.

The hamster Nipah infection work was largely done in Lyon, France, together with

staff from INSERM and UBIVE of the Pasteur Institute. Prof KL Lam and Dr V Deubel

played key roles in kindly inviting me to participate in this project. For help in the scientific

vii

work, I am thankful to Dr V Deubel, Dr F Wild, Dr MC Georges-Courbot, Grosjean I,

Blanquier B, Dr Brisson C, Dr Fevre-Montange M, Dr A Bernard and Dr M Chevallier.

They were involved in various ways, in the infection experiments in the biosafety level 4

lab, tissue preparation, immunohistochemistry, in situ hybridization and molecular work.

The hamster Hendra infection work that followed was greatly assisted by Dr V Guillaume,

Dr B Horvat and Dr F Wild who provided the infected animals for pathological analysis.

I owe a great debt to my friends and colleagues, Prof CT Tan, Prof KJ Goh, Prof

HT Chong and other neurologists involved in the Malaysian outbreak. They generously

provided me with the essential clinical input and helpful discussions that was so critical to

the analysis of the pathological data. Many of the autopsies were also obtained on the

strength of their persuasion and caring relationship with the deceased and their next-of-kin.

Great credit is due the relatives of the deceased for recognising the need for autopsy to

investigate the cause of death despite such difficult circumstances.

The virologists, Dr KB Chua and Professor SK Lam, most willingly provided me

much needed laboratory data and other information to complete the study. Tanaka-san and

Dr T. Sata from the National Institute of Infectious Diseases, Japan, helped with the

electron microscopy and immunoelectron microscopy. For the preparation of large brain

sections, I have to thank Mr KK Chan, Addenbrookes Hospital, United Kingdom and Dr H

Kojima, Tokyo Metropolitan Institute for Neuroscience, Japan. The primary antibodies so

critical to the success of immunohistochemistry were generously donated by Dr P Hooper

(CSIRO, Australia) and Dr SR Zaki (CDC, Atlanta). Dr Linfa Wang provided the Hendra

viral plasmids for which I am happy to acknowledge.

viii

I am grateful to Prof LM Looi for agreeing to be my supervisor despite her

enormous work load. Her wise counsel at the height of the Malaysian outbreak is very

much appreciated. I would also like to thank my former students Dr KC Ong and Dr KC

Yaiw and JW Chong for help with the development of the probes and in situ hybridisation

work. Mr YX Chin helped me with the finer technical aspects of digital image processing

and publishing, and P Anada Raj, the Bahasa Malaysia version of the abstract.

Much of the research was supported by the Malaysian government grants, 06-02-03-

0743, 06-02-03-0000PR0060/04 and other grants. I am grateful to the Ministry of Science,

Technology and Innovation for their generosity. In addition, I received travel grants from

the French Embassy, Kuala Lumpur to go to Lyon, France, for which I am very thankful.

Last but not least, I wish to thank my father Mr PC Wong who throughout my life

has always been most supportive of my academic endeavours (as opposed to monetary

pursuits), even though, he himself was not fortunate enough to attend a regular school,

much less a university. To my extended family of friends, thank you for the support. To Dr

Z Hasan, still my best friend after more than 20 years despite various trials and tribulations,

I thank you.

ix

TABLE OF CONTENTS

Page

Preface

Abstract

i

Acknowledgement

iv

List of Figures

xv

List of Tables

xviii

Lists of Abbreviations and Symbols

xix

Chapter 1 Introduction

1.1 Emerging Viruses and Outbreaks

1

1.2 Viral Spread into the CNS

4

1.3 Pathology of Viral Encephalitis

6

1.4 Virology of Henipavirus

8

1.5 NiV Transmission to Humans

10

1.6 Clinical Manifestation of Human NiV Infection

11

1.7 Laboratory and Radiological Investigations in Human NiV Infection

12

1.8 Prognosis, Complications and Sequelae in Human NiV Infection

15

1.9 Acute NiV Infection in Animals

18

2.0 Pteropid Bats as Reservoir Hosts of Henipaviruses

19

2.1 Objectives

24

x

TABLE OF CONTENTS

Page

Chapter 2 Materials and Methods

2.1 Human Acute NiV Infection

25

2.1.1 Demographic and Clinical data

25

2.1.2 Autopsy Tissues

25

2.1.3 Antibodies, Cell and Tissue Controls

26

2.1.4 IHC

26

2.1.5 ISH

26

2.1.6 EM

30

2.1.7 NiV Antibody Detection Assays

30

2.1.8 Virus Isolation and Identification

30

2.2 Human Relapsed or Late-Onset

NiV Encephalitis

31

2.2.1 Demographic and Clinical Data

31


31

2.2.3 IHC

32

2.2.4 ISH

32

2.2.5 EM and IEM

33

2.2.6 Large Brain Section Topographic Study

33

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TABLE OF CONTENTS

Page

2.3 Human HeV Infection

34


34


34

2.3.3 IHC

35

2.3.4 ISH

35

2.4 Hamster Acute NiV Infection

37

2.4.1 Virus Stock and Titration

37

2.4.2 Animal Infection Experiments

37

2.4.3 Virus Isolation and Titration

39

2.4.4 NiV Antibody Testing

39

2.4.5 RT-PCR

39

2.4.6 Hamster Tissues

40

2.4.7 IHC

40

2.4.8 ISH

40

2.4.9 EM and IEM

41

2.5 Hamster Acute HeV Infection

41


41


41

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TABLE OF CONTENTS

Page

2.5.3 IHC

42

Chapter 3 Results


43

3.1.1 Demographic and Clinical Features

43

3.1.2 Pathological Features

43

Blood vessels 48 CNS 50 Lung 58 Kidney 58 Lymphoid organs 59 Heart 59 Other organs

59

3.1.3 EM

65

3.1.4 IHC 65 Antibody specificity 65 Blood vessels 65 CNS 67 Non-CNS organs

67

3.1.5 NiV Antibody Detection Serological Assays

68

3.1.6 Correlation of IHC and Serological Test Results

68

3.1.7 Temporal Evolution of Lesions and Presence of Viral Antigens in the CNS

71

3.1.8 Virus Isolation

71

3.1.9 ISH

72

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TABLE OF CONTENTS

Page

3.2 Human Relapsed/Late-Onset NiV Encephalitis

74


74

3.2.2 Pathological Features 74 CNS 74 Non-CNS organs

83

3.2.3 IHC and ISH

83

3.2.4 EM and IEM

86

3.3 Human Acute HeV Infection

90

3.3.1 Pathological Features 90 Blood vessels 90 CNS 90 Non-CNS organs

96

3.4 Human Relapsed HeV Encephalitis

101


101


104

3.5.1 Animal Infection Experiments: Survival and LD

50

104

3.5.2 Viral Isolation and Viral Genome Detection

106

3.5.3 Pathological Features 106 Blood vessels 106 CNS 109 Non-CNS organs

112

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TABLE OF CONTENTS

Page


119


119

Chapter 4 Discussion 124


124


130


136


137


138


142

Chapter 5 Conclusions 143

Chapter 6 References 145

Appendix A: Hybridisation Solution for ISH

160

B: Anti-Nipah Antibody Assay in Human Samples

160

C: Nipah Virus Culture and Identification in Human CSF

161

D: Nipah Virus Stock and Titration

162

E: Anti-Nipah Antibody Assay in Hamster Samples

162

F: List of Publications

163

xv

LIST OF FIGURES

Page

Figure 1.1 Nipah virus outbreaks in Malaysia and Singapore

5

Figure 1.2 Cell culture of Nipah virus

9

Figure 1.3 Brain MRI in human Nipah encephalitis

14

Figure 1.4 Sequelae and complications of Nipah virus infection

17

Figure 1.5 World map showing distribution of pteropid or fruit bats

21

Figure 1.6 Likely routes of henipavirus transmission from animals to humans

23

Figure 3.1 Vascular pathology and viral immunolocalisation in human acute Nipah virus infection

52

Figure 3.2 CNS pathology and viral immunolocalisation in human acute Nipah virus infection

54

Figure 3.3 CNS pathology and viral immunolocalisation in human acute Nipah virus infection.

56

Figure 3.4 Pulmonary pathology and viral immunolocalisation in human acute Nipah virus infection

60

Figure 3.5 Pathology and viral immunolocalisation in spleen and kidney in human acute Nipah virus infection

62

Figure 3.6 Pathology and viral immunolocalisation in lymph node, heart and adrenal gland in human acute Nipah virus infection

64

Figure 3.7 Ultrastructure of Nipah virus inclusions as seen in the CNS

66

Figure 3.8 Temporal relationship of IgM and IgG in cerebrospinal fluid and serum in fatal human acute Nipah virus infection

69

Figure 3.9 Temporal distribution of microscopic lesions and viral antigens in the central nervous system in fatal human acute Nipah virus infection

73

xvi

LIST OF FIGURES

Page

Figure 3.10 Macroscopic features of brain sections and distribution of lesions in human relapsed/late-onset Nipah virus encephalitis

77

Figure 3.11 CNS pathology in human relapsed/late-onset Nipah virus encephalitis

81

Figure 3.12 CNS pathology and virus immunolocalisation in human relapsed/late-onset Nipah encephalitis

84

Figure 3.13 CNS pathology in resolving acute Nipah virus encephalitis

87

Figure 3.14 Ultrastructure of infected neurons in human relapsed/late-onset Nipah virus encephalitis

89

Figure 3.15 Vascular and CNS pathology in human acute Hendra virus infection

94

Figure 3.16 CNS pathology in human acute Hendra virus infection

97

Figure 3.17 Pathology in the lung, heart, kidney and lymph node in human acute Hendra virus infection

99

Figure 3.18 CNS pathology in human relapsed Hendra virus encephalitis

102

Figure 3.19 Survival graphs of hamsters infected by Nipah virus via different routes and doses (LD50

study) 105

Figure 3.20 Gel Electrophoresis Results of Reverse Transcriptase-Polymerase Chain Reaction in Hamster Tissues with Acute Nipah Virus Infection by Intranasal Route (LD50

Study)

108

Figure 3.21 Vascular and parenchymal pathology in hamster acute Nipah virus infection

110

Figure 3.22 CNS pathology in hamster acute Nipah virus infection

114

Figure 3.23 Pathology in lung, kidney and spleen in hamster acute Nipah virus infection

116

Figure 3.24 Ultrastructure of infected neurons in Hamster acute Nipah virus infection

118

Figure 3.25 Pathology of hamster acute Hendra virus infection

121

xvii

LIST OF FIGURES

Page

Figure 4.1 Summary of the dual pathogenetic mechanisms of acute Nipah virus infection

128

Figure 4.2 Hypothesis for the pathogenesis of human relapsed/late-onset Nipah virus encephalitis

132

xviii

LIST OF TABLES

Page

Table 2.1 Nipah Virus Nucleoprotein Gene Specific Primers and

Reverse Transcription-Polymerase Chain Reaction (RT-PCR) Conditions

28

Table 2.2 Hendra Virus Nucleoprotein Gene Specific Primers and Polymerase Chain Reaction (PCR) Conditions

36

Table 3.1 Clinical and Laboratory Data of Fatal Human Cases of Acute Nipah Virus Infection

44

Table 3.2 Frequency of Clinical Symptoms and Signs in Human Acute Nipah Virus Infection

47

Table 3.3 Frequency of Necrosis, Vasculitis and Viral Antigens in Major Organs in Human Acute Nipah Virus Infection

49

Table 3.4 Frequency of Microscopic Features in the Central Nervous System in Fatal Human Cases of Nipah Virus Infection

51

Table 3.5 Concordance of Immunohistochemistry and Serological Assays as Diagnostic Tests in Fatal Human Cases of Acute Nipah Virus Infection

70

Table 3.6 Demography, Clinical and Pathological Data of Fatal Human Cases of Relapsed/Late-onset Nipah Virus Encephalitis

75

Table 3.7 Demography, Clinical and Pathological Data of Fatal Human Cases of Human Hendra Virus Infection

92

Table 3.8 Reverse transcriptase-Polymerase Chain Reaction (RT-PCR) Analysis, Virus Culture and Pathological Lesions in Hamster Nipah Virus Infection

107

xix

LIST OF ABBREVIATIONS

BCIP 5-Bromo-4-Chloro-Indolyl Phosphate BSL-4 Biosafety level 4 CSF Cerebrospinal fluid DAB 3, 3’-Diaminobenzidine DIG Digoxigenin DNA Deoxyribonucleic acid dUTP Deoxyuridine triphosphate nucleotide EDTA Ethylenediaminetetraacetic acid ELISA Enzyme-linked immunosorbent assay EM Electron microscopy H&E Haematoxylin and eosin HeV Hendra virus IEM Immunoelectron microscopy IHC Immunohistochemistry IN Intranasal IP Intraperitoneal ISH In situ hybridisation JE Japanese encephalitis LD Lethal dose in 50% of animals 50 MRI Magnetic resonance imaging NBT Nitroblue tetrazolium NiV Nipah virus PBS Phosphate buffered saline PCR Polymerase chain reaction pfu Plaque forming unit RNA Ribonucleic acid rpm Rounds per minute RT Reverse transcriptase SSPE Subacute sclerosing panencephalitis TBS Tris-buffered saline LIST OF SYMBOLS

M molar mg milligram ml millilitre µm micron

1

CHAPTER 1: INTRODUCTION

1.1 Emerging Viruses and Outbreaks

The last few decades saw the emergence and re-emergence of many human pathogenic

viruses from practically all major families of viruses. Many of these outbreaks were

zoonoses, due either to known or novel viruses that have jumped the species barrier to

infect humans. One of the most devastating zoonosis to date is caused by the human

immunodeficiency virus, which is believed to have originated from African primates

(Holmes, 2001). Influenza virus (H5N1) derived from avian species have caused serious

morbidity and mortality in far fewer people but nonetheless has already posed a potentially

serious health threat to millions of people (Peiris et al., 2004, CDC, 1997). A new

hantavirus called Sin Nombre, emerged to spread from rodents to humans to cause the

hantavirus pulmonary syndrome in the Americas (Nichol et al., 1993, Zaki et al., 1995).

Zoonotic viral encephalitides due to known arboviruses continue to cause deaths in

endemic countries and also spread to other previously unaffected areas. Thousands of

Japanese encephalitis (JE) virus infections, transmitted from birds, still occur in the Indian

subcontinent and elsewhere, despite the availability of vaccines (Kabilan et al., 2004). West

Nile virus, another known bird-transmitted arbovirus, recently emerged in New York state,

USA, to cause severe disease in humans and animals (Lanciotti et al., 1999, CDC, 2007). In

a short span of a decade, it has now spread to the entire country thus extending dramatically

its previous known range in Africa and the Middle East.

As far as newly emerging or novel viruses are concerned, one of the most important

natural mammalian hosts is the bat. Forty viruses have been isolated from bat species in as

many years recently. These include Ebola virus, Australian bat lyssavirus, SARS

2

coronavirus, Menangle virus, Tioman virus, Hendra virus (HeV) and Nipah virus (NiV)

(Eaton, 2001, Wong et al., 2007, Calisher et al., 2006). Because of the ability of bats to fly

to large areas of their habitat, theoretically they may to be particularly effective for virus

dissemination. However, not all bat-derived viruses have been shown to be pathogenic. Of

the pathogenic viruses, the highest human mortalities are probably due to infections by

Ebola virus, SARS coronavirus and NiV.

Menangle virus, Tioman virus, Hendra virus (HeV) and Nipah virus (NiV) all

belong to the family Paramyxoviridae, subfamily Paramyxovirinae, and comprised the

latest additions to this medically important family of viruses. Older paramyxoviruses such

canine distemper and measles, though not bat-derived, have long been associated with

animal and human diseases. The Menangle virus and Tioman virus (genus: Rubulavirus)

were discovered in 1997 and 1999, respectively (Chua et al., 2001b, Philbey et al., 1998).

Both viruses can cause disease in pigs and seroconversion in humans (Yaiw et al., 2008,

Philbey et al., 1998, Yaiw et al., 2007). Human Menangle virus infection is associated with

a relatively mild flu-like illness with no fatalities reported so far. Tioman virus was

discovered in the same bats that yielded the first NiV isolates in Tioman Island (Figure 1.1)

but so far no known human disease has been associated with it. However, there was

serological evidence of neutralising antibodies to Tioman or Tioman-like viruses in a few

Tioman islanders (Yaiw et al., 2007).

HeV was first isolated following an outbreak in horses and 2 humans in the town of

Hendra, Australia in 1994 (Selvey et al., 1995, Murray et al., 1995). Both patients had

worked with HeV-infected horses. One of them died from the infection, while the other

survived. After the first outbreak, more outbreaks in horses and humans were reported

(Hanna et al., 2006, O'Sullivan et al., 1997, Rogers et al., 1996, Field et al., 2000). The

third human case died from the disease in 1995 but he was believed to have first contracted

3

the infection in 1994, even before the first outbreak occurred and HeV was discovered

(O'Sullivan et al., 1997, Hanna et al., 2006). A non-fatal, fourth human case was reported in

2004 (Hanna et al., 2006). More recently, 2 further human cases, one of them fatal, have

been reported, making a total of 6 cases with 3 fatalities so far (Hanna et al., 2006,

ProMED-mail, 2008). All the human cases have had close contact with infected horses.

Indeed, there is now considerable evidence that horses are the intermediate hosts for viral

transmission to man (McCormack et al., 1999, Williamson et al., 1998).

The first reported NiV outbreak started around September of 1998 in the pig farms

just outside Ipoh, a town of about half a million people in the northern part of peninsular

Malaysia (CDC, 1999a). From this epicentre (Figure 1.1), there were about 27 patients

infected with 15 fatalities, 9 of whom were subsequently confirmed to have NiV infection

at postmortem (CDC, 1999b). The outbreak spread to several farms in the south following

the inadvertent transport of infected pigs from Ipoh (Mohd Nor et al., 2000). This area that

included the neighbourhoods of Sikamat, Kampung Sungai Nipah, Kampung Sawah and

Bukit Pelanduk was to become the second - and most severely affected - epicentre of the

outbreak. The virus was named “Nipah” after Kampung Sungai Nipah (Nipah river village)

whose patients’ specimens yielded the first virus isolates. It is estimated that this 2nd

Several infected pigs exported to an abattoir in Singapore spread the infection to 11

abattoir workers (CDC, 1999a, Paton et al., 1999). Later from around Sepang and Sungai

Buloh, several more cases were reported (Figure 1.1). Published sources have quoted a

prevalence of 265 cases of acute NiV encephalitis with 105 fatalities in Malaysia (Parashar

et al., 2000). If asymptomatic or mildly symptomatic, non-encephalitic seroconvertors that

number about 89 cases (Tan et al., 2002) are included, the total number is probably more

that 350 cases (Wong et al., 2002). Sadly, political, logistic, diagnostic and other

epicentre involved more than 180 people (CDC, 1999a).

4

difficulties may prevent the true figures from being known. After 1999, in Malaysia,

Singapore and other countries of Southeast Asia, there are no further reports of new NiV

infections. Unfortunately, beginning 2001, several recurrent outbreaks of NiV-like viral

encephalitides reported in Bangladesh and India were later confirmed to be caused by NiV

(Hossain et al., 2008, Harit et al., 2006). So far, these outbreaks in the Indian subcontinent

have involved more than 120 people.

1. 2 Viral Spread into the CNS

Neurotrophic viruses usually spread into the CNS by 2 classical routes (Cassady and

Whitley, 1997). The first is a haematogenous route and subsequent crossing of the cerebral

vascular blood-brain-barrier or crossing of blood vessels in the choroid plexus. In the

former, virus gains access into the parenchymal cells directly, whereas in the latter, virus

first gets into the CSF and thence into the CNS parenchyma. Neurotrophic arboviruses such

as JE, West Nile and St Louis encephalitis and paramyxoviruses such as measles spread by

the haematogenous route. These viruses appear to be able to cross the blood-brain-barrier

without causing vasculitis.

The second possible route is via retrograde peripheral nerve transmission as

exemplified by rabies and herpes simplex (Cassady and Whitley, 1997). Rabies virus

spreads from peripheral nerves to the CNS from the site of infected animal bite while

herpes simplex is believed to enter the CNS via the nerve endings of the olfactory bulb in

the cribriform plate. It then appears to spread by neural pathways from olfactory bulb to the

cingulate gyri and temporal lobes.

Tioman Island

*

1st Epicentre

2nd Epicentre

A

B C

Figure 1.1Nipah virus outbreaks in Malaysia and Singapore. A: Map of peninsular Malaysia showing the 1st and 2nd epicentres of Nipah virus outbreaks in pig farms, and subsequent spread to Singapore. Tioman Island is shown by the asterisk. A pig farm in the 1st epicentre (B) and the village of Sungai Nipah in the 2nd epicentre (C). Courtesy of Dr KB Chua and Prof C T Tan.

5

6

1.3 Pathology of Viral Encephalitis

The general pathological features of the most common forms of viral encephalitis are well

known for many years. Typically, there will be cell death, cell alterations, inflammation,

glial reaction and viral inclusions (Booss and Esiri, 1986). The major target in most viral

encephalitides is the neuron. Degenerate neuronal features include swelling or shrinkage

with eosinophilia of cytoplasm and nuclear pyknosis. Glial cell may also undergo viral

cytolysis in some virus infections but the process is often less conspicuous than in neurons.

If parenchymal cell necrosis is massive, it may be resemble infarction macroscopically as in

the case of herpes simplex encephalitis. Other rarer cell alterations include neurofibrillary

tangles in some cases of SSPE and herpes simplex encephalitis, and bizarre astrocytes in

progressive multifocal leukoencephalopathy caused by the JC virus.

Acute inflammation sets in after a few days of infection and usually begins with

infiltration of neutrophils in the early stages, followed by macrophages, lymphocytes and

still later, plasma cells. Neuronophagia refers to the surrounding of infected or degenerate

neuron by these inflammatory cells. Aggregates of microglial cells, lymphocytes and a few

astrocytes may remain after the infected cell remnant has disappeared giving rise to the

characteristic microglial nodule or star (Booss and Esiri, 1986). The parenchyma becomes

oedematous producing a spongy appearance and inflammatory cells begin to appear around

venules. After a week or more, perivascular cuffing by inflammatory cells becomes more

prominent.

Viral inclusions or inclusion bodies are characteristic features but are not found in

all forms of viral encephalitides. They consist of viral proteins and other components and

may be found in the cytoplasm, nucleus or both. Nuclear inclusions are found in measles,

herpes simplex, cytomegalovirus, varicella zoster and JC virus infections. The presence of

7

viral inclusions is often good evidence of viral infection, especially if they are shown to

contain viral antigens/RNA. Viral inclusions may be found in viral encephalitides due to

paramyxoviruses (e.g. measles and canine distemper virus), herpesvirus (e.g. herpes

simplex and cytomegalovirus), rabies and JC virus. No viral inclusions are found in

encephalitides caused by enteroviruses (e.g. poliovirus and enterovirus 71) and arboviruses

(e.g. JE, West Nile virus, Tick-borne encephalitis virus and St Louis encephalitis virus).

The distribution and relative intensity of inflammatory lesions in the CNS as shown

by magnetic resonance imaging (MRI) or pathological studies may provide useful clues to

the diagnosis of viral encephalitis. Good examples are herpes simplex encephalitis

(temporal lobe, cingulate gyrus), poliovirus and enterovirus 71 encephalitis (spinal cord

anterior horn, brainstem, hypothalamus and cerebellar dentate nucleus), arboviral

encephalitis (thalamus, basal ganglia, brainstem) (Booss and Esiri, 1986, Wong et al.,

2008).

Among the paramyxoviruses that can cause CNS disease, measles virus is perhaps

the most interesting. An acute infection may be complicated by post-infectious

encephalomyelitis, the result of an autoimmune phenomenon against oligodendroglia in the

central nervous system (CNS) (Esiri and Kennedy, 1997). This phenomenon which has also

been described following other viral infections, is characterised pathologically by

perivenous demyelination. More rarely, measles inclusion body encephalitis (or subacute

measles encephalitis) can occur a few weeks after an acute measles infection in patients

who are immunocompromised, e.g. patients who have concomitant lymphoid malignancies

(Esiri and Kennedy, 1997). Subacute sclerosing panencephalitis (SSPE) has also been

causally linked to the measles virus but it usually occurs several years after the acute

infection. Pathologically, both measles inclusion body encephalomyelitis and SSPE has

been shown to be due to direct infection of neurons and other parenchymal cells in the

8

CNS. Both cytoplasmic and nuclear viral inclusions and nucleocapsids can be found in

these conditions. Moreover, SSPE is thought to be associated with measles virus mutations

(Cattaneo et al., 1989).

1.4 Virology of Henipavirus

Because HeV and NiV share a high genomic homology and other characteristics that make

them distinct from other paramyxoviruses, a new genus Henipavirus (Hendra + Nipa

However, the genome size of henipaviruses of about 18.2 kb is much larger than the

more usual 15.1 to 15.9 kb of other members of Paramyxovirinae. Compared to the

genome of HeV, at 18,246 nucleotides, NiV is 12 nucleotides longer (Harcourt et al., 2001).

There is a high degree of nucleotide homology in the open reading frames of the various

genes of HeV and NiV that exceeds 70%, and a high amino acid identity of more than 80%

in most genes (Harcourt et al., 2000). For example, the N gene nucleotide homology

between HeV and NiV is 78%, but these viruses have no more than 49% similarity

compared with other members of the subfamily (Chua et al., 2000a).

h) was

created to accommodate them in the family Paramyxoviridae and subfamily

Paramyxovirinae (Wang et al., 2000). Like other paramyxoviruses, henipaviruses are

enveloped viruses (Figure 1.2), have negative-strand RNA genomes (Wang et al., 2001) and

form syncytia in cell cultures. The general genomic structure possesses 6 transcription units

encoding 6 major structural proteins viz., nucleoprotein (N), phosphoprotein (P), matrix

protein (M), fusion protein (F), attachment protein (G) and large protein (L) or RNA

polymerase, in the order 3’-N-P-M-F-G-L-5’.

1

Figure 1.2Cell culture of Nipah virus. Fuzzy-type nucleocapsids (arrows) within cell and budding enveloped virus particles (arrowheads) emerging from cell surface. Courtesy of Dr. K.B. Chua.

9

10

1.5 NiV Transmission to Humans

It has now been confirmed, at least in the Malaysia/Singapore outbreak, that direct contact

with pigs or fresh pig products was responsible for viral transmission to humans. Parashar

et al., reported that among 110 NiV-infected patients a significantly higher number of 59%

versus 24% of community farm controls reported increased numbers of sick or dying pigs

on the farm. Moreover, patients were significantly more likely than controls (86% versus

50%) to perform activities requiring direct contact with these animals (Parashar et al.,

2000). There were also reports of infection, albeit at a lower rate, among abattoir workers

and pork sellers (Sahani et al., 2001, Premalatha et al., 2000, Chew et al., 2000).

Among army personnel involved with culling of infected pigs, and who could have

had physical contact with these animals despite protective gear, 6 out of 1412 personnel

investigated seroconverted. Of these, 2 soldiers were reported to have developed NiV

encephalitis (Ali et al., 2001). Widespread surveillance of pig populations to detect infected

pigs and culling of sick pigs stopped the epidemic (CDC, 1999b). In Singapore the banning

of pig imports from Malaysia, and abattoir closure also stopped the outbreak there (Chew et

al., 2000, CDC, 1999b). Possible viral transmission to humans from other animals e.g. cats

and dogs, have been suggested in a small number of patients (Parashar et al., 2000, Tan et

al., 1999). There was a higher prevalence in males especially from the Chinese ethnic

group, as this group more than others, were traditionally involved in pig farming (Goh et

al., 2000).

Viral transmission to Malaysian health care workers was thought to be generally

low (Mounts et al., 2001). In a large survey of 288 health care workers, only 3 were found

to have IgG antibodies but IgM and serum neutralisation tests were negative, and thus these

cases were thought to be false positives. However, a nurse who had previously cared for an

11

NiV-infected patient, subsequently seroconverted but remained asymptomatic. Her brain

MRI showed a few discrete lesions typically seen in acute NiV encephalitis (Tan et al.,

2000, Tan and Tan, 2001). Therefore, in Malaysia human-to-human transmission is very

low but still possible as exemplified by this case, and the fact that virus could be isolated

from patients’ secretions (Chua et al., 2001a).

In contrast, evidence from the Bangladesh/India outbreaks showed a high incidence

of human-to-human transmission either to health care workers or other people in contact

with infected patients e.g. family members (Harit et al., 2006, Hsu et al., 2004, Gurley et

al., 2007). Interestingly, no animal has been positively identified as possible intermediate

hosts so far, although in one outbreak, sick patients were reported to be more likely to have

contact with a sick cow (Hsu et al., 2004). Contaminated date palm sap, a local delicacy in

Bangladesh has been implicated in some cases (Luby et al., 2006).

1.6 Clinical Manifestation of Human NiV Infection

The incubation period was difficult to determine with certainty but if duration of fever is

any indication, it appears to range from a few days to 2 weeks (Goh et al., 2000, Chong et

al., 2000). A significant number of people exposed to the virus appeared to become

symptomatic after exposure. It is estimated that the proportion of symptomatic versus

asymptomatic seroconvertors is roughly three to one (Tan et al., 1999). A wide spectrum of

clinical manifestations ranging from milder symptoms of fever, headache and drowsiness,

to a severe, fatal acute encephalitic syndrome has been reported (Lee et al., 1999, Goh et

al., 2000, Chua et al., 1999, Paton et al., 1999). In a clinical analysis of 90 patients

diagnosed with acute NiV encephalitis treated at the University of Malaya Medical Centre,

Malaysia, the main presenting features were fever, headache, dizziness, vomiting, and

12

reduced level of consciousness (Goh et al., 2000). In fact, more than 50% of patients with

acute NiV encephalitis were found on clinical examination to have some degree of reduced

consciousness.

Distinctive clinical signs such as areflexia, hypotonia, abnormal pupillary response,

tachycardia, hypertension, abnormal doll’s eye reflex and segmental myoclonus, suggested

involvement of the brainstem and upper cervical cord. Absent or reduced tendon reflexes

with hypotonia were seen in 56% of patients especially in patients with reduced

consciousness. Segmental myoclonus, which occurred in 32% of patients, was

characterised by focal, rhythmic jerking of muscles. The diaphragm and other muscles in

the arms, legs, neck and face were prominently involved. Meningism was noted in 28 %,

while seizures mainly in the form of generalised tonic-clonic convulsions, were recorded in

23% of patients. Electroencephalography most commonly showed continuous diffuse,

symmetrical slowing with or without focal discharges (Chew et al., 1999).

There appeared to be involvement of the respiratory tract in relatively few patients

with only 14% reported to have unproductive cough (Goh et al., 2000). In another series

from the Seremban hospital, 24% of patients had abnormal findings in the chest x-rays but

none had a primary lung disease (Chong et al., 2000). In the Singapore series of 11 patients,

3 were clinically thought to have atypical pneumonia with abnormal chest x-rays (Paton et

al., 1999).

1.7 Laboratory and Radiological Investigations in Human NiV Infection

Not surprisingly, cerebrospinal fluid (CSF) examination revealed abnormal findings in

many patients (Goh et al., 2000, Lee et al., 1999). In a large series, more than 75% of

patients showed elevated protein levels and/or elevated white cell counts on repeated CSF

13

examination (Goh et al., 2000). Both protein levels and white cell counts were elevated in

39% of patients at first examination. Glucose levels were generally within normal limits.

Thrombocytopenia was a feature in 30-66 % of patients while leukopenia was

present in 11-60 %. Alanine and aspartate aminotransferases were elevated in 33-61 % and

42-60 % of patients respectively, but blood urea, creatinine, and electrolyte levels remained

normal in most patients (Chong et al., 2000, Goh et al., 2000). Thrombocytopenia and

leukopenia were also reported in the Singapore series (Paton et al., 1999). Specific anti-NiV

IgM and IgG antibodies, critical to the diagnosis of NiV infection, could be detected in the

serum and CSF in many patients. For IgM seroconversion, by day 4 it was about 65%, and

by day 12, 100%. IgM persisted for at least 3 months in most patients. For IgG, there was

100% seroconversion by day 25 (Ramasundram et al., 2000).

Brain MRI proved to be a useful diagnostic aid in acute NiV encephalitis (Sarji et

al., 2000, Lim et al., 2000). Typically, in acute NiV encephalitis the T2-weighted and

FLAIR brain MRI showed multiple, disseminated, small discrete hyperintense lesions

mainly in the subcortical and deep white matter, and occasionally in the cortex (Figure 1.3).

The lesions measured about 2-7 mm in diameter. At a later phase of uncomplicated acute

encephalitis, these lesions were also seen and generally did not enlarge, although there may

be a reduction in size (Sarji et al., 2000, Lim et al., 2002). Interestingly, cerebral cortex

lesions may become more prominent (Lim et al., 2002). In 3 Bangladeshi patients with

apparent acute NiV encephalitis and available brain MRI findings, only 1 patient showed

the same discrete hyperintense lesions while surprisingly, the other 2 showed multiple

confluent lesions (Quddus et al., 2004).

14

Figure 1.3 Brain MRI in human Nipah encephalitis. A: Axial brain MRI (fluid attenuated inversion recovery) shows multiple discrete hyperintense lesions in the white and grey matter of a patient with acute Nipah encephalitis. B: Axial brain MRI findings in a patient with relapsed Nipah encephalitis showing confluent lesions involving primarily the cortical grey matter. Courtesy of Dr KJ Goh.

15

In some patients where viral culture was attempted, virus could be isolated from

CSF, urine, and throat and nasal secretions (Chua et al., 2000b, Chua et al., 2001a). Viruses

isolated from these different sites have practically identical genomes (Chan et al., 2001).

The cytopathic effect in cell cultures showed giant syncytia formation as would be expected

of paramyxoviruses (Chua et al., 1999). In infected cell cultures, using ultrastructural

features such as the type and distribution of nucleocapsid aggregates and viral envelope

differences, it is possible to distinguish NiV, HeV and other paramyxoviruses from each

other. Within infected cells the combination of single fringed viral envelopes and

peripherally distributed type 1 nucleocapsids is characteristic of NiV (Hyatt et al., 2001).

Direct viral visualisation by electron microscopy (EM) in negatively-stained preparations of

CSF specimens was also found to be useful for diagnosis but may not be able to distinguish

NiV from other paramyxoviruses (Chow et al., 2000).

1.8 Prognosis, Complications and Sequelae in Human NiV Infection

Overall, poor prognostic factors are not well known but there were suggestions that in acute

NiV encephalitis, brainstem involvement, presence of virus in the CSF and diabetes

mellitus conferred a poorer prognosis (Goh et al., 2000, Chong et al., 2001, Chua et al.,

2000b).

The mortality of acute NiV encephalitis is about 40% in Malaysia (Parashar et al.,

2000) and about 70% in Bangladesh/India (Hossain et al., 2008, Harit et al., 2006). In many

of the Malaysian patients who recovered there were no apparent serious sequelae (Goh et

al., 2000). Nonetheless, more subtle neuropsychiatric sequelae have never been

investigated. In one rare case who recovered from a coma, the patient was able to walk and

16

communicate, but later developed a fatal intracerebral haemorrhage while still in the ward

(Goh et al., 2000).

Most unexpectedly, a small number of patients suffered a second or even a third

neurological episode following apparent complete recovery. These “relapsed NiV

encephalitis” patients constitute about 8% of the total number of survivors (Tan et al.,

2002). Symptoms appeared after an average of about 8 months following viral exposure. In

addition about 3 % who were either asymptomatic or only had mild non-encephalitic acute

illness also developed similar neurological episodes for the first time several months later

(“late-onset NiV encephalitis”). Clinical, radiologic and preliminary pathological findings

suggested that relapsed and late-onset NiV encephalitis are essentially the same disease

process, but distinct from acute NiV encephalitis (Tan et al., 2002, Sarji et al., 2000). The

major clinical differences include a significantly lower incidence of fever, headache,

areflexia, coma, abnormal pupils, segmental myoclonus and meningism in relapsed/late-

onset encephalitis. Conversely, focal neurological signs and seizures were significantly

higher compared to acute NiV encephalitis. The T2-weighted and FLAIR brain MRI in

relapsed/late-onset encephalitis showed patchy, confluent areas of cortical lesions (Figure

1.3). The sequelae and complications of acute NiV infection are summarised in Figure 1.4.

17

Figure 1.4 Sequelae and complications of Nipah virus infection

Nipah Virus Infection

Asymptomatic or Mild Symptoms

Acute Encephalitic Syndrome (+/- pulmonary symptoms)

Late-Onset or Relapsed Encephalitis

Recovery +/- Neurologic Deficit

Massive Cerebral Haemorrhage

Death

Symptoms

≈ 3%

≈ 18%

40-70%

rare

≈ 8%

18

1.9 Acute NiV Infection in Animals

NiV-infected pigs develop a distinctive clinical syndrome called “Porcine Respiratory and

Encephalitis Syndrome” or “Barking Pig Syndrome” (Mohd Nor et al., 2000). As the name

suggests, pigs developed a characteristic loud barking cough, which differs from other

known porcine respiratory diseases. The prominence of this symptom appears to contrast

with humans. Fever was common and neurological signs included paralysis, abnormal

movement and gait. Many pigs however may remain asymptomatic or, having developed

clinical signs and symptoms, recovered to a large extent (Mohd Nor et al., 2000).

Apart from pigs, other animals including the cat, dog and horse were reported to be

susceptible to infection (Daniels et al., 2001, CDC, 1999b, Hooper et al., 2001, Johara et

al., 2001). Clinical manifestations in one infected dog were reported to consist of fever,

respiratory distress, conjunctivitis, and mucopurulent nasal and conjunctival discharges;

signs which apparently resembled canine distemper (Hooper et al., 2001).

In both natural and experimental infection, the main pathology in the pig was found

in the respiratory system and meninges (Hooper et al., 2001). There was evidence of

tracheitis, peri-bronchial inflammation and pneumonia. The lungs showed numerous

macrophages, neutrophils and multinucleated cells within alveoli and bronchioles.

Eosinophilic intracytoplasmic inclusions, confirmed by immunohistochemistry (IHC) to

contain viral antigen, could be found in these cells. Free antigen was also detected on the

bronchial epithelium. Although vasculitis was rare, syncytial cells were seen in small blood

vessels. Meningitis was characterised by oedema, vasculitis, and infiltration of

lymphocytes, plasma cells and macrophages. Viral antigen could be localised to the

arachnoid membrane. Encephalitis on the other hand was rare, and when they were found

19

consisted of mild perivascular lymphocytic cuffing and glial foci. No necrotic foci were

apparent.

Pathological findings in NiV-infected dog, cat and horse are less well characterised

since there were far fewer pathological specimens available for study (Hooper et al., 2001).

Nonetheless, there were suggestions that systemic vasculitis occurs in all these animals. In

addition, non-suppurative meningitis and brain parenchyma rarefaction was also reported in

the dog and horse. In the experimentally infected cat, the respiratory system was found to

be as severely affected as in the pig. Experimental infection in the guinea pig, showed

marked involvement of blood vessels, urinary bladder, female reproductive tract, meninges,

ependymal cells, and to a lesser extent the neural parenchyma and lung (Torres-Velez et al.,

2008). Nonetheless viral antigens were immunolocalised to neurons and neuronal viral

inclusions were evident. Overall, the pathology in the animal studies seems to suggest that

none of the animals studied so far had prominent CNS involvement, which is the hallmark

of severe human disease.

The pathological findings in the respiratory system could explain the severe

pulmonary symptoms in the pig, and support the suggestion that aerosol spread of NiV

from pig to human is an important route of transmission. Viral transmission via urine from

the cat, dog and guinea pig may also be important since viral antigen was detected in the

kidney and urinary tract of these animals, and virus could be isolated from cat urine

(Hooper et al., 2001, Torres-Velez et al., 2008).

2.0 Pteropid Bats as Reservoir Hosts of Henipaviruses

The range and distribution of pteropid or fruit bats includes Southeast Asia, parts of China,

Japan, Oceania, the Indian subcontinent, Australia and Africa (Figure 1.5). There is now

20

direct and indirect evidence that the fruit bat is a reservoir host of NiV in Malaysia,

Cambodia, Thailand, China, India, Bangladesh, Madagascar and western Africa (Olson et

al., 2002, Reynes et al., 2005, Chua et al., 2002, Wacharapluesadee et al., 2005, Epstein et

al., 2008, Hayman et al., 2008, Hsu et al., 2004, Lehle et al., 2007, Li et al., 2008). Direct

evidence is by virus isolation or virus RNA detection, and indirect evidence is the presence

of anti-NiV antibodies. Many species of pteropid bats including Pteropus hypomelanus, P.

vampyrus, P. giganteus and P. lylei were found to be naturally infected. In Malaysia, NiV

was isolated from P. hypomelanus from Tioman Island (Figure 1.1). For HeV, the reservoir

bat species are P. poliocephalus and P. alecto (Halpin et al., 2000). Apart from Australia,

fruit bats in nearby Papua New Guinea may also harbour HeV (Halpin et al., 1999), but this

virus has not been isolated in other countries so far.

NiV may have been accidentally transmitted to pigs via fruits half-eaten by bats and

dropped off near pigsties where they can be eaten by pigs. Indeed, viruses have been

isolated from such fruits (Chua et al., 2002). Since bats can shed virus in their urine

(Middleton et al., 2007), direct contamination of food or water consumed by pigs could

represent another mode of transmission. The mode of HeV transmission from bats to horses

remained unclear but pasture or feed contaminated by bat urine and other fluids may play a

role in transmission (Hanna et al., 2006, Williamson et al., 1998). Whatever the mode of

transmission from bats, pigs and horses appear to be the main intermediate/amplifying

hosts for henipaviruses. In the case of NiV, the close proximity of pigs in many Malaysian

pig farms probably contributed significantly to pig-to-pig transmission.

21

Fig 1.5 World map showing distribution of Pteropid or fruit bats in Asia, Oceania, Africa and Australia (shown in grey).

Asia

Australia

Africa

Oceania

22

Since in Bangladesh intermediate hosts did not appear to play an important role, it is

believed that direct bat-to-human transmission occurred via contaminated food such as date

palm juice (Luby et al., 2006) and half-eaten fruits. As jars used to collect date palm sap are

hung onto trees overnight, bats that come to feed from the jars could contaminate it.

Although bat-to-human transmission has not been investigated in India, the modes of

transmission may be similar to Bangladesh given that the affected area, Siliguri, is close to

the outbreak areas in neighbouring Bangladesh, and thus share similar demographic, social

and environmental characteristics (Harit et al., 2006). Figure 1.6 summarises the likely

modes of henipavirus transmission in Malaysia, Bangladesh and Australia.

Pteropid bats

Human

Nipah Virus

Hendra Virus

Human Human

Pig

cHorse Human

b

Figure 1.6Likely routes of henipavirus transmission from animals to humans. (a) In Malaysia bat-to-pig Nipah virus transmission could be via contaminated half-eaten fruits dropped into pig farms and were eaten by pigs. (b) In Bangladesh direct bat-to-human Nipah virus transmission is via contaminated date palm juice and possibly other foods. (c) In Australia bat-to-horse Hendra virus transmission is believed to be via contaminated pasture. Note: The pig and horse act as intermediate or amplifying hosts. Human-to-human transmission was best documented in Bangladesh and India.

a

23

24

2.1 Objectives

Pathological studies of novel, emerging infections such as NiV are critical to the

understanding of the clinical manifestations, complications and pathogenesis. At present

there is still much that is not known about the pathology and pathogenesis of henipavirus

infection in general and NiV infection in particular. Moreover, the relapsing nature of NiV

relapsed/late-onset encephalitis is intriguing. Its study may uncover new mechanisms of

viral neuropathogenesis.

Overall, a more thorough understanding of the pathology in humans and animal

models could improve histopathological diagnosis by identifying cells/tissues most often

susceptible to infection. Moreover, distinct pathological features may be identified that

could aid diagnosis. A good animal model for NiV infection that could be used to test and

develop effective therapies and vaccines and other management strategies might ultimately

benefit patients.

The specific objectives of this study are:

1. To study the pathological changes in human NiV infection with special emphasis on the

CNS.

2. To investigate the pathogenesis of acute and relapsed human NiV infection.

3. To establish and study a golden hamster model for acute NiV infection.

4. To study and compare HeV infection in humans and golden hamster with NiV

infection.

25

CHAPTER 2: MATERIALS AND METHODS



A series of 31 fatal cases of acute NiV infection was studied. These cases had positive IgG

and/or IgM serology or positive virus culture. This series comprised all the cases that were

autopsied from late-1998 to mid-1999, and were drawn from five hospitals in Malaysia.

More than 100 patients with the infection died, so these autopsies represent about 30% of

all fatalities.

Fifteen cases were from the Seremban Hospital (Table 3.1, Case 1-15), 3 were from

the Kuala Lumpur Hospital (Case 16-18), 9 were from the Ipoh Hospital (Case 19-27) and

4 were from the University of Malaya Medical Centre (Case 28-31). Medical records from

the various hospitals were systematically reviewed, and demographic, clinical, and other

data were extracted.


Of the 31 autopsies, 28 were full autopsies and 3 autopsies were limited to the brain (Cases

13, 18 and 29, Table 3.1). Tissues were fixed in 10% buffered formalin from a few days to

several weeks and extensively sampled for routine processing and paraffin embedding.

Four µm sections of tissues were placed on Fischer Plus slides (Fischer Scientific,

Pittsburgh, PA), deparaffinised, and then rehydrated in serial graded alcohol and stained

with haematoxylin and eosin (H&E).

26

2.1.3 Antibodies, Cell and Tissue Controls

The antibody initially used for NiV antigen detection was anti-HeV hyperimmune mouse

ascitic fluid (Centers for Disease Control and Prevention [CDC], Atlanta, Georgia, USA).

Subsequently, hyperimmune mouse ascitic fluid against NiV was generated at the CDC and

reactivity was compared with that of the mouse anti-HeV antibody. The specificity and

sensitivity of these antibodies in the IHC analyses were tested using Vero E6 cells (Vero

clone CRL 1586; American Type Culture Collection, Rockville, MD) that were uninfected

or infected with HeV or NiV. Antibody specificities were further confirmed by testing

specimens from patients with JE, measles, eastern equine encephalitis, enterovirus 71 and

influenza, and tissue culture cells infected with western equine encephalitis virus, measles

virus, and La Crosse encephalitis virus.

Negative antibody controls in the IHC analysis included replacing primary antibody

with normal mouse ascitic fluid or with the primary antibody absorbed with NiV antigens.

Because there was a high initial suspicion of JE, specimens from all patients were also

stained for JE viral antigens by using a cross-reactive Flavivirus antibody.

2.1.4 IHC

Tissue blocks were chosen for IHC after slides of the H&E stained specimens were

reviewed. The IHC was based on a method described previously for Hantavirus (Zaki et al.,

1995). Four µm sections were deparaffinised and rehydrated through graded alcohol and

distilled water. They were predigested by 0.1 mg/ml proteinase K (Boehringer-Mannheim

Corporation, Indianapolis, USA) in 0.6 M Tris (pH 7.5)/0.1 CaCl2 for 15 minutes at room

27

temperature and blocked with normal swine serum. Primary antibodies were applied for 1

hour at room temperature. Optimal conditions for primary antibody and digestion

conditions were previously determined by titration experiments. For anti-HeV and anti-NiV

antibodies, the dilutions were 1:4000 and 1:2000, respectively. This step was followed by

sequential application of biotinylated link antibody, alkaline phosphatase-conjugated

streptavidin, and napthol fast red according to manufacturer’s protocol (LSAB2 Universal

Alkaline Phosphatase Kit; Dako Corporation, Carpinteria, USA). Sections were then

counterstained in Mayer haematoxylin (Fischer Scientific, Pittsburgh, USA) and mounted

with an aqueous mounting medium (Faramount, Dako Corp). Specimens from all cases

were tested with anti-HeV antibody and selected cases with anti-NiV antibody.

2.1.5 ISH

For ISH, digoxigenin (DIG)-labeled DNA probes and riboprobes were generated from a

228 bp, RT-PCR (reverse transcriptase-polymerase chain reaction) product using the

primers in Table 2.1 (Chua et al., 2000a). This probe targets the NiV N gene. The RT-PCR

product was cloned in the pdrive cloning vector (Qiagen PCR cloning kit, Qiagen Inc.,

Valencia, California, USA) according to the manufacturer’s protocol. DNA probes were

prepared directly from plasmids containing the 228 bp, RT-PCR product by another PCR

that incorporates DIG-labeled 11-dUTP (Boehringer Mannheim, Germany) in the reaction.

For riboprobes, plasmids containing the correct insert in both orientations were linearised

with the restriction endonuclease Hind III, and transcribed to produce sense and anti-sense

riboprobes, respectively, using the DIG RNA labeling kit (Roche Diagnostics, Mannheim,

Germany). The riboprobes were treated with DNase (15 min, 37 0C) then purified by

ethanol precipitation before use.

28

Table 2.1 Nipah Virus Nucleoprotein Gene Specific Primers and Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) Conditions

Primers

5’-CTGCTGCAGTTCAGGAAACATCAG-3’ 5’-ACCGGATGTGCTCACAGAACTG-3’

RT-PCR mix

Primers (10pmol/µl) 5 µl each dNTP (10mM) 4 µl Buffer (5x) 10 µl Dithiotreitol (100mM) 2 µl RNAse inhibitor (5 units/µl) 1 µl Titan enzyme mix 1 µl RNA template 10 – 20 µl H2

O to final volume of 50 µl

RT-PCR conditions

500

94C 30 min

0

94C 5 min

0

50C 1 min x 30 cycles

0


0


0

4 C 10 min 0

C hold

29

The ISH method was slightly modified from a previous method (Jessie et al., 2004).

Dewaxed tissue sections were pretreated with hydrochloric acid followed by 0.1mg/ml

proteinase K in 100 mM Tris /50mM EDTA/0.05% sodium dodecyl sulphate, pH 8.0 buffer

(15 min, 370C). This is followed by a wash in 1% glycine/phosphate buffered saline (PBS)

and two washes with PBS. For the ISH procedure using DNA probes (dilution 1:100), the

hybridisation solution containing the probe was laid onto the slide and preheated to 990C

for 15 min to denature the probe. Then the slides were incubated overnight at 420C in a

moist chamber. For the ISH procedure using riboprobes (dilution 1:50), the preheating step

was omitted and hybridisation allowed to proceed as before but at 450

Post-hybridisation steps included sequential washings with decreasing

concentrations of SSC (twice for 15 min in 6x solution, 42

C overnight. The

standard filtered hybridisation solution consisted of 6x sodium chloride/sodium citrate

(SSC), 5x Denhardt’s solution, denatured salmon sperm DNA (100µg/ml) and 5% dextran

sulphate in formamide (Killen and O'Sullivan, 1993). (Appendix A)

0

C; once for 15 min in 2x

solution, room temperature). This was followed by 10% blocking solution (Boehringer

Mannheim, Germany) for 30 min at room temperature (Komminoth, 1996). The slides were

then incubated with alkaline phosphatase-conjugated, anti-DIG Fab fragments (Roche

diagnostics, Mannheim, Germany) diluted 1:5000 in Tris-NaCl buffer for at least 30 min.

This was followed by washes in the same buffer (2X, 15 min each) and NBT/BCIP solution

(Roche diagnostics, Mannheim, Germany) according to manufacturer’s protocol. The

colour reaction was stopped after about 45 min. The slides were counterstained with Mayer

haematoxylin and coverslipped in an aqueous medium. For negative controls, duplicate

assays using negative tissues (similar to those used in IHC) and anti-sense riboprobes, and

an assay that omitted the probe were included.

30

2.1.6 EM

Formalin-fixed brain tissues were post-fixed with 1% osmium tetroxide in 0.2 M phosphate

buffer, en bloc stained with 4% aqueous uranyl acetate, dehydrated through a graded series

of alcohol and propylene oxide, and embedded in a mixture of epon substitute and Araldite

(Mollenhauer, 1964). Ultrathin sections were stained with 4% aqueous uranyl acetate and

Reynold’s lead citrate.

2.1.7 NiV Antibody Detection Assays

NiV antibodies were detected by IgM capture ELISA and IgG ELISA and inactivated HeV

virus antigens. These assays were done at the Department of Medical Microbiology,

University of Malaya. The tests followed methods previously described for Ebola virus

(Ksiazek et al., 1999). (Appendix B)

2.1.8 Virus Isolation and Identification

Virus isolation and identification in the CSF was attempted in 8 patients by use of a

previously described method (Chua et al., 2001a). Virus was cultured in Vero cells and

detected by immunofluorescence by the Department of Medical Microbiology, University

of Malaya (Appendix C).

31

2.2 Human Relapsed or Late-Onset NiV Encephalitis


Three fatal cases of relapsed/late-onset NiV encephalitis infection, 1 case each from Kelang

Hospital, Seremban Hospital and the University of Malaya Medical Centre (Cases 1 to 3,

Table 3.6) autopsied from 1998 to 2001 were studied. In addition, a patient with severe

acute NiV encephalitis who was comatose on admission to the University of Malaya

Medical Centre, and died about 8 months later without regaining consciousness or

developing relapsed/late onset encephalitis, was included in this study (Case 4, Table 3.6).

Available clinical information and medical records were systematically reviewed and

demographic, clinical, and other data were extracted.


Of the 4 autopsies, 2 were full autopsies, and 2 “brain-only” autopsies (Table 3.6).

However, no spinal cord tissues were available for study in any of the cases. Tissues were

fixed in 10% buffered formalin for more than two weeks and extensively sampled for

routine processing and paraffin embedding. Four µm sections of tissues were placed on

glass slides deparaffinised, rehydrated and stained with H&E as before. Selected CNS

slides were stained with luxol fast blue/H&E.

32

2.2.3 IHC

IHC was performed on all small and some large tissue sections using a standard

immunoperoxidase technique modified from a previous IHC method (Jessie et al., 2004).

After deparaffinisation, dehydration and microwaving (990C, 20 min, citrate buffer 0.1M,

pH 6.0), the anti-NiV rabbit polyclonal antibody (1:1000) (Gift from Dr P Hooper,

Australia) or the anti-HeV mouse polyclonal antibody (1:500 to 1:1000) (Gift from Dr SR

Zaki, CDC, USA) was added to tissue sections and incubated for 2 hours at room

temperature or overnight at 40

C. This was followed by biotinylated secondary antibody

(DAKO, Denmark), avidin-biotin complex linked to peroxidase, and 3, 3’-

Diaminobenzidine (DAB; Sigma, USA), with Tris-buffered saline (TBS) washes in

between steps. The secondary antibody was either swine anti-rabbit or rabbit anti-mouse

depending on the primary antibody used. The slides were routinely counterstained with

Harris haematoxylin and mounted. For negative controls, duplicate assays using the above

mentioned tissues (Section 2.1.4) were performed. In addition, an assay that omitted the

primary antibody was also done.

2.2.4 ISH

A standard ISH assay using NiV-specific DNA probes was performed as described above

(Section 2.1.5). For negative controls, duplicate ISH assays using some of the negative

tissues as in IHC, and assays that used anti-sense riboprobes or omitted the probe altogether

were included.

33

2.2.5 EM and IEM

For EM, selected 1 mm3

For IEM, 1 mm

formalin fixed CNS tissues from Cases 1 and 2 (Table 3.6) were

routinely processed for epon embedding as described above (Section 2.1.6). Ultrathin

sections were counterstained with uranyl acetate and lead citrate, and viewed with a Hitachi

7650 transmission electron microscope at 80 kv.

3

formalin-fixed, CNS tissues were processed in LR White

(ProSciTech, Australia) according to the manufacturer’s protocol, with the omission of the

osmium tetroxide post-fixation step. Ultrathin sections (75nm) were placed on 200 mesh

nickel grids, covered with Formvar and coated with carbon. After a PBS wash for 5 min,

the blocking solution (Block Ace, Dainihonseiyaku, Japan) was added for 10 min followed

by rabbit anti-NiV polyclonal antibodies (1:100 dilution; gift from Peter Hooper, Australia)

for 2.5 hr. Following another PBS wash, a goat anti-rabbit polyclonal secondary antibody,

labeled with 10nm gold (BB International) was added and incubated for 2 hr. Following

PBS and distilled water washes, the grid was air dried and postfixed in 1% osmium

tetroxide for 10 min, and then washed with distilled water. Finally the grids were incubated

with 2% uranyl acetate and Millonig lead for 10 and 1 min respectively and viewed as

before. The entire procedure was done at room temperature.

2.2.6 Large Brain Section Topographic Study

34

To plot the distribution of inflammation and/or necrosis, large brain sections were studied

for Cases 1 and 2 (Table 3.6). Slides with H&E stained, 8 µm thick, large brain tissue

sections were overlaid with 4 mm2

square grids printed on transparent plastic sheets. The

brain sections were then examined under a light microscope and semi-quantitated for

inflammation by manually marking on the grids where inflammation (perivascular cuffing,

microglial nodules, parenchymal inflammation, necrosis) was found. Squares overlying

more or less confluent lesions that cover more than half the square area were marked. In

addition, isolated small perineuronal vacuolation was also indicated.

2.3 Human HeV Infection


So far only 6 cases have been reported and the demographic and clinical data from 4

published cases (Selvey et al., 1995, O'Sullivan et al., 1997, Hanna et al., 2006) are

summarised in Table 3.7.


Formalin-fixed, paraffin-embedded tissues of major organs from autopsies of the 2 fatal

cases of HeV infection (Cases 1 and 2, Table 3.7) were obtained from Drs T Robertson and

BB Ong (Queensland Pathology & Scientific Services, Australia). Tissue sections were

sectioned 5 µm and routinely stained with H&E. These organs/tissues included brain, lung,

35

heart, kidney, liver and spleen (Cases 1 and 2, Table 3.7); pancreas, thyroid, adrenal, lymph

node and intestine (Case 1 only), and spinal cord (Case 2 only).

2.3.3 IHC

A standard immunoperoxidase protocol was followed as described above (Section 2.2.3)

using the primary mouse anti-HeV polyclonal antibody (1:1000 dilution; gift from Dr SR

Zaki, CDC, USA), with slight modifications in that this assay utilised the ENVISION

detection method (DakoCytomation, Denmark) instead. The rest of the procedure was

similar to that described. The IHC assay to identify macrophages/microglia is similar

(Section 2.2.3) but the anti-CD68 antibody (clone PGM1, Dako) is used as the primary

antibody

2.3.4 ISH

DNA probes were prepared from a plasmid containing the whole nucleoprotein gene of

HeV (courtesy of Dr LF Wang, Australia), by performing a PCR that incorporated DIG-

11dUTP to produce a DIG-labeled DNA probe of 271 bp, as described above (Section

2.1.5). The primers and the PCR conditions are listed in Table 2.2. The same ISH protocol

(Section 2.1.5) was used on all tissues except for the non-CNS tissues of Case 1 (Table

3.7), as tissues were insufficient for this purpose.

For both IHC and ISH assays, positive controls using HeV-infected hamster tissues

were used. Negative control tissues included normal CNS tissues and CNS tissues infected

by Enterovirus 71 and measles virus. For both assays, duplicate procedures that omitted the

primary antibody and probe respectively were performed as additional negative controls.

36

Table 2.2 Hendra Virus Nucleoprotein Gene Specific Primers and Polymerase Chain Reaction (PCR) Conditions

Primers

5’-GCA-ATG-GCT-GAC-AGA-GAT-GA-3’ 5’- GCT-CGA-GGC-CCT-ATT-TCT-CT-3’

PCR mix

Primers (0.4 µm each) 2 µl dNTP (10mM each) 1 µl Taq buffer (10 x) 5 µl MgCl2Taq polymerase (Fermentas) 0.5 µl

(25 mM) 6 µl

DNA template (10ng) 1 µl H2

O to final volume of 50 µl

PCR conditions

95°C 3 min 940

56°C 1 min x 35 cycles C 30 s

72°C 30 s x 35 cycles 72°C 10 min x 35 cycles

4 0

C hold

37


In order to identify small animals that may be susceptible to NiV infection and that may be

used as infectious disease models, several groups of animals viz., the golden hamster

(Mesocricetus auratus), guinea pig and Swiss mice were experimentally infected.

2.4.1 Virus Stock and Titration

NiV isolated from a patient’s CSF was received in the “Jean Mérieux” biosafety level 4

(BSL-4) laboratory in Lyon, France, from Dr KB Chua and Dr SK Lam (University of

Malaya, Malaysia) after 2 passages in Vero cells. Virus stock was obtained after a third

passage on Vero cells conducted under BSL-4 containment. Virus stock was titrated by Dr

V Duebel (Appendix D). The final virus stock was 2 x 107

plaque forming units (pfu)/ml.


Altogether 3 sets of animal studies were done. In the first study, preliminary testing for

susceptibility to NiV infection was performed on 2 groups of animals comprising 5 mice, 2

guinea pigs and 2 hamsters each. Four week-old, female Swiss mice (Charles River,

L’Arbresle, France), 4 month-old, male Hartley guinea pigs (Charles River), and 2 month-

old male golden hamsters (Janvier, Le Fenest St Isle, France) were used in this experiment

in which each group was inoculated either by the intranasal (IN) or the intraperitoneal (IP)

route. For the IN route, 30 µl of virus stock (6 x 105 pfu) was given to each animal, while

for the IP route 0.5 ml (107 pfu) was inoculated. The animals were housed in ventilated

38

containment equipped with Hepa filters in the BSL-4 lab, and observed for signs of

infection.

Based on the results of the first study, a second study was then performed on adult

hamsters (7-14 weeks old) using IN and IP inoculation routes to determine the lethal doses

needed to kill 50% of the animals (LD50). Groups of 6 hamsters were infected with 10-fold

dilutions of NiV stock and observed twice daily throughout 4 weeks. The LD50

In order to investigate the possibility of on-going reinfection between animals

housed together in the same cage contributing to mortality, a third study was done. In this

study, 2 hamsters infected by IP route with 10

was

calculated based on an established method (Reed and Muench, 1938). The mice and guinea

pigs from the first study were not studied further.

5

Suitable tissue specimens from the first and second studies including blood, brain,

lung, heart, liver, spinal cord, spleen and kidney were collected from a total of 12 hamsters

which had died recently (≤12 hours) or were terminally moribund. The latter were

anaesthetised with ketamine and xylazine, and exsanguinated by cardiac puncture and

necropsied. Urine was collected from the bladder whenever possible. Animals discovered

dead after more than 12 hours were not studied.

pfu of virus were placed 3 days after

inoculation into the same cage as 4 uninfected hamsters. The animals were observed and

blood samples obtained for serology after 30 days.

Tissues were frozen at –800C for virus culture RT-PCR analysis. For

histopathological studies, tissues were fixed in 10% buffered formalin for at least 15 days

before routine tissue processing and paraffin embedding outside the BSL-4 lab. Tissues

from the nasal passage and cervical lymph nodes were also dissected out from formalin-

fixed carcasses for routine processing and paraffin embedding only.

39

For EM, fresh or formalin-fixed tissues were fixed in 3% glutaraldehyde in 0.1 M

phosphate buffer pH 7.4 for a few hours and transferred to phosphate buffer. Similarly,

tissues for IEM were fixed in 2% paraformaldehyde/0.05% glutaraldehyde, and transferred

to buffer. In addition, EM and IEM tissues that were initially not formalin fixed, were

gamma-irradiated (2 x 106

Blood samples were collected by cardiac puncture at sacrifice or obtained from the

retro-orbital sinus in surviving animals. All the infection experiments were performed by

Dr V. Deubel and his team in a BSL-4 laboratory.

rads) to further ensure non-infectivity.

2.4.3 Virus Isolation and Titration

Infectious virus in urine and other tissues was detected by Vero cell culture by Dr V

Deubel. A small fragment of tissue was mechanically-crushed (Mini-beadbeater; Biospec,

Bartlesville, USA) twice for 30 seconds each, in a 2 ml tube containing 0.5 ml of sterile

glass beads and 0.5 ml of Dulbecco’s minimum essential medium, and centrifuged at 3000

rpm for 5 min at 4 0

C. The supernatant was cultured for virus as described (Appendix D).

2.4.4 NiV Antibody Testing

Sera of hamsters were tested by ELISA for NiV antibodies by Dr V Duebel. (Appendix E)

2.4.5 RT-PCR

Total RNA was extracted from 20 µl of serum and urine, and from mechanically-crushed,

fresh frozen tissues using an RNA extraction kit (QIAamp Viral RNA Mini Kit; Qiagen

40

Inc., Valencia, California, USA). After the lysis step, about 2 µg of extract was used in a

RT-PCR protocol (Titan One Tube RT-PCR System; Roche Diagnostics, Mannheim,

Germany) to detect the NiV N gene. The specific primers used and the reaction conditions

are shown in Table 2.1.


Formalin-fixed, paraffin-embedded tissues were microtomed 3 µm thick, placed on glass

slides, and stained with hemalin-phloxine-safranin stain for light microscopy.

2.4.7 IHC

Tissue sections, 3 µm thick, were placed on silanised slides and deparaffinized as before.

Antigen was retrieved by thermic treatment (pH 6.0 citrate buffer, 96-980

C for 40 min).

The IHC procedure (immunoperoxidase method) described above used the rabbit

polyclonal anti-NiV antibody (Section 2.1.4). The controls were as described.

2.4.8 ISH

The ISH procedure used NiV-specific riboprobes and controls as described (Section 2.1.5).

41

2.4.9 EM and IEM

Formalin- or glutaraldehyde-fixed tissues were post-fixed, dehydrated and embedded in

epon (Ladd, UK). Ultra-thin sections (70 nm) were counterstained as described (Section

2.1.6). Grids were examined in a Jeol 1200 EX transmission electron microscope at 60 kv.

Immunogold staining was performed on paraformaldehyde/glutaraldehyde-fixed tissue

embedded in LR White (Inland, France) as before (Section 2.2.5) except that were TBS/1%

albumin was used as the blocking step and for incubation with primary antibody.



Two sets of young adult hamsters were infected with HeV. The first set consisted of 10

hamsters that were experimentally infected by IP route with a viral dose of 103 pfu (or 100

LD50). Two animals were sacrificed daily from 1 to 5 days post-infection, respectively. A

second set of 4 animals were infected with a higher 105 pfu (or 10000 LD50

) dose by IP

route and sacrificed 2 animals at a time, at 2 and 4 days post-infection, respectively. All the

infection experiments were performed by Dr V. Guillaume in a BSL-4 laboratory.


Tissues from sacrificed animals were fixed in 10% paraformaldehyde for 15 days or more,

and routinely processed for paraffin embedding. These tissues included brain, lung, heart,

liver, spleen, kidney and intestine. Sections were prepared as before and stained with H&E.

42

2.5.3 IHC

The IHC (immunoperoxidase) assay utilised the anti-HeV antibody and ENVISION

method as described (Section 2.3.3).

43

CHAPTER 3: RESULTS



The age of patients in this study ranged from 13 to 75 years (mean, 43 years; median, 46

years) and the male-to-female ratio was 28:3 (Table 3.1). The prodrome, defined as the time

from fever onset to the day of hospital admission, averaged 3.3 days (range, 1-7 days). The

duration of illness, defined as the time from fever onset to death, averaged 9.5 days (range,

2-34 days). Four patients survived >14 days before death.

Clinical symptoms and signs are summarised in Table 3.2. All patients had fever.

More than 70% of patients complained of drowsiness, headache, and disorientation or

confusion. The most frequent clinical sign among patients was reduced consciousness. Case

31 (Table 3.1) was recovering in the hospital ward when he developed massive fatal

intracerebral haemorrhage.


The macroscopic features were non-specific. In the CNS, lesions cannot be identified with

any degree of confidence. Only 2 of 10 brains examined showed unequivocal herniation.

Case 29 (Table 3.1) had cerebellar tonsil herniation and Case 31 had uncal herniation and

showed a large intracerebral clot in the frontal lobe with intraventricular extension and

Duret haemorrhages in the midbrain and pons. In general non-CNS organs were grossly

unremarkable, but some showed oedema, congestion and focal haemorrhage.

Table 3.1 Clinical and Laboratory Data of Fatal Human Cases of Acute Nipah Virus Infection

Case no.

Age (year) /Sex

Prodrome (no. days)

Total duration of illness

*

(no. days)

Serology

**

Virus Isolation

Immunohisto-chemistry

CSF Serum

IgM

IgG IgM IgG

1

41/M 3 10 nd nd + - + +

2

52/M 6 8 - - + - + +

3

24/M 5 7 - - + - + +

4

65/M 2 6 - - + - nd +

5

22/M 4 8 nd nd + - + +

6

27/M 4 10 nd nd + - nd +

7

54/M

1 2 + - + - nd +

8

30/M 4 6 + - + - nd +

9

42/M 5 6 nd nd + - + +

10

46/M 1 6 + - + + nd +

11 20/M 4 11 + - + + nd +

(Table 3.1, cont.)

44

Case no.

Age (year) /Sex

Prodrome (no. days)


*

(no. days)

Serology

**

Virus Isolation


CSF Serum

IgM

IgG IgM IgG

12

71/M 3 7 + - + + nd +

13

49/M 2 7 + - + - nd +

14

44/M

5 14 + - nd nd nd -

15

13/F 2 5 nd nd nd nd nd +

16

36/M 3 6 - - nd nd nd +

17

50/F 3 3 - - + - nd +

18

49/F 3 7 nd nd + - nd +

19

54/M 4 31 nd nd + + nd -

20

39/M 3 17 nd nd + + nd -

21

46/M 2 3 nd nd + - nd +

22

37/M 4 7 nd nd + - nd +

23

53/M 1 3 nd nd + - nd +

45 (Table 3.1, cont.)

46

Case no.

Age (year) /Sex

Prodrome (no. days)


*

(no. days)

Serology

**

Virus Isolation


CSF Serum

IgM

IgG IgM IgG

24

51/M 7 9 + + + - nd +

25

29/M 3 8 nd nd + - nd +

26

55/M 2 25 + + + - nd +

27

75/M 2 4 nd nd - - nd +

28

51/M 3 7 + - + - + +

29

52/M 7 11 + + + - + +

30

34/M 3 8 + - - - nd +

31

31/M 2 34 + + - - - -

% Pos-itive

87

27

86

18

88

88

* Prodrome, onset of fever to admission. Total duration of illness, onset of fever to death. ** Positive, +; Negative, - ; nd = not done (no specimens were available)

47

Table 3.2 Frequency of Clinical Symptoms and Signs in Human Acute Nipah Virus Infection %

Symptoms

Fever 100 Drowsiness 88 Headache 82 Disorientation/confusion 76 Giddiness 61 Myalgia 54 Cough/ Respiratory symptoms 40 Convulsion 28 Vomiting 19

Signs Reduced consciousness 89 Segmental myoclonus 50 Hyporeflexia/ areflexia 50 Seizure 40 Cranial nerve palsy 29 Pyramidal signs 21 Nystagamus/ cerebellar signs 17 Meningism 10 Dysphasia 5

48

Blood vessels

The distribution of histopathological lesions and immunostaining is shown in Table

3.3. Extensive involvement of blood vessels in the CNS, lung, heart and kidney was

observed in acute NiV infection. However, blood vessels in the CNS were the most

severely involved (83%) followed by pulmonary vessels (64%). Typically, small arteries,

arterioles, capillaries and venules showed evidence of vasculitis. Vasculitis was not found

in medium-sized vessels (e.g. renal artery and vein, anterior and middle cerebral arteries) or

large arteries (e.g. aorta and pulmonary trunk).

Vasculitis was characterised by various degrees of segmental endothelial

destruction, mural necrosis and karyorrhexis (Figure 3.1 A, B, D, E; Figure 3.2, B; Figure

3.6, C, F). Mural necrosis often appeared fibrinoid. Sometimes there was only focal

endotheliitis. Inflammatory cell infiltration of vascular walls by neutrophils and

mononuclear cells was usually focal and either partial or transmural (Figure 3.1 B, D, E).

Thrombosis was found in both inflamed and uninflamed vessels (Figure 3.1 D, Figure 3.2

B; Figure 3.5 G). Necrosis and haemorrhage adjacent to vasculitic or thrombotic vessels

were frequently seen (Figure 3.1 F).

Syncytial or multinucleated giant endothelial cells were seen in blood vessels of

various organs (Figure 3.1 B, C, F-H). In the CNS they were found in 28% of the cases

(Table 3.4), mostly in patients whose duration of illness ranged from 6 to 15 days. The

syncytia typically consisted of several overlapping or sharply molded nuclei with moderate

to abundant cytoplasm. Some syncytia appeared rather bizarre or protruded prominently

into the vascular lumen (Figure 3.1 C), and may be accompanied by mild vasculitis (Figure

3.1B).

49

Table 3.3 Frequency of Necrosis, Vasculitis and Immunostaining of Viral Antigens in Major Organs in Human Acute Nipah Virus Infection Pathological findings

Brain no. (%)

Lung no. (%)

Heart no. (%)

Kidney no. (%)

Spleen no. (%)

Necrosis*

27/29(93%)

** 17/28 (61%)

1/28 (4%)

9/28 (32%)

10/23 (43%)

Vasculitis

24/29 (83%)

18/28 (64%)

9/28 (32%)

7/28 (25%)

0/23 (0%)

Viral antigens 26/31 (84%)

7/28 (25%)

4/23 (17%)

6/24 (25%)

1/20 (5%)

* Necrosis was in the form of parenchymal necrotic plaques in brain, fibrinoid alveolar necrosis in the lung, and fibrinoid glomerular necrosis in kidney. In the spleen, acute necrotic inflammation was found in the area of the periarteriolar lymphoid sheath. **

The percentage of cases involved was calculated by using as the numerator the number of cases with one or more findings. The denominator is the total number of cases for which tissues were available for study.

50

CNS

In the CNS, the main pathological findings were vasculitis, thrombosis,

parenchymal necrosis and presence of viral inclusions. Vascular involvement in grey and

white matter was seen throughout the CNS. The spinal cord was examined in 8 cases and

showed similar pathological changes in 3 cases as observed elsewhere in the CNS. The

olfactory bulb was examined in 9 cases and did not show any significant pathology.

Common histopathological lesions and their relative frequency in the CNS are shown in

Table 3.4.

Plaques with various degrees of necrosis were found in both the grey and white

matter (Figure 3.2 A-C). These necrotic plaques were round or oval with diameters that

ranged from about 0.2mm to ≥ 5 mm. Vasculitis, thrombosis and various degrees of

parenchymal oedema and inflammation were frequently found in the vicinity of these

necrotic plaques (Figure 3.2 A, B). The inflammatory cellular infiltrate consisted of

neutrophils, macrophages, lymphocytes, plasma cells and reactive microglia.

Microabscesses were found occasionally. In some necrotic plaques, there could be a

predominance of foamy macrophages. Elsewhere in the parenchyma, focal neuronophagia,

microglial nodule formation and perivascular cuffing were seen (Figure 3.3 E, F). Overall,

parenchymal inflammation was present in 66% of cases (Table 3.4).

Perineuronal microcystic degeneration was most commonly seen in the vicinity of

necrotic plaques (Figure 3.2 E; Figure 3.3 G). Microcystic change with no adjacent necrotic

plaques was also occasionally seen. In the white matter, damaged axons occasionally

formed axonal spheroids similar to those seen in diffuse axonal injury. No large geographic

infarctions of the type associated with occlusion of medium-sized or larger arteries, such as

the middle cerebral artery, were observed.

51

Table 3.4 Frequency of Microscopic Features in the Central Nervous System in Fatal Human Cases* of Acute Nipah Virus Infection

Histopathological lesion Frequency

Necrotic plaque 93%

Perivascular cuffing 90%

Thrombosis 90%

Vasculitis 83%

Parenchymal inflammation 66%

Viral inclusions 62%

Meningitis 55%

Endothelial syncytia 28%

* Brain tissues from 2 cases (Case 14, 15, Table 3.1) had severe freezing artifact that precluded adequate histopathological examination.

1

*

A B

C D

E F

G H

52

(Figure 3.1, cont.)

53

Figure 3.1 Vascular pathology and viral immunolocalisation in human acute Nipah virus infection. A: Vasculitis in a lung artery. Note focal and transmural mixed inflammatory infiltrate (arrow). B: High power magnification of another pulmonary vessel showing endothelial syncytia (arrow) and adjacent mural inflammation. C: Bizarre endothelial syncytia (arrow) protruding into the lumen of an uninflamed meningeal vessel. D: Cerebral vessel showing vasculitis and thrombosis. E: Cerebral venule showing endothelial ulceration (arrow) associated with inflammatory cellular debris. F: Cerebral haemorrhage (*) adjacent to vessel with a multinucleated syncytium (arrow). G. Higher power magnification showing endothelial origin of syncytium (arrow) seen in F. H: Positive immunostaining is seen in the cytoplasm of endothelial syncytia (arrow) and lining (arrowhead). H&E stains, A-G; immmunoalkaline phosphatase with napthol fast red substrate and haematoxylin counterstain, H. Original magnifications: x 40 (A); x 100 (F); x 200 (E); x 400 (B-D, G, H)

1

**

A B

DC

E F

HG

*

54(Figure 3.2, cont.)

55

Figure 3.2 CNS pathology and viral immunolocalisation in human acute Nipah virus infection. A: Well-circumscribed, necrotic plaque (arrow) associated with a vasculitic and thrombotic arteriole (arrowhead). B: Higher power magnification of the arteriole seen in A showing complete obstruction (arrowhead). C: Concentric immunostaining pattern of viral antigens around a necrotic plaque. D: Higher power magnification showing Nipah virus antigens in perikaryon and neuronal process (arrows). E: Perineuronal microcystic degeneration with positive immunostaining (arrow). F: Rare glial cell in the white matter showing immunostaining. G and H: The pencil bundles of Wilson (*) in the putamen are immunonegative even though adjacent neurons are positive for viral antigens. H&E stains, A, B; immmunoalkaline phosphatase with napthol fast red substrate and haematoxylin counterstain, C-H. Original magnification: x 40 (C); x 100 (A); x 200 (B, G); x 400 (D-F, H).

1

*

A B

DC

E F

HG


57

Figure 3.3 CNS pathology and virus localisation in human acute Nipah virus infection. A: Typical eosinophilic viral inclusions in the cytoplasm of several neurons (arrows). B: The viral nature of these inclusions is evidenced by immunostaining of Nipah viral antigens. C: Less-common neuronal inclusions occupying most of the nucleus (arrows) and pushing the chromatin to the periphery. D: These inclusions are also immunostained for viral antigens. E and F: As in other viral encephalitides, there is significant parenchymal inflammation including neuronophagia (E, arrows) and perivascular cuffing (F). G and H: Nipah virus RNA demonstrated in neurons in microcystic areas (arrows). H&E stains, A, C, E, F; immmunoalkaline phosphatase with napthol fast red substrate and haematoxylin counterstain, B, D; in situ hybridization, haematoxylin counterstain, G, H. Original magnifications: x 100 (F); x 200 (E, G); x 400 (A-D, H).

58

Viral inclusions were found in the cytoplasm and nuclei of neurons and mostly near

vasculitic vessels or necrotic plaques. Cytoplasmic inclusions are usually small, discrete,

eosinophilic, and sometimes multiple. (Figure 3.3 A, B). Nuclear inclusions were less

commonly found and occupied most of the nucleus except for a thin rim of chromatin at the

periphery (Figure 3.3 C, D). Although we found inclusions in 62% of our cases (Table 3.4),

in many instances they were found in only a few neurons after extensive search.

Lung

In the lung, vasculitis was seen in 64% of cases (Table 3.3; Figure 3.1 A, B) and

multiple foci of fibrinoid necrosis were found in 61% of cases (Figure 3.4 A). Fibrinoid

necrosis often involved several adjacent alveoli, and was frequently associated with small

vessel vasculitis. Multinucleated giant cells with nuclear inclusions were occasionally noted

in alveolar spaces adjacent to necrotic areas (Figure 3.4 C). Alveolar haemorrhage,

pulmonary oedema and aspiration pneumonia were often encountered. Histopathological

changes in bronchiolar epithelium were uncommon except in one case in which a large

bronchus showed severe transmural inflammation and ulceration (Case 22, Table 3.1).

Kidney

Focal glomerular fibrinoid necrosis, with or without thrombosis and periglomerular

inflammation was seen in 32% of cases (Figure 3.5 E, F; Table 3.3). In some cases,

glomeruli were totally destroyed by inflammation. Vasculitis, thrombosis and interstitial

inflammation were occasionally seen. Syncytial formation involving the periphery of the

glomerulus and tubular epithelium was rarely observed (Figure 3.5 G, H). No syncytia were

seen in transitional epithelium of the renal calyx and pelvis. Large, wedge-shaped infarction

was not observed.

59

Lymphoid organs

The spleen showed white pulp depletion and focal, acute necrotising inflammation

in the periarteriolar sheaths (Figure 3.5 A, B). In addition, large prominent multinucleated

giant cells with intranuclear inclusions (Figure 3.5 D) were seen in the parenchyma of 1

case (Case 22, Table 3.1). No vasculitis or large wedge-shaped infarction was observed.

Lymph nodes showed large reactive mononuclear cells, occasional necrosis and

haemophagocytosis. Rarely, multinucleated giant cells were encountered in cells lining the

subcapsular sinusof lymph nodes (Figure 3.6 A, B).

Heart

Vasculitis was noted in the heart in 32% of cases (Table 3.3; Figure 3.6 C). A large

myocardial infarction associated with vasculitis was found in one patient who had been

comatose for >2 weeks (Case 20, Table 3.1). In another patient who survived more than a

month (Case 31), focal myocardial fibrosis associated with vasculitis was also noted.

Other organs

Rare, focal vasculitis was occasionally seen in small arteries in the mesentery,

adrenal gland (Figure 3.6 F), and pancreas. No significant pathology was observed in the

liver, thyroid, stomach and skeletal muscle.

1

A B

DC

E F

HG


61

Figure 3.4 Pulmonary pathology and viral immunolocalisation in human acute Nipah virus infection. A: Focal alveolar necrosis and inflammation. B: These necrotic lesions show prominent immunostaining of viral antigens (arrow). C: Multinucleated giant cell (arrow) with nuclear inclusions in the alveolar space. D: Viral nature of giant cells as evidenced by immunostaining for Nipah virus antigens (arrows). E and F: Nipah virus antigens in tunica media of small arteries (arrows). G and H: Rare instance of immunostaining of bronchiolar epithelium (G, arrow). H&E stains, A, C; immmunoalkaline phosphatase with napthol fast red substrate and haematoxylin counterstain, B, D, E-H. Original magnification: x 100 (E, G); x 200 (A, B); x 400 (C, D, F, H).

1

A B

C D

E F

HG


63

Figure 3.5 Pathology and viral immunolocalisation in spleen and kidney in human acute Nipah virus infection. A: Prominent lymphoid necrosis and depletion in the spleen. B: Splenic necrosis is particularly evident in the periarteriolar sheath. C: Same areas showing viral immunostaining (arrow). D: Multinucleated giant cell with nuclear inclusions as seen in the spleen parenchyma. E: Fibrinoid necrosis of glomerulus (arrow) and surrounding inflammation. F: Prominent endothelial immunostaining in glomerular capillaries. G: Affected glomerular capillaries are often thrombosed (arrowhead). Very rarely syncytia may be seen arising from tubular epithelium (arrow). H: Multinucleated syncytium seen at the edge of a glomerulus (arrows). H&E stains, A, B, D, E, G, H; immmunoalkaline phosphatase with napthol fast red substrate and haematoxylin counterstain, C, F. Original magnifications x 40 (A); x 100 (C, E); x 200 (B, D, F, G); x 400 (H).

1

A

D

E F

B

Figure 3.6Pathology and viral immunolocalisation in lymph node, heart and adrenal gland in human acute Nipah virus infection. A. Low and high-power magnification of lymph node showing multinucleated giant cells (arrows) in the subcapsular sinus and viral inclusion (arrowhead). C: Severe myocardial arteritis. D: Positive immunostaining in the endothelium of a small blood vessel in the myocardium. E: Rare cardiac myocyte showing immunostaining of viral antigens. F: Peri-adrenal arteritis. H&E, A-C, F; immmunoalkaline phosphatase with napthol fast red substrate and haematoxylin counterstain, D, E. Original magnifications: x 40 (C); x 100 (F), x 200 (A); x 400 (B, D, E) 64

C

65

3.1.3 EM

EM examination of CNS tissues showed consisting of smooth, filamentous nucleocapsids

associated with dense amorphous material (Figure 3.7). They were generally difficult to

locate and mostly seen in neuronal bodies and dendritic processes and occasionally within

endothelial cells.

3.1.4 IHC

Antibody specificity

Anti-HeV and anti-NiV antibodies showed similar specificity reacting with viral

antigens in formalin-fixed, infected Vero E6 cells. After absorption with excess NiV antigen

these antibodies failed to react with infected cells. No staining was seen when uninfected

cell controls were reacted with either antibody. Tissues from other types of viral

encephalitides, including JE, were negative when tested with anti-HeV and anti-NiV

antibodies. All the NiV cases were negative when tested with anti-flavirus antibodies.

Blood vessels

Immunostaining for NiV antigens was seen in blood vessels of most organs,

particularly in those with vasculitis. No vascular staining was found in spleen or liver.

Vascular staining was seen mainly in endothelium, endothelial syncytia (Figure 3.1 H;

Figure 3.5 F; Figure 3.6 D), and smooth muscle of the tunica media (Figure 3.4 E, F). No

staining was present in medium-size or larger arteries. There was no apparent difference in

vascular staining in the grey or white matter of the CNS.

Figure 3.7Ultrastructural appearance of Nipah virus inclusions as seen in the CNS. A to D: Characteristic intracytoplasmic nucleocapsid viral inclusions (arrows) within infected neurons (A, B) and endothelial cells (C, D). Details of filamentous nucleocapsids (arrowheads) and associated dense material are seen in the high power magnifications (B, D). Original magnifications: x 12,000 (A, C); x 115,000 (B, D). Courtesy of C. Goldsmith.

66

67

CNS

In the CNS, apart from blood vessels, the most prominent staining was seen in

neurons associated with necrotic plaques or vasculitis. However, not all plaques even in

neuronal areas were associated with viral antigen staining. Neuronal staining at the

periphery of necrotic plaques was usually in the form of either concentric or eccentric rings

(Figure 3.2 C). The occasional remaining neurons in plaques or microcystic areas showed

positive staining for viral antigens (Figure 3.2 E). Occasionally, plaque-like areas of

neuronal staining with no evidence of necrosis or vasculitis were seen. Cytoplasmic and

nuclear staining were observed as granular or homogeneous in the neurons and peripheral

processes of the perikaryon (Figure 3.3 B, D). Immunostained round or oval granules

corresponded to cytoplasmic viral inclusions as seen with H&E (Figure 3.3 B).

Immunostaining was seen in 3 of 8 of the spinal cords examined and was localised to areas

with parenchymal necrosis, inflammation and syncytial cell formation.

Viral antigens in glial cells (astrocytes or oligodendrocytes) were rarely observed

(Figure 3.2 F). This relative sparing of glial cells was clearly evident in the putamen, where

pencil bundles of Wilson (white matter tracts) immediately adjacent to neurons positive for

viral antigens showed no immunostaining (Figure 3.2 G, H). Similarly, there was general

sparing of the white matter tracts in the anterior pons in contrast to pontine nuclei

immunostained for viral antigens. Focal immunostaining of meninges and ependyma was

seen in several cases; the choroid plexus was negative for viral antigens in all cases.

Non-CNS organs

Localisation of viral antigen was clearly seen in the non-CNS tissues, although to a

lesser extent (Table 3.3). Twenty five percent of cases were IHC-positive in the lung and

kidney, compared with 84% in the CNS. In the lung, viral antigens were usually found in

the areas of fibrinoid necrosis and in blood vessels (Figure 3.4 D-F). Viral antigens were

68

also noted inside multinucleated giant cells in or lining the alveolar space in 3 cases (Figure

3.4 B). Only 1 case (Case 22, Table 3.1) had bronchial inflammation and unequivocal

staining of the bronchiolar epithelium (Figure 3.4 G, H).

In the kidney, glomerular capillaries, small blood vessels and syncytial cells in the

periphery of the glomerulus exhibited viral antigen staining (Figure 3.5 F). In the heart,

IHC staining was found mainly in the blood vessels. In 1 case, there was focal staining of

cardiac myocytes adjacent to an area of capillary involvement (Figure 3.6 D, E). NiV

antigen was identified in occasional macrophages and multinucleated giant cells in the

spleen (Figure 3.5 C) and lymph node. No immunostaining of viral antigens was seen in the

liver.

3.1.5 NiV Antibody Detection Serological Assays

NiV IgM antibodies were more often detected than IgG in CSF (87% versus 27%) and

blood (86% versus 18%) (Table 3.1). In the single largest group of 18 patients with a

duration of illness of 6 to 10 days, IgM was found in the serum of 94% and the CSF of

64%, and IgG was found in 12% and 9%, respectively. IgM antibodies generally appeared

earlier than IgG in the serum before CSF (Figure 3.8).

3.1.6 Correlation of IHC and Serological Test Results

All cases in this study were examined by IHC, and serology was performed on serum or

CSF of all but 1 case. IHC analysis showed 87% of cases to be positive for viral antigen

and 93% of cases had positive NiV antibody results in the serum and/or CSF (Table 3.5).

There was an 80% concordance between positive IHC and serology.

69

Figure 3.8

Temporal relationship of IgM and IgG in cerebrospinal fluid (CSF) and serum in fatal human acute Nipah virus infection. Cases are grouped into 4 subgroups by duration of illness: 0-5 days (n=6), 6-10 days (n=18), 11-15 days (n=3) and 16-35 days (n=4). The percentage of positive cases for each type of immunoglobulin was calculated using as numerator the number of patients within each subgroup with a positive antibody level. The denominator is the total number of patients in that subgroup. Key: Black lines, serum. Grey lines, CSF

70

Table 3.5 Concordance of Immunohistochemistry and Serological Assays as Diagnostic Tests in Fatal Human Cases* of Acute Nipah Virus Infection

Test results

Positive cases/

total tested

% Positive

cases

Serology (either serum or cerebrospinal fluid)

IgM positive 28/30 93 IgG positive 8/30 27 IgM and/or IgG positive

28/30 93

Immunohistochemistry (IHC)

26/30 87

Combined Serology and IHC Both serology and IHC-negative 0/30 0 Both serology and IHC-positive 24/30** 80 Either serology or IHC-positive 30/30 100

* Correlation of IHC and serology was done in 30 cases; one case (Case 15, Table 3.1) was excluded because serology was not done. ** In 4 cases (Case 14, 19, 20, 31, Table 3.1) the IHC were negative but serology was positive. In 2 cases, the IHC was positive with negative serology (Cases 16 and 27).

71

Discrepancies between IHC and serological assays were found in 6 cases; 4 of these cases

were IHC negative and serology positive, and 2 cases were IHC positive and serology

negative. If both tests were performed together, confirmation of NiV infection could be

obtained in all the cases.

3.1.7 Temporal Evolution of Lesions and Presence of Viral Antigens in the CNS

The temporal evolution of histopathological lesions and viral antigens in the brain in acute

fatal NiV infection is shown in Figure 3.9. Vasculitis, thrombosis, necrotic plaques, and

syncytia peaked between 6 and 10 days after fever onset. On the other hand, parenchymal

inflammation and perivascular cuffing were most severe 11-15 days after fever onset and

were found in >60% of tissue sections of cases with duration of illness >16 days.

Viral antigens were more commonly detected in tissues 6 to 10 days after the fever

onset and gradually decreased throughout time (Figure 3.9). Tissues of all 18 patients with

a 6 to 10 day duration of illness were positive by IHC. In contrast, among the 4 patients

whose duration of illness was >16 days, only 1 (Case 26, Table 3.1) was IHC positive.

3.1.8 Virus Isolation

Virus isolation from the CSF was attempted in 8 cases and successful for all but 1, which

was also IHC-negative (Case 31, Table 3.1),

72

3.1.9 ISH

Viral RNA was detected mainly in neurons (Figure 3.3 G, H) and some blood vessels in the

inflamed areas.

73

Figure 3.9

Temporal distribution of microscopic lesions and viral antigens in the central nervous system in fatal human acute Nipah virus infection. Cases were grouped into 4 subgroups by duration of illness: 0-5 days (n=6), 6-10 days (n=18), 11-15 days (n=3) and 16 to 35 days (n=4). The percentage of a particular histopathological lesion found in each subgroup was calculated by using as the numerator the number of CNS sections with at least one positive lesion found therein. The denominator is the total number of CNS sections in that subgroup.

Key: Black, viral antigens. Red, necrotic plaque. Light blue, thrombosis. Green, parenchymal inflammation. Pink, vasculitis. Dark blue, perivascular cuffing. Brown, syncytium.

74



The demographic and clinical features of the 3 patients with relapsed/late-onset NiV

infection are summarised in Table 3.6. The average age was 27 years and the incubation

period ranged from about 3 to 22 months.


In Cases 1 and 2 (Table 3.6), macroscopically apparent confluent brain lesions consisted of

varying degrees of softening and necrosis in the cerebral cortex and subcortical neuronal

areas such as the thalamus, putamen, globus pallidus and caudate nucleus (Figure 3.10 A,

C). The grey/white junction in the cerebral cortex may be indistinct. In addition, in Case 2,

small discrete blood clots of up to 1 cm were also noted. Case 3 was partly autolysed

especially in the brainstem and cerebellum. The anterior part of the left frontal lobe was

particularly soft and necrotic. In the case of resolving acute NiV encephalitis with no

evidence of relapsed/late-onset encephalitis (Case 4, Table 3.6), there was cerebral atrophy

and ventricle enlargement (Figure 3.10 E). In the cerebral grey and white matter, multiple

small cystic lesions were noted (Figure 3.13 A).

CNS

Extensive confluent brain lesions were found in the 2 cases of relapsed/late-onset

encephalitis (Cases 1 and 2, Table 3.6), mainly in neuronal areas including the cortical grey

matter, caudate nucleus, putamen, globus pallidus, amygdala and hippocampus (Figure 3.10

B, D). Case 3 had confluent lesions limited to the left frontal cortex.

Table 3.6 Demography, Clinical and Pathological Data of Fatal Human Cases of Relapsed/Late-Onset Nipah Virus Encephalitis Case no.

Age (year) /sex

Clinical presentation Incubation period

Light microscopy Positive IHC* and/or ISH tissue staining

Pathological/clinical

diagnosis CNS Non-CNS

1

24/M In January 2001 had reduced unconsciousness, abnormal behavior, hallucinations, fever and vomiting Brain CT: Sulci and gyri effacement, hypodense lesions in right temporal- parietal lobes. Worked in pig farm (2nd

epicentre). In early 1999 had high fever for 1 week but apparently recovery. Nipah IgG serology positive

≈22 months

Brain: Perineuronal vacuolation, vacuolated hypocellular lesions merging with extensive areas of severe neuronal loss and inflammation. No vasculitis. Other organs not available for study

Brain (neurons, macrophages, glial cells)

na Late-onset Nipah encephalitis

2

26/M In March 1999 had fever, paralysis and seizures. Died 11 days later. Apparently had same medical problems 3 months ago after having worked in a pig farm (2nd

≈3 months

epicentre). Brain CT: Cerebral oedema, small discrete haemorrhages in cerebral cortex. Nipah IgG serology positive

Brain: Perineuronal vacuolation, vacuolated hypocellular lesions merging with extensive areas of severe neuronal loss and inflammation. Prominent reactive blood vessels in lesions; No vasculitis. Other organs: No vasculitis or significant pathology

Brain (neurons, ependyma, macrophages, glial cells)

Negative staining

Relapsed Nipah encephalitis

(Table 3.6, cont.) 75

Case no.

Age (year) /sex


Light microscopy Positive IHC* and/or ISH tissue staining

Pathological/clinical

diagnosis CNS Non-CNS

3

30/F In March 1999, worked in a pig farm (2nd

epicentre). Asymptomatic but seroconverted in April 1999. Brain MRI and cerebrospinal fluid were normal. In June 1999 had fever, headache, vomiting and seizures for 3 days. Brain MRI showed confluent right frontal lobe lesions in grey matter. Died about 3 weeks later. Nipah IgG and IgM serology positive

≈3 months Brain: Perineuronal vacuolation, vacuolated hypocellular lesions merging with extensive areas of neuronal loss and increasing inflammation in frontal lobe. No vasculitis Other organs: No vasculitis or significant pathology

Brain (neurons mainly)

Negative staining

Late-onset Nipah encephalitis

4

41/M In March 1999 had fever, reduced consciousness, inappropriate speech, tetraplegia and meningism. Ventilated >1 month. In coma with spontaneous eye opening, limb movements until cardiorespiratory arrest about 8 months later. Worked in pig farm (2nd

epicentre). Brain MRI (May 1999): Moderate cerebral atrophy and disseminated multiple small cystic lesions (pinpoint to 1 cm). Nipah IgG serology positive

na** Brain: Disseminated, discrete, small cystic lesions consisting mainly of foamy macrophages and reactive glial cells.

Negative staining

na Resolving Acute Nipah encephalitis; no evidence of relapse/ late-onset encephalitis

* IHC, immunohistochemistry; ISH, in situ hybridization. ** Incubation period for relapsed/late-onset encephalitis is not available (na) 76

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77

78

Figure 3.10 Macroscopic features of brain sections and distribution of lesions in human relapsed/late-onset Nipah virus encephalitis. A: Axial brain section of Case 2 (Table 3.6) shows mainly cerebral cortex softening and effacement of the grey-white junction. B: As indicated by the large dots in the same brain section, there were extensive, confluent lesions in the cerebral cortex, thalami and caudate nuclei with relative sparing of the occipital lobes. C: Coronal brain section of Case 1 shows softening and necrosis mainly in the right fronto-temporal lobes, putamen and globus pallidus. D: There were extensive, confluent lesions in right fronto-temporal lobe, putamen and globus pallidus and left temporal lobe and globus pallidus. E: Coronal brain section of Case 4 shows cerebral atrophy and multiple small cystic lesions. F: Disseminated small discrete cystic lesions mainly in the grey and white matter of the cerebral cortex; confluent lesions were not observed. Key: In B and D, the large dots represent confluent lesions. The small dots represent areas with perineuronal vacuolation. In F, the squares show the relative size of the cystic lesions.

79

A characteristic feature in the involved areas of brain was a spherical or band-like,

pale vacuolated necrotic lesion comprising varying numbers of eosinophilic, degenerate or

necrotic neurons and scanty inflammatory cells in a mildly or moderately vacuolated

neuropil (Figure 3.11 A, C; Figure 3.12 A). This lesion was focal and discrete or more

extensive and confluent, e.g. involving more than half the cortical grey matter thickness or

large areas of the putamen or caudate nucleus. The edge of the lesion was often, but not

invariably, accentuated by irregular vacuoles of varying sizes that merged into a subtler

peripheral zone beyond that consisted of the occasional eosinophilic or degenerate neurons

within a mildly vacuolated neuropil. This zone was contiguous with normal appearing cells

still further into the periphery (Figure 3.11 C, Figure 3.12 A). In some areas, vacuolated

necrotic lesions formed more complex, wave-like or concentric patterns (Figure 3.11 A, B).

In presumably more advanced lesions, neuronal loss was extensive and the

parenchyma was replaced by pale or eosinophilic necrotic material, a varying number of

macrophages, other inflammatory cells and reactive blood vessels (Figure 3.12 B). In many

areas, especially in the cortical grey matter, these lesions were in continuity or merged into

the more complex vacuolated necrotic lesions (Figure 3.11 A). Advanced lesions with

severe or near total neuronal loss, numerous reactive blood vessels and macrophages were

particularly prominent in Case 2 (Table 3.6)

Overall, the degree of inflammation was mild in the vacuolated necrotic lesions. In

more advanced lesions, significant parenchymal infiltration and perivascular cuffing and

focal haemorrhage or small blood clots were observed. Blood clots of up to 1 cm were

noted in Case 2. Neuronophagia and prominent microglial nodules were rarely observed.

There was no evidence of vasculitis or thrombosis in all the CNS tissues examined (Figure

3.12 F).

80

In many neuronal areas, especially cortical grey matter, small clusters (mostly 100

to 250µ) of perineuronal vacuolation or microcystic degeneration were observed either

adjacent or well away from vacuolated necrotic lesions (Figure 3.10 B, D). The vacuoles

were empty or filled with finely granular material, or found around normal or slightly

degenerate neurons (Figure 3.12 C). Perineuronal vacuolation was also observed near or

coalescing with the larger vacuolated necrotic lesion.

White matter involvement immediately adjacent and continuous with several more

severely affected cortical areas was noted (Figure 3.11 A). There was general loss of

oligodendrocytes and myelin staining, and increased number of foamy macrophages and

other glial cells (Figure 3.12 H). In the caudate nucleus and putamen, involved pencil

bundles of Wilson appeared paler and were surrounded by eosinophilic, degenerate

neurons. Typical perivenous demyelination was absent.

Viral inclusions could be found in many neuronal areas but were less obvious in the

vicinity of vacuolated pale or more advanced lesions. In general, cytoplasmic eosinophilic

viral inclusions, some very large and prominent, were found mainly in neuronal perikarya

or in the neuropil (Figure 3.12 D). Nuclear inclusions were rare. Some cells in the inflamed

and demyelinated white matter areas also contained viral inclusions.

The brain of the patient with resolving acute NiV encephalitis (Case 4, Table 3.6)

showed relatively discrete slit-like, oval or spherical lesions in the cortical grey matter,

grey/white junction, white matter proper, caudate nucleus, globus pallidus, putamen and

brainstem (Figure 3.13 A, B, D, E). The lesions appeared to be randomly distributed

(Figure 3.10 F) and ranged from about 250 µ to 3.8mm in largest diameter. No confluent or

necrotic lesions of the type seen in the relapsed/late onset cases were noted.

1

*

A

B C

*


82

Figure 3.11 CNS pathology in human relapsed/late-onset Nipah virus encephalitis. A: Large brain section showing confluent cerebral cortex lesions that consist of vacuolated necrotic lesions that are spherical, concentric (arrow) or wavelike. These lesions merge with more complex or severely affected regions (*). Adjacent white matter areas may also be affected (arrowheads). B: Higher power magnification of concentric vacuolated necrotic lesions. C: Spherical vacuolated necrotic lesion characterised by neuronal loss and degeneration, and mild inflammation. Luxol fast blue/H&E stains, A; H&E stains, B and C. Original magnifications: x 1 (A); x 40 (B); x 100 (C).

83

The typical cystic lesion was characterised by loss of neurons in the neuronal areas

and glial cells in the white matter, and their replacement by varying number of foamy

macrophages, lymphocytes, plasma cells and new blood vessels (Figure 3.13 B-E). In some

lesions, sheets of macrophages were observed while in other lesions macrophages were less

prominent. In the adjacent brain parenchyma some degree of reactive gliosis and

perivascular lymphocytic cuffing may be observed (Figure 3.13 B, C). More subtle lesions

consisted of very focal, mild neuronal loss and a few reactive astrocytes. There was no

evidence of vasculitis.

Non-CNS organs

Non-CNS tissues from Cases 2 and 3 (Table 3.6) showed no evidence of vasculitis,

parenchymal necrosis, inflammation, viral inclusions in the organs examined.

3.2.3 IHC and ISH

NiV antigen and RNA were detected in all the affected areas of the CNS but mainly in

neurons or immediately adjacent to vacuolated necrotic lesions (Fig 3.12 E, F, H) in all the

3 relapsed/late-onset encephalitis cases. In the more advanced lesions and in the white

matter immediately adjacent to these lesions, inflammatory cells such as

macrophages/microglia, and astrocytes, oligodendrocytes, ependymal cells appeared to be

positive for IHC and ISH as well (Fig 3.12 G). Ependymal cells were also positive. IHC

and ISH assays were negative in the CNS tissues from Case 4 (Table 3.6) and all the non-

CNS organs available for study. In particular, blood vessels were all negative.

A B

C

E F

HG

D

(Figure 3.12, cont.) 84

85

Figure 3.12 CNS pathology and virus immunolocalisation in human relapsed/late-onset Nipah encephalitis. A: Band-like, vacuolated necrotic lesion (arrow) in the lower half of cortical grey matter. B: Advanced inflammatory lesion in the lower cortical grey matter characterised by severe loss of parenchymal cells and replacement by reactive blood vessels, inflammatory cells and reactive astrocytes (arrow). C: Perineuronal vacuolation. D: Eosinophilic viral inclusions in neuronal cytoplasm (arrows). E: Neuronal viral antigens are mainly immunolocalised to the periphery of a vacuolated necrotic lesion. The centre of the lesion (arrow) shows severe neuronal loss with only occasional foci of viral antigens. F: High power magnification showing viral antigens in neuronal perikarya and processes. G: Macrophage or glial cells immunopositive for viral antigens in the white matter. H: Virus RNA demonstrated in neurons. Adjacent blood vessel shows no evidence of vasculitis or thrombosis (arrow). H&E stains, A-D; immunoperoxidase with diaminobenzidine substrate and haematoxylin counterstain, E-G; in situ hybridisation with and haematoxylin counterstain (H). Original magnifications: x 40 (A); x 100 (B); x 200 (C, E); x 400 (D, F, G, H)

86

3.2.4 EM and IEM

In relapsed/late-onset encephalitis brain tissues, nucleocapsids (Figure 3.14 A) were

demonstrated, and viral inclusions within neuronal cytoplasm were decorated with gold

particles in the IEM assay (Figure 3.14 B-D). In some nuclei, a sprinkling of gold particles

could also be detected decorating nucleocapsid-like structures (Figure 3.14 B, D).

A B

C

D E

*


88

Figure 3.13 CNS pathology in resolving acute Nipah virus encephalitis. A. Macroscopically detectable discrete cystic lesion in the cerebral cortex (arrow). B. Discrete cystic lesion in cerebral cortex with adjacent perivascular cuffing (arrow). C. The discrete cystic lesion comprises mainly foamy macrophages (*) and other inflammatory cells with adjacent reactive gliosis (arrows). D: Discrete lesion consisting mainly of reactive blood vessels and some inflammatory cells. E: Subtle, discrete inflammatory lesion in the white matter (arrow). H&E stains, B-E; Original magnifications: x 40 (B); x 100 (D, E); x 200 (C).

1

A B

C D

Figure 3.14Ultrastructure of infected neurons in human relapsed/late-onset Nipah virus encephalitis. A. Nucleocapsids (arrows) in neuronal cytoplasm. B - D: Viral inclusions (arrows) labeled by gold particles in the immunoelectron microscopy assay using anti-Nipah antibodies. Focal labeling is also seen some nuclei (B and D (*)). Original magnifications: x 100,000 (A-D)

89

90


The clinical features of all 4 published cases are summarised in Table 3.7. Case 1 is the

patient with acute HeV infection who did not present with clinical encephalitis.


Blood vessels

The light microscopic features are summarised in the Table 3.7. Vasculitis and

endotheliitis were observed in blood vessels in the brain, lung, kidney and heart (Figure

3.15 A, B; Figure 3.17 A, E). In contrast to perivascular cuffing, vasculitis was

characterised by karyorrhexis and intramural or subendothelial inflammatory cells.

However, no convincing endothelial syncytia were found. Occasional thrombotic plugs

were detected in the pulmonary blood vessel, and rarely in glomerular capillaries (Figure

3.17 F). Viral antigens were observed in some vascular endothelium, and possibly smooth

muscle cells, in the brain (Figure 3.15 C), lung and kidney (Figure 3.17 G). Choroid plexus

showed no evidence of vasculitis or virus.

CNS

In the brain parenchyma, there were both discrete necrotic or more subtle vacuolar

plaques, in the cerebellum (Figure 3.15 E; Figure 3.16 A), grey and white matter of the

cerebral cortex (Figure 3.15 D, H), corpus callosum, hippocampus, thalamus, external

capsule and globus pallidus. The most obvious necrotic plaques were found in the white

matter consisting of eosinophilic axonal spheroid-like material (Figure 3.15 H). The

necrotic/vacuolar plaques comprised neuropil vacuolation, mild parenchymal infiltration by

mononuclear inflammatory cells and mild perivascular cuffing. In addition, neuronal loss

91

could be evident in neuronal areas (Figure 3.15 D; Figure 3.16 A). In the cerebellar

molecular layer, vacuolar plaques were paler staining and consisted of small fine vacuoles

(Figure 3.15 E). Occasional neuronal bodies at the periphery of a plaque may show

cytoplasmic eosinophilia (Figure 3.15 E). Eosinophilic cytoplasmic viral inclusions were

detected in neurons in the hippocampus (pyramidal layer) (Figure 3.16 F) and thalamus,

and ependymal cells (Figure 3.16 E). There were no nuclear inclusions detected. Overall,

by light microscopy, parenchymal and meningeal inflammation, and perivascular cuffing

were mild, and prominent microglial nodules were not observed. However, CD68 IHC

showed an increased number of microglial cells/macrophages in and around

necrotic/vacuolar plaques (Figure 3.15 G, Figure 3.16 C) and surrounding some neurons,

neuronophagia (Figure 3.15 D).

Viral antigens and/or RNA were detected mainly in neurons (soma and processes),

neuropil (Figure 3.16 B, G, H; Figure 3.15 F) and occasionally, in the ependyma (Figure

3.16 I) and subependymal cells. Single or plaque-like groups of positive neurons were

observed in the cerebellum (dentate nucleus, molecular and granular layers), cerebral

cortex, midbrain (periaqueductal grey area, substantia nigra), hippocampus (pyramidal

layer, dentate gyrus, entorhinal cortex) and thalamus (Figure 3.15 F; Figure 3.16 B; Figure

3.16 I). Positive neurons were often, but not always, found in or near necrotic/vacuolar

plaques. Conversely, not all plaque-like neuronal virus antigen positivity was associated

with necrosis or prominent vacuolation (Figure 3.16 I). Necrotic plaques in the white matter

were not associated with viral antigens/RNA.

Table 3.7 Demography, Clinical and Pathological Data of Fatal Human Cases of Hendra Virus Infection Case no.

Age (year) /sex


a

Light microscopy Positive IHC b Pathological and/or ISH tissue staining

/clinical diagnosis

CNS

Non-CNS

1 49/M Influenza-like illness, complicated by respiratory and renal failure, and cardiac irritability. Died about 12 days after admission to hospital

≈6 days Brain: Vasculitis and necrotic/vacuolar plaques in the cerebrum and cerebellum. Mild meningoencephalitis Lung: Vasculitis, parenchymal inflammation and necrosis. Heart and kidney: Vasculitis, mild inflammation

Brain (Neurons, ependyma, blood vessels)

Lung (type II pneumocytes, blood vessels) Kidney (glomeruli, capillaries, tubules)

Acute Hendra infection with encephalitis c

2 35/M Presented initially with headache, drowsiness and meningitis. After 13 months readmitted with fever, seizures, neurological deficits and coma. Died after 25 days of hospital admission

≈12 days (13 mo.)

Brain: Confluent inflamed areas, neuronal loss, gliosis, mainly in cerebral cortex; no vasculitis Other organs: No vasculitis or significant pathology

Brain (Neurons mainly; possibly also glial and inflammatory cells)

Negative Relapsing Hendra encephalitis

(Table 3.7, cont) 92

Case no.

Age (year) /sex


a

Light microscopy Positive IHC b Pathological and/or ISH tissue staining

/clinical diagnosis

CNS

Non-CNS

3 40/M Influenza-like illness, myalgia, headache and vertigo of 5 days duration. Recovered apparently well after 6 weeks

≈5 days na na d na Acute Hendra infection

4 Adult/F

Cough, sore throat, fever, lethargy, and lymphadenopathy. Recovered and apparently well

≈7 days na na na Acute Hendra Infection

a Information (age, sex, clinical presentation, incubation period) compiled from references (Selvey et al, 1995; O’Sullivan et al, 1997; Hanna et al, 2006). Note: The 5 and 6th case were reported in July 2008 (ProMed-mail, 2008) b IHC, immunohistochemistry; ISH, in situ hybridization. ISH was not performed on non CNS tissues of Case 1 due to insufficient tissues. c Acute encephalitis based on pathological evidence; clinical encephalitis was not reported. d

na, tissues not available for study

93

1

A B

DC

E F

HG


95

Figure 3.15 Vascular and CNS pathology in human acute Hendra virus infection. A: Endotheliitis in a meningeal vessel characterised by subendothelial infiltration of inflammatory cells (arrows). B: Cerebral vasculitis characterised by intramural inflammatory cells and necrosis. C: Immunolocalisation of viral antigens in meningeal capillary endothelial cells (arrows). D: Necrotic plaque in the cerebral cortex. E: Subtle vacuolar plaque (arrows) in the cerebellum molecular layer consists of fine neuropil vacuolation with adjacent eosinophilic Purkinje cells (arrowhead). F: Viral antigens in a similar cerebellum vacuolar plaque seen as fine neuropil granular immunopositivity. G: CD68-positive macrophages/microglia in the vicinity of a vacuolar plaque in the cerebellum. H: White matter necrotic plaque consists of eosinophilic, axonal spheroid-like material. H&E stains: A, B, D, E, H; immunoperoxidase with diaminobenzidine substrate and haematoxylin counterstain, F, G. Original magnifications: x 40 (D); x 100 (E, G); x 200 (A, F, H); x 400 (B, C).

96

Non-CNS organs

In the lung, there was severe parenchymal inflammation, necrosis, intra-alveolar

macrophages/inflammatory cells, prominent type II pneumocyte proliferation and

occasional alveolar membranes (Figure 3.17 B, C). The kidney showed the rare focal

glomerulitis (Figure 3.17 F) and focal inflammation around necrotic tubules.

There was evidence of emperipolesis and the occasional bizarre multinucleated giant cell in

the lymph nodes (Figure 3.17 H). Viral antigens were demonstrated in alveolar type II

pneumocytes, intra-alveolar macrophages (Figure 3.17 D) and the occasional glomeruli

(Figure 3.17 G) and tubules

1

A B

DC

E F

H

G

I


98

Figure 3.16 CNS pathology in human acute Hendra virus infection. A-D: Necrotic/vacuolar plaque in cerebellum dentate nucleus. A. Necrotic/vacuolar plaque shows neuronal loss, coarse neuropil vacuolation, mild inflammation and surviving neurons (arrows). B: Neurons (arrows) and neuronal processes (arrowhead) immunostained for viral antigens. C: Inflammatory cells comprised mainly CD68-positive macrophages/microglia. D: Neuronophagia, neurons surrounded by CD68-positive macrophages/microglia (arrows). E. Eosinophilic viral inclusions in ependyma (arrows). F. Eosinophilic cytoplasmic viral inclusions in neurons (arrows) in the hippocampus pyramidal layer. G: Granular, virus antigens in neuronal cytoplasm (arrowhead) and nucleus (arrow). H: Virus RNA in neurons from the same area in F. I: Plaque-like cluster of neurons and adjacent ependymal cells (arrow) immunostained for viral antigens in the midbrain periaqueductal area. H&E stains, A, E, F; immunoperoxidase with diaminobenzidine substrate and haematoxylin counterstain, B-D, G, I. Original magnifications: x 40 (I); x 100 (A, C); x 400 (B, D-H).

1

A B

DC

E F

HG


100

Figure 3.17 Pathology in the lung, heart, kidney and lymph node in human acute Hendra virus infection. A. Vasculitis and endotheliitis in an artery in the lung (arrow). B. Focal necrosis and inflammation of lung parenchyma. C: High power magnification of B shows intra-alveolar inflammatory cells, type II pneumocyte proliferation and a multinucleated giant cell (arrow). D. Viral antigens are immunolocalised to type II pneumocytes and alveolar macrophages. E. Myocardial vasculitis characterised by intramural inflammation (arrow). F. Focal glomerulitis and capillary thrombosis (arrow) in the kidney. G: Viral antigens are demonstrated in capillary endothelium and the edge of the glomerulus. H. Multinucleated giant cell (arrow) in the lymph node. H&E stains, A-C, E-H; immunoperoxidase with diaminobenzidine substrate and haematoxylin counterstain, D, G. Original magnifications: x 40 (B); x 200 (A, E-H); x 400 (C, D).

101


The clinical features are summarised in Table 3.7 (Case 2).


Only the CNS tissues, and mainly the cerebral cortex, showed evidence of inflammation

although very focal areas in the pons, cerebellum and spinal cord were also inflamed.

Inflammatory lesions were usually extensive and confluent, and consisted of intense

infiltration of macrophages, lymphocytes and some plasma cells, with prominent

perivascular cuffing (Figure 3.18 A-C). There was severe neuronal loss, glial proliferation

and an increased number of reactive blood vessels. More discrete, plaque-like, but

otherwise similar inflammatory lesions, were occasionally observed. There was no

evidence of vasculitis, endothelial syncytia or thrombosis, and viral inclusions were not

prominent. No convincing vacuolated necrotic lesions similar to that seen in relapsed/late-

onset NiV encephalitis were identified.

Within inflamed areas, focal viral antigens/RNA were demonstrated mainly in

surviving neurons (Figure 3.18 D, E), and possibly in some glial/inflammatory cells as

well. Overall, inflammation was much more extensive than viral antigens/RNA. Severe

meningitis was found over many areas of the cerebral cortex (Figure 3.18 A). All the non-

CNS organs were uninflamed, and in particular, no vasculitis, endothelial syncytia or viral

inclusions and antigens/RNA were detected.

1

B C

ED


A

103

Figure 3.18 CNS pathology in human relapsed Hendra virus encephalitis. A: Severe meningoencephalitis consisting of large confluent areas of inflammation in the cerebral cortex (arrows). B: Encephalitis is characterised by severe necrosis, neuronal loss, parenchymal inflammation, reactive blood vessels and perivascular cuffing. C. High power magnification of inflamed areas shows mainly macrophages, lymphocytes and some plasma cells, and severe neuronal loss. D: Viral antigens immunolocalised to mainly neurons, and possibly glial and inflammatory cells as well. E: Virus RNA was detected in neuron-like cells (arrows). H&E stains, A-C; immunoperoxidase with diaminobenzidine substrate and haematoxylin counterstain, D; in situ hybridisation with haematoxylin counterstain, E. Original magnifications: x 40 (A); x 100 (B); x 200 (C); x 400 (D, E).

104

3.5 Hamster acute NiV infection

3.5.1 Animal Infection Experiments: Survival and LD

50

In the first study, none of the Swiss mice inoculated by either the IN or IP route developed

any clinical signs. Only guinea pigs that were infected by IP route, and therefore received

107

Figure 3.19 shows the dose-survival graphs of hamsters in the second study (LD

infectious viral particles showed transient fever and weight loss after 5 to 7 days but

they recovered. Golden hamsters infected by both routes showed difficulties with

movement and balance, and rapidly died 5 to 8 days after infection.

50)

that were inoculated with serial dilutions of viruses, viz., 1 to 104 pfu by IP route and 10 to

106 pfu by IN route. The time interval between infection and appearance of clinical signs

and death was shorter in IP-infected hamsters. They died 5 to 9 days post-infection and <24

hours after the appearance of tremor and limb paralysis. Conversely, the majority of IN-

inoculated animals showed a progressive deterioration presenting with imbalance, limb

paralysis, lethargy, muscle twitching and breathing difficulties in the final stages. The

majority of animals died between 9 and 15 days post-infection. However, 6 animals died

later, one at day 18, two at day 21 and three at day 29. The LD50

In animals surviving more than 30 days post-infection, and that were inoculated

with lower viral doses (1 and 10 pfu/animal for IP route; 10 and 10

of animals by IP and IN

route was, respectively, 270 pfu and 47,000 pfu for each animal.

2 pfu/animal for IN

route) there was no seroconversion. In fact, none of these animals died or showed any signs

of illness. In contrast, surviving animals infected with higher viral doses, and that were kept

in the same cages as animals given the same doses and died, had high levels of antibody.

Nonetheless, these survivors showed no clinical signs of illness.

105

0

1

2

3

4

5

6

7

1 2 3 4 5 6 7 8 9 10 11 12 13

No.

of s

urvi

ving

ani

mal

s*1 & 10

102

103

104

Survival in days

0

1

2

3

4

5

6

7

1 3 5 7 9 11 13 15 17 19 21 23 25 27 29

Survival in days

No.

of s

urvi

ving

ani

mal

s

*10 & 102

103 104

105

106

* Dose expressed asnumber of plaque formingunits/animal

(Figure 3.19, cont.)

106

Figure 3.19 Survival graphs of hamsters infected by Nipah virus via different routes and doses (LD50

study)

In the transmission study (third study) in which uninfected animals were housed

together with infected animals, none of the uninfected animals showed evidence of disease

or seroconversion.

3.5.2 Viral Isolation and Viral Genome Detection

In general, RT-PCR of various animal autopsy specimens showed that NiV genome could

be detected in most tissues and urine (Table 3.8; Figure 3.20). Serum was the notable

exception in that it was uniformly negative for viral genome. Because of this, viral culture

was not attempted on serum. Where both these tests were performed, the range of tissues

positive for viral culture correlated well with RT-PCR, although the percentage for

positivity was lower for viral culture especially in IN-infected hamsters (Table 3.8).


Blood Vessels

Vascular pathology was found in brain, lung, liver, kidney and heart. In large blood

vessels the more florid changes were characterised by focal, transmural fibrinoid necrosis

with surrounding inflammation (Figure 3.21 A, B; Figure 3.23 D). However, vasculitis was

more subtle with fewer inflammatory cells (Figure 3.21 G; Figure 3.23 A) and very focal

nuclear pyknosis and karyorrhexis (Figure 3.21 E).

107

Table 3.8 Reverse transcriptase-Polymerase Chain Reaction (RT-PCR) Analysis, Virus Culture and Pathological Lesions in Hamster Nipah Virus Infection Tissue / Specimen

No. of animals with pathology / total no. of animals (%)

No. of positive animals / total no. of animals (%)

Intranasal Route

Intraperitoneal Route

RT-PCR

Virus culture

RT-PCR

Virus culture

Blood

na* 0/5 (0%) nd ** 0/2 (0%) nd

Urine

na 3/3 (100%) 0/2 (0%) 2/3 (67%) 1/1 (100%)

Brain

8/12 (67%) 4/5 (80%) 1/5 (20%) 3/3 (100%) 2/3 (67%)

Spinal cord

0/7 (0%) na na 3/3 (100%) 2/2 (100%)

Lung

4/12 (33%) 4/5 (80%) 0/5 (0%) 3/3 (100%) 2/3 (67%)

Kidney

2/12 (17%) 4/5 (80%) 1/5 (20%) 3/3 (100%) 2/3 (67%)

Spleen

1/12 (8%) 5/5 (100%) 0/5 (0%) 3/3 (100%) 1/3 (33%)

Liver

4/12 (33%) 5/5 (100%) 0/5 (0%) 3/3 (100%) 2/3 (67%)

Heart

3/12 (25%) 3/5 (60%) 0/5 (0%) 3/3 (100%) 0/3 (0%)

* na = tissues not available or not suitable for analysis. ** nd = tissues available but analysis not done because RT-PCR is negative Note: The LD50

of animals by intranasal and intraperitoneal routes was respectively 47,000 and 270 plaque forming units for each animal.

1

Animal 4 Animal 3

Heart

Kidney

Spleen

Brain

Liver

Lung

Serum

Heart

Kidney

Spleen

Brain

Liver

Lung

Serum

+ control

Figure 3.20Gel Electrophoresis Results of Reverse Transcriptase-Polymerase Chain Reaction in Hamster Tissues with Acute Nipah Virus Infection by Intranasal Route (LD50 Study)

108

109

Multinucleated syncytia arising from the endothelium were encountered in one hamster that

died 8 days after IP-inoculation (Figure 3.21 C, D). Thrombosis could be found in the

lumen of some vessels (Figure 3.23 E). Viral antigen and genome as demonstrated by IHC

and ISH, respectively, localised to endothelial cells and syncytia (Figure 3.21 D, F; Figure

3.23 F) and underlying smooth muscle of the tunica media in blood vessels. Viral

nucleocapsids were also detected in the vascular wall.

CNS

The brain was the most severely affected (67%) in terms of vascular and

parenchymal lesions, compared with other organs (Table 3.8). Apart from vasculitis,

the most striking features were in neurons found in the vicinity of vasculitis. Affected

neurons showed numerous eosinophilic inclusion bodies in the cytoplasm (Figure 3.22 C,

D). These inclusions, as well as neuronal cytoplasm with no obvious inclusions, and

neuronal processes, were often positive for both viral antigen and RNA (Figure 3.22 F, H,

I). Very rarely, there may be apparent cytoplasmic fusion of adjacent neurons (Figure 3.22

D) Ultrastructurally, these inclusions were composed of defined masses of filamentous

nucleocapsids of the fuzzy type typically associated with paramyxoviruses (Figure 3.24 A).

These inclusions were immunogold-labeled by NiV-specific antibodies (Figure 3.24 B).

Nuclear inclusions could not be found but there was evidence of nuclear IHC positivity

(Figure 3.22 H).

A B

DC

E F

HG


111

Figure 3.21 Vascular and parenchymal pathology in hamster acute Nipah virus infection. A: Hepatic artery showing focal, transmural fibrinoid necrosis with surrounding inflammation. B: Artery in kidney showing focal transmural fibrinoid necrosis (arrow) with surrounding inflammation. C: Endothelial multinucleated syncytia (arrows) in a small pulmonary artery. D Virus RNA demonstrated in endothelial syncytia (arrows) and vascular smooth muscle (arrowheads) in the same tissue as C. E: Necrosis and karyorrhexis (arrow) in a cerebral vessel. F: Viral antigens immunolocalised to endothelium (arrowhead) and smooth muscle (arrows) in a meningeal blood vessel. G: Mild, focal endotheliitis in the aorta (arrow). H: Myocardial necrosis (arrow) with adjacent inflammation (arrowhead). H&E stains, A-C, E, G, H; immunoperoxidase with diaminobenzidine substrate and haematoxylin counterstain, F; in situ hybridisation with haematoxylin counterstain, D. Original magnifications: x 100 (G); x 200 (A, B, H); x 400 (D); x 1000 (C, E, F);

112

Other parenchymal changes included focal areas with evidence of

ischaemia/infarction and oedema (Figure 3.22 B). Parenchymal and meningeal

inflammation (Figure 3.22 E) were generally mild, and only occasionally were perivascular

cuffing and neuronophagia observed. Rarely, IHC positivity was noted in ependymal lining

(Figure 3.22 G), and in mononuclear cells found in the meninges and choroid plexus. The

choroid plexus lining epithelium however was negative for viral antigen and genome. IHC

and ISH positivity was not observed in the white matter.

Non-CNS organs

In the lung, small discrete nodular or more confluent areas of parenchymal

inflammation, often associated with vasculitic vessels, could sometimes be observed

(Figure 3.23 A to D). Inflammatory cells consisted mainly of a varying mixture of

macrophages, neutrophils and lymphocytes. Multinucleated giant cells and inflammatory

cells positive for NiV by IHC and ISH were rare. Fibrinoid necrosis of lung parenchyma

was rare and focal. Bronchitis, multinucleated syncytia or other evidence of NiV infection

of bronchial epithelium were not found.

Glomerular lesions in the kidney were rare but the most florid lesions had

thrombotic plugs in the glomerular capillaries, peripheral multinucleated syncytia, and

surrounding inflammation (Figure 3.23 E). Viral antigen was detected only in the

occasional glomerulus and tubule (Figure 3.23 F). In the kidney of several animals, the

covering epithelium of the renal papilla that project into the calyx consistently

demonstrated the presence of viral antigen (Figure 3.23 G) but ISH was negative in the

same epithelium.

The rare focus of necrosis was noted in the spleen but no vasculitis or

multinucleated giant cells were observed. IHC and ISH were occasionally positive in

periarteriolar lymphoid cells (Figure 3.23 H). There appeared to be no specific liver

113

parenchymal lesions. In the heart, myocarditis associated with infarction was only rarely

observed (Figure 3.21 H). No inflammation or viral antigen was detected in lymph nodes or

nasal epithelium.

In general, the spectrum and severity of pathological lesions in the blood vessels

and the other organs showed no significant difference between IP- and IN-inoculated

animals.

A B

DC

E F

G

H I


115

Figure 3.22 CNS pathology in hamster acute Nipah virus infection. A: Small vessel vasculitis (arrow) characterised by mild inflammation in the vicinity of infected neurons. B: Focal parenchymal ischaemia, infarction and perineuronal microscytic areas (arrows). C: Neurons with eosinophilic cytoplasmic viral inclusions (arrows). D: Neurons showing viral inclusions (arrows) and probable cytoplasmic fusion (arrowhead). E: Mild meningitis (arrow). F: Immunolocalisation of viral antigens to neurons (arrows) and processes near a vasculitic vessel (arrowhead). G: Immunolocalisation of viral antigens to ependyma (arrows) and subependymal cells. H: Viral antigens demonstrated in neuronal nuclei (arrows), and in cytoplasmic viral inclusions (arrowhead). I: Virus RNA is shown in neuronal cytoplasm (arrows). H&E stains, A-E; immunoperoxidase with diaminobenzidine substrate and haematoxylin counterstain, F-H; in situ hybridisation with haematoxylin counterstain, I. Original magnifications: x 100 (E); x 200 (A, B, F, G, I); x 400 (C, D, H).

Scale=1mm

A B

DC

E F

HG

(Figure 3.23, cont.)

116

117

Figure 3.23 Pathology in lung, kidney and spleen in hamster acute Nipah virus infection. A: Macroscopically evident, nodular inflammatory lesions (arrows) on lung surface. B: Discrete nodular inflammatory lesions in the lung parenchyma (arrows). C: Severe inflammation in lung parenchyma adjacent to necrotic blood vessels (arrow). D: Higher power magnification of C showing intra-alveolar inflammatory cells and necrotic vessels (arrow). E: Glomerulitis characterised by thrombotic plugs (arrows), inflammation and syncytial formation (arrowhead) at the periphery of the glomerulus. F: Viral antigens are immunolocalised to renal tubule (arrowhead) and glomerulus (arrow). G: Viral antigens found in epithelium covering the papilla in the kidney (arrow). H: Lymphoid cells in the splenic white pulp immunopositive for viral antigens. H&E stains, B-E; immunoperoxidase with diaminobenzidine substrate and haematoxylin counterstain, F-H. Original magnifications: x 25 (B); x 40 (C); x 100 (D, H); x x 200 (E-G).

N

A

B

N

Figure 3.24Ultrastructure of infected neurons in hamster acute Nipah virus infectionA: Cytoplasmic viral inclusion (arrows) composed of nucleocapsids in a neuron. B: Immunogold-labeled neuronal viral inclusion (arrows) demonstrated by immunoelectron microscopy with specific anti-Nipah antibodies. N = nucleus. Scale bars, 0.5 micron

118

119

3.6 Hamster Acute HeV infection


In the group of hamsters infected with a lower dose of HeV (103

At 2 days post-infection, focal, small amounts of viral antigens were noted in lung,

kidney and spleen. Significantly larger amounts of viral antigens began to appear from 3-5

days post-infection, in the lung, brain, kidney, liver, heart and spleen. In the lung, IHC

staining was predominantly in inflammatory aggregates, alveolar walls and blood vessels

(Figure 3.25 D, E). In the brain, large plaque-like neuronal/neuropil immunopositivity for

viral antigens could be found in the cerebellum (Figure 3.25 J) and cerebral cortex.

Neuronal staining was the most prominent but viral antigens could also be detected in the

meninges, blood vessel (Figure 3.25 K) and ependyma. In the kidney, viral antigens were

mainly confined to glomeruli (Figure 3.25 F), tubules (Figure 3.25 G) and occasional blood

vessel (Figure 3.25 H). Viral antigens were immunolocalised in the heart and aorta to

endocardium (Figure 3.25 L) and endothelium, respectively. In the liver, immunopositivity

pfu), from 1-2 days post-

infection, only scattered foci of mild inflammation were found in the lung (Figure 3.25 A)

and spleen at 2 days post-infection. From 3 days post-infection onwards, the lungs showed

better defined nodular aggregates of inflammatory cells in the parenchyma (Figure 3.25 B).

However all the other organs (brain, heart, kidney, liver and spleen) did not show

significant inflammation. From 4-5 days post-infection hyalinised necrotic blood vessels

were observed in the lung, liver (Figure 3.25 N), kidney and spleen, but not in the brain or

aorta. Increasing numbers of endothelial syncytia were also seen in the pulmonary vessels

(Figure 3.25 C) but endothelial syncytia were not prominent in the other organs. A few

necrotic plaques were observed in the brain (Figure 3.25 I) and spleen.

120

was mainly in blood vessels and endothelial lining of sinusoids (Figure 3.25 M) while in

the spleen, the lymphocytes, blood vessels, mesothelial cells were positive.

The histopathological lesions and IHC findings in the animals infected with a high

dose (105 pfu) of HeV were similar to those infected with 103 pfu but generally, the lesions

were more florid in the former.

A B

C D

E F

HG


I J

K L

M N


123

Figure 3.25 Pathology of hamster acute Hendra virus infection. A: Subtle nodular lung parenchyma inflammation (arrows) is observed at 3 days post-infection. B: Nodular inflammation in the lung (arrows) is more prominent at 5 days post-infection. C: Endothelial syncytia in small pulmonary artery (arrows). D: Viral antigens immunolocalised to nodular inflammatory lesions in the lung. E: Endothelial lining (arrows) and syncytium (arrowhead), and intra-alveolar cells are immunopositive for viral antigens. F: Immunolocalisation of viral antigens in a kidney glomerulus. G: Immunolocalisation of viral antigens in renal tubules. H. Renal vascular walls positive for viral antigens (arrows). I: Necrotic plaque in brain parenchyma characterised by neuronal loss and minimal inflammation. J: Plaque-like neuronal/neuropil immunopositivity for viral antigens. K: Viral antigens immunolocalised to meningeal vessel wall (arrows). L: Viral antigen immunolocalised to aorta wall (arrows). M: Liver sinusoid endothelial lining and syncytium (arrow) immunopositive for viral antigens. N: Hepatic vasculitis characterised by fibrinoid necrosis and surrounding inflammation (arrow). H&E stains, A-C, I, N; immunoperoxidase with diaminobenzidine substrate and haematoxylin counterstain, D-H, J-M. Original magnifications: x 2.5 (A); x 40 (B); x 100 (D, I, L); x 200 (G, H, K, M, N); x 400 (C, E, F, J).

124

CHAPTER 4: DISCUSSION


The outbreaks of NiV and HeV infections in Asia and Australia, respectively, are prime

examples of emerging zoonotic infectious diseases that continue to occur worldwide. They

also underscore the fact that epidemic viral encephalitides other than the well-known

arboviral encephalitis are still very important diseases despite modern scientific advances.

In many of these outbreaks, pathologists, as part of a multidisciplinary team, played key

roles in identifying the causative agent or describing the pathogenic processes (Zaki and

Paddock, 1999, Schwartz et al., 1995).

In the NiV outbreak, the clinical and laboratory speculation initially centered on JE

virus as the causative agent; however, several features of the outbreak argued against this

possibility. First, JE appeared unlikely in an outbreak that affected mainly adults rather than

children in an area of sporadic JE transmission. Second, most if not all patients had a

history of direct contact with pigs. Third, there was clustering of cases with higher infection

rates in the same households than would be expected with JE. Fourthly, many patients had

been previously vaccinated against JE with a standard vaccine and were therefore likely to

have developed protective immunity against the virus (Chua et al., 1999). Finally, despite

massive efforts to reduce the population of mosquitoes in the farms in the outbreak areas

and widespread desertion by farm workers, the number admitted to hospitals continued to

rise, providing further evidence that the outbreak was not due to JE.

The diagnosis of NiV infection, suspected by history and clinical manifestations,

can be supported by characteristic histological findings. These findings include syncytial

giant cell formation, vasculitis and viral inclusions. Other CNS changes observed,

125

including perivascular cuffing, parenchymal inflammation and neuronophagia, are rather

non-specific features and can be found in other acute viral encephalitides (Esiri and

Kennedy, 1997). From the diagnostic standpoint, perhaps the most unique finding is the

presence of syncytial multinucleated endothelial cells. To our knowledge, this feature has

not been described in other infective encephalitides. However, this feature occurred in only

28% of the cases and cannot be used as a sensitive criterion for diagnosis of NiV infection.

There is a higher likelihood of identifying these cells in small blood vessels of the CNS of

patients who succumbed early to the disease. The presence of multinucleated giant cells of

non-endothelial origin found in the kidney at the edge of the glomerulus also seems to be a

unique, albeit relatively rare, feature of NiV infection. Characteristic multinucleated giant

cells were also seen in the lung, spleen and lymph nodes; however these cells can also be

seen in measles virus, respiratory syncytial virus, parainfluenza virus, herpesvirus, and

other infections and therefore are not unique to NiV infection (Zaki and Bellini, 1997,

Delage et al., 1979, Delage et al., 1984, Read et al., 1980, Worrell and Cockerell, 1997).

Widespread vasculitis with predominant CNS involvement is a useful

histopathological feature that may suggest the diagnosis of NiV infection. Many infectious

agents can cause vasculitis, including herpesviruses, rickettsiae, and Neisseria (Somer and

Finegold, 1995). However, the histopathological features are somewhat different in these

infections. In rickettsial encephalitis (Walker and Dumler, 1997), vasculitis is usually more

subtle and CNS necrosis less prominent than seen in NiV encephalitis. Varicella-zoster and

herpes simplex infections may be associated with granulomatous angiitis, a feature not seen

in NiV infection (Fukumoto et al., 1986, Schmitt et al., 1992).

Finally, the diagnosis of NiV infection can be supported by the histopathological

and ultrastructural appearance and distribution of characteristic viral inclusions. However,

similar inclusions can be seen in other paramyxoviral infections (Zaki and Bellini, 1997)

126

and therefore unequivocal diagnosis can be made only by laboratory tests such as IHC,

virus isolation, PCR and serology.

The utility of IHC as a diagnostic modality was established by correlation with

results of virus isolation and serological assays. Virus can be isolated from the CSF,

respiratory secretions and urine of patients (Chua et al., 2002) but should be done in BSL- 4

laboratory facilities. The combination of IHC and serological tests established the diagnosis

for all cases regardless of duration of illness. Four seropositive and IHC-negative cases had

duration of illness of 14 days or more, emphasizing the important role of serology in the

diagnosis and suggesting that in most cases viral antigens are cleared by 2 weeks after

infection. Conversely, the 3 IHC-positive cases with negative or unavailable serological

results underscore the importance of IHC in the diagnosis of fatal NiV infection (Table 3.5).

CNS is the optimal tissue for IHC analysis because it was 3 to 4 times more likely to be

positive for NiV than were lung or kidney tissues, the next most likely organs to be positive

(Table 3.3)

Several lines of evidence allowed for precise characterisation of viral tropism and

its consequences. Epithelial cell involvement, although not common, included bronchiolar

mucosa, renal tubules, and podocytes at the edge of glomerulus. Endothelial cells and

neurons had a remarkably high viral load, as evidenced by immunostaining of NiV

antigens. The ultrastructural finding of inclusions in endothelial and neuronal cells is

consistent with replication in these sites (Goldsmith et al., 2002, Hyatt et al., 2001). The

ISH studies also confirmed the involvement of neurons in viral replication. In contrast to

neurons, glial cells, such as ependyma, oligodendrocytes and astrocytes were far less

susceptible. Our data showed that small vessels, such as small arteries, arterioles, venules,

and capillaries, were more prone to vasculitis and thrombosis than larger vessels.

Differences in endothelial susceptibility to viral infection could certainly account for the

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different frequencies of vasculitis in various organs: CNS, 80% of cases, followed by the

lung, heart, and kidney with 62%, 31%, and 24%, respectively.

Widespread vasculitis, a key event in the pathogenesis of NiV infection, seems to be

a consequence of infection of vascular endothelial and smooth muscle cells (Figure 4.1).

Vasculitis resulted in thrombosis, vascular occlusion, ischaemia and microinfarction.

Overall, the frequency of vasculitis seemed to be proportional to necrosis and necrotic

plaques, particularly in the CNS and lung. However, the necrotic plaques and the acute

encephalitis syndrome may stem from both direct neuronal infection and ischaemic injury

especially in the neuronal areas such as the cerebral grey matter and brain stem (Figure

4.1). This dual pathogenetic mechanism for tissue injury in the CNS, and indeed in the

other organs, appears to be unique in viral pathogenesis. This sequence of pathological

events is supported by the concomitant increase in frequency of syncytia, vasculitis,

thrombosis, necrotic plaques, and viral antigen in the CNS (Figure 3.9). In contrast to

necrotic plaques in neuronal areas, plaques in the white matter are likely to be caused

mainly by microinfarction since glial cells are far less susceptible to NiV infection than

neurons. Thrombosis was usually associated with vasculitis; thrombus in uninflamed

vessels could represent thromboembolism or thrombus propagation.

The pattern of vasculitis and viral antigen distribution suggests endothelial infection

occurs before transmural spread to the underlying smooth muscle in the tunica media.

Finding of numerous infected neurons in association with necrotic plaques and vasculitic

vessels suggests that a breach of the blood-brain-barrier may facilitate virus escape and

extravascular CNS infection (Figure 4.1). In SSPE, endothelial infection is thought to

facilitate measles virus entry into the brain even though vasculitis is absent (Cosby and

Brankin, 1995). Once neurons are infected, inter-neuronal spread along neuronal pathways

may contribute to viral dissemination.

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Figure 4.1 Summary of the dual pathogenetic mechanisms of acute Nipah virus infection. The primary site for virus replication that leads to viraemia may be the lung and lymphoid organs like in measles infection.

Nipah Virus Replication (? site)

Viraemia

Vascular Infection (endothelial/smooth muscle)

Vasculitis

Thrombosis & Obstruction

Virus Escape Damaged Vessel

Ischaemia & Infarction

Parenchymal Infection

Acute Nipah Infection

129

The appearance of similar pathological lesions in several organs at the same time

suggests an early viraemic phase that follows primary viral replication (Figure 4.1). In

measles infection, primary viral replication occurs in respiratory tract mucosa and lymphoid

organs and is followed by a cell-associated viraemia (Griffin and Bellini, 1996). In this

series of fatal acute NiV cases, the extensive lymphoid necrosis and immunostaining of

lymphoid and respiratory tissues suggest that these tissues can also be similarly involved in

primary replication. Although unlikely to be a primary replication site, endothelium may act

as a site of secondary viral replication and amplification of viraemia. The temporal

sequence of antibody rise first in serum and then in the CSF (Figure 3.8) provides indirect

evidence that viraemia occurs before CNS infection and probably reflects the fact that

induction of antigen-specific, antibody-secreting B cells first occurs in the peripheral

lymphoid tissues. Virus entry into the CNS is very likely via the haematogenous route, as

there is no evidence for transnasal infection of olfactory bulb like in herpes simplex

encephalitis.

CNS predilection, as evidenced by pathological changes and viral

immunolocalisation, correlates well with the encephalitic syndrome reported in fatal and

non fatal cases (Chua et al., 1999, Paton et al., 1999). In this series of acute NiV fatal cases,

predominant clinical features included drowsiness, disorientation, confusion, segmental

myoclonus, and areflexia. Clinically, non-fatal cases also undergo a similar, less severe

acute encephalitic syndrome (Goh et al., 2000). Brain MRI studies of non-fatal and fatal

cases show similar scattered, small, and discrete lesions that probably represent necrotic

plaques (Lim et al., 2000, Sarji et al., 2000). The absence of large wedge-shaped infarctions

in the cerebrum may be related to absence of thrombo-occlusion of larger cerebral blood

130

vessels. Certainly, there was a paucity of vasculitis in large arteries such as the middle

cerebral artery.

In our study, CNS pathological changes were more severe than histopathological

changes in other organs. This finding also correlates with the lower frequency of non-CNS

clinical manifestations. Large infarcts were also not found in non-CNS organs, with the

possible exception of the single case of large myocardial infarct assumed to have resulted

from coronary artery vasculitis. A pulmonary syndrome was reported in some cases from

Singapore (Paton et al., 1999) and 40% of cases described in this study had cough or

respiratory symptoms (Table 3.2). These symptoms could be related to pulmonary lesions,

such as necrosis, oedema and haemorrhage. The immunolocalisation of viral antigens to

bronchiolar epithelium, albeit in only 1 patient, could explain why virus could be isolated

from tracheal secretions (Chua et al., 2001a). Similarly, renal glomerular and tubular

involvement could account for detectable virus in patient urine.

The natural history of acute NiV infection is still unclear. Acute encephalitis

probably occurs in a significant proportion of cases after exposure to virus (Figure 1.4).

Mortality from acute NiV encephalitis may be as high as 40-70% (Goh et al., 2000, Harit et

al., 2006, Hsu et al., 2004) and is more likely in patients with brainstem signs and virus

isolated from CSF (Chua et al., 2000b). Approximately 15% of patients with acute

encephalitic syndrome develop residual neurologic deficits (Goh et al., 2000). Other

survivors may develop relapsed/late-onset encephalitis.


One of the most interesting complications of acute NiV infection is relapsed/late onset

encephalitis. In particular, the repeated neurological relapse that may occur in some patients

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is intriguing. Fortunately, as clinical data (Tan et al., 2002) and Case 4 (Table 3.6) shows,

this complication is not common nor invariable. The unequivocal presence of viral

nucleocapsids, inclusions, antigens and RNA and other features confirm the suggestion that

relapsed and late-onset encephalitis have identical pathology and both are the result of

relapsed infection rather than post-infectious encephalitis (Tan et al., 2002). Moreover,

there was no evidence of perivenous demyelination. Interestingly, the amount and relative

size of CNS viral inclusions generally exceeded that found in acute NiV cases suggesting

that viral replication and the consequent accumulation of viral inclusions may have

proceeded for a longer period of time. This is consistent with the long incubation period in

our cases of 3-22 months, and with a larger series of patients where the average incubation

period was about 8 months (Tan et al., 2002).

The absence of vasculitis in both CNS and non-CNS organs suggests that viral

relapse in the CNS was de-novo rather than a reinfection from viral foci from outside the

CNS (Figure 4.2). It is assumed that if the relapsed virus originated from outside the CNS,

viraemia would have to occur first to deliver the virus into the CNS in the same way as

acute NiV encephalitis. The total absence of vasculitis and/or virus in the blood vessels

provided indirect evidence that this did not happen because vascular endothelium/smooth

muscle, being highly susceptible to NiV, would likely be infected if there was viraemia. If

this hypothesis is correct, it is assumed that viral foci introduced during the acute CNS

infection were not eliminated by the immune response.

The reasons for viral relapse in relapsed/late-onset NiV encephalitis are unknown.

Virus mutation as in the case of SSPE where mutations in the M (matrix) and other genes

(Cattaneo et al., 1989) is one possibility.

Acute Encephalitis/Mild Infection

Figure 4.2Hypothesis for the pathogenesis of human relapsed/late-onset Nipah virus encephalitis. Recurrent viral spread from residual foci results in confluent inflammatory lesions as opposed to the discrete necrotic plaques in acute Nipah encephalitis.

Virus Foci Disseminatedby Viraemia

De Novo Recurrence fromResidual Virus Foci

132

Relapsed/Late-onsetEncephalitis/

133

Alterations in the host’s immune response, as in the case of measles inclusion body

encephalitis that results from immune suppression associated with lymphoid malignancies,

suggests that immune suppression may play a role in viral recurrence (Esiri and Kennedy,

1997). Interestingly, acute measles infection itself is associated with host immune

suppression (Griffin and Bellini, 1996), and being a paramyxovirus as well, NiV could

possibly cause immune suppression that might impact on subsequent development of

relapsed/last onset encephalitis. The possible association of acute NiV infection and

immunosuppression should be investigated in the hamster model. Further work is also

needed to investigate the pathogenesis of relapsed/late-onset NiV infection. Unfortunately,

there is currently no animal model for relapsed/late-onset NiV encephalitis.

The spherical or band-like, vacuolated necrotic lesion that is characterised by mild

to moderate neuronal degeneration and mild inflammation, is assumed to progress to the

complex vacuolated necrotic lesions and confluent parenchymal damage with severe

neuronal loss and inflammation. The absence of vasculitis-induced thrombosis, and the

demonstration of viral antigens and RNA mainly in neurons, strongly suggest that direct

neuronal infection alone may have caused these lesions. Vacuolation within or at the edge

of the vacuolated necrotic lesion may be due to oedema and/or neuronal degeneration. The

wave-like or concentric vacuolated necrotic lesions may arise from adjacent overlapping or

“colliding” lesions. As far as we are aware, these lesions have not been described in other

viral encephalitides. If these complex lesions are proven to be unique in relapsed/late onset

NiV encephalitis, their distinct morphology may be useful diagnostic features. The presence

of perineuronal vacuolation near or coalescing with vacuolated necrotic lesions suggests

that the former may be a precursor lesion. Morphologically, perineuronal vacuolation

resemble the neuronal vacuolation/microscytic areas seen near necrotic plaques in acute

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NiV encephalitis (Figure 3.2 E and Figure 3.3 G). In both instances, viral antigen and/RNA

were also demonstrated.

Of interest in the relapsed/late onset encephalitis cases is the apparent greater

involvement of the white matter especially in areas adjacent to lesions in the cerebral cortex

and the white matter tracts in the putamen. Compared with acute NiV encephalitis, many

more macrophages and possibly other inflammatory cells as well as glial cells such as

astrocytes and oligodendrocytes were positive for IHC and ISH within these areas. The

identity of these infected, non-neuronal cells and the reason why they appear to support

viral replication more readily than in acute NiV encephalitis will require further

investigation.

Clinically, relapsed NiV encephalitis is probably different from SSPE, as the latter

does not present with initial acute encephalitis before panencephalitis sets in years later.

Late-onset encephalitis arguably resembles SSPE more as the acute infections in both cases

are often mild and non-encephalitic. Nonetheless, relapsed/late onset encephalitis is not

invariably fatal like SSPE, and may even recur over several years (Chong and Tan, 2003,

Tan et al., 2002). Measles inclusion body encephalitis may also have some resemblance to

late-onset NiV encephalitis as both present within weeks of the acute infections, but so far

there is no reported overt lymphoid or other malignancies associated with the latter.

Nonetheless, immunosuppression could still play a role in late-onset NiV encephalitis.

The confluent inflammatory lesions in relapse/late-onset NiV encephalitis correlates

very well with the brain MRI findings (Goh et al., 2000, Sarji et al., 2000), and in fact

resembles the more typical MRI findings in other viral encephalitides such as herpes

simplex encephalitis, SSPE and measles inclusion body encephalitis. We believe that

confluent lesions are the result of progressive neuronal cell-to-cell viral spread via the

complex multi-connections of neuronal processes (Figure 4.2). Interestingly, 2 Bangladeshi

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patients with apparent acute NiV encephalitis presented with brain MRI changes that were

confluent hyperintense lesions rather than discrete lesions as expected (Quddus et al.,

2004). One possibility is that these 2 cases were really relapsed/late-onset NiV encephalitis

that happened to have a much shorter incubation period than normal. In this scenario, even

before the acute encephalitis phase has subsided, it merged imperceptibly with the

developing relapsed encephalitis. In the largest series of 22 relapsed/late-onset encephalitis

published so far (Tan et al., 2002), there was 1 patient who had episodes of neurological

relapse, the first at 9 days and the second at 6 weeks from the initial acute encephalitis. If

this hypothesis is correct, the factors that led to viral recurrence may have kicked in earlier

than usual in these 2 patients.

In the case of resolving acute NiV encephalitis, the discrete cystic lesions most

likely originated from necrotic plaques and represented various stages of healing. The

vasculitis that must have been present about 8 months ago appears to have long subsided,

but focal perivascular cuffing remains. The random distribution of these lesions in both

grey and white matter indirectly suggests that there may be no predilection for particular

blood vessels and/or neurons, at least in the cerebral hemispheres. The cerebral atrophy

indicates that there was considerable parenchymal damage in this patient following acute

encephalitis.

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Pathological findings in the two cases (Case 1 and 2, Table 3.7) confirmed that HeV was

neuronotropic and could cause CNS infection giving rise to acute and relapsed encephalitis

in the same way as NiV infection. In the patient with acute HeV infection without apparent

clinical encephalitis (Case 1 Table 3.7), mild vasculitis, meningitis, parenchymal

inflammation, necrotic/vacuolar plaques, and neuronal infection were found in the CNS,

features that have not been previously reported in acute HeV infection.

The necrotic/vacuolar plaques in acute HeV encephalitis may be equivalent to the

necrotic plaque of acute NiV encephalitis in that, like the latter, it may be the result of a

combination of neuronal infection and microinfarction following vasculitis-induced

thrombosis. However, thrombus-occluded cerebral blood vessels were apparently absent,

although there was vasculitis, and viral antigens could be demonstrated in cerebral vessels

and neurons. Vasculitis was also noted in the lung, heart and kidney, and vascular thrombi

were identified in the lung and glomeruli. Multinucleated endothelial syncytia were not

observed in this HeV case. The reasons for these apparent differences from acute NiV

infection could include the rarity of multinucleated endothelial syncytia (only found in 28

% of acute NiV encephalitis), a relatively mild CNS involvement, disintegration of

thrombotic plugs over time or inadequate sampling. Within or near the discrete white

matter necrotic plaques, there was no evidence of infection of glial or other cells

whatsoever, suggesting that microinfarction may be solely involved in their pathogenesis as

in the case of acute NiV encephalitis. If this is correct, focal infarction and death of

oligodendroglial cells surrounding axons may be responsible for this phenomenon.

Like NiV infection, the widespread vasculitis and simultaneous multisystem

involvement suggested that viraemia must have occurred, spreading the infection to

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multiple organs. Neuronal and other parenchymal cell infection was probably facilitated by

vasculitic damage to the blood brain barrier and other vessels. It is difficult to determine if

clinical encephalitis was missed as a result of the patient’s severe clinical condition at

admission, or whether clinical encephalitis was not severe enough to be detected.

Apart from blood vessels, the target cells/tissues in acute HeV infection seemed to

be similar to NiV infection. Viral inclusions and antigens/RNA in neurons and ependyma

in particular, suggest that active viral replication occurs in these cells. Likewise, type II

pneumocytes and alveolar macrophages may be able to support viral replication. Overall,

like NiV, involvement of glial cells appears to be rare in HeV infection. IHC was found to

be very useful to better demonstrate CD68-positive macrophages/microglial cells in

necrotic/vacuolar plaques, as the inflammation was mild and unconvincing by light

microscopy in many areas. This was not done in NiV encephalitis, as inflammation was

much more obvious.


The second fatal case of HeV infection (Table 3.7) with a relapse of neurological

manifestations 13 months after acute disease seems to be similar to relapsed NiV

encephalitis. The pathological findings confirmed that the patient had recurrent infection

rather than post-infectious encephalitis. At variance to a previous report (O'Sullivan et al.,

1997), we found no evidence of multinucleated endothelial syncytia or vasculitis in any of

the organs examined, nor evidence of viral antigens/RNA in the non CNS organs. Hence,

we postulate that recurrence occurred in much the same way as relapsed/late-onset NiV

encephalitis (Figure 4.2). After 13 months, vasculitis present initially, subsided and healed,

but the virus infection recurred to spread throughout the CNS. So far virus has not be

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isolated from either relapsed HeV or relapsed NiV encephalitis (O'Sullivan et al., 1997, Tan

et al., 2002).

As relapsed NiV encephalitis could occur even after 4 years or more (Chong and

Tan, 2003), it is theoretically possible that survivors of acute HeV infection could still

develop encephalitis long after 13 months. Fortunately, relapsed/late onset NiV encephalitis

is not uniformly fatal, suggesting that HeV encephalitis may not be invariably fatal as well

(Tan et al., 2002). Nonetheless, long-term follow-up of Cases 3 and 4 (Table 3.7) is

essential, and could be invaluable to determine future disease course. Since the brain MRI

changes (O'Sullivan et al., 1997) correlates well with the confluent pathological lesions in

relapsed HeV it may be a useful tool for long term follow-up of survivors


Of the 3 animal species, mouse, guinea pig and hamster that were inoculated with NiV the

hamster appeared to be the most susceptible. Depending upon the route and dose most of

the infected hamsters developed severe illness. Studies of tissues obtained from infected

hamsters suggested that it is a suitable animal model for acute NiV infection, demonstrating

most of the characteristics found in human acute NiV infection.

Hamsters could be infected by either IP or IN routes but infection by the IP

appeared to kill animals faster than the IN route. Furthermore, far lower IP doses were

required to kill the same number of animals as shown by the widely disparate LD50 doses

between IP- and IN-infected animals. This is probably not surprising because IN-inoculated

NiV presumably had to penetrate the mucosal barrier of the aerodigestive tract before

infection could take place, whereas IP-inoculated NiV theoretically could enter the

systemic circulation directly. The absence of detectable antibodies in the sera of animals

139

surviving more than 30 days post-infection, and which were either IP-inoculated with ≤10

pfu/animal or IN-inoculated with ≤102

Histopathological studies of infected hamster tissues showed that blood vessels and

particularly those in the CNS, developed vasculitis characterised by necrosis and intramural

inflammation. Evidence of direct viral infection of the vessel wall, including the

endothelium and smooth muscle, was provided by the presence of endothelial

multinucleated syncytia formation, and the detection of viral nucleocapsid, antigen and

RNA in the vascular wall. All these vascular features confirmed the findings in acute

human NiV infection. A notable exception could be hepatic vasculitis, which was not

reported in human infection. Moreover, neuronal infection as evidenced by viral inclusions,

and antigen/RNA is also very similar to the human infection confirming that the CNS is a

major target in acute NiV infection. The apparent cytoplasmic neuronal fusion (Figure 3.22

D) could be a very rare example of multinucleated neuronal syncytium analogous to

endothelial syncytium. This was not found in our cases of human NiV encephalitis but

multinucleated giant cells have been reported in measles inclusion body encephalitis (Esiri

and Kennedy, 1997).

pfu/animal, suggested that these animals could not

be infected with these low doses.

Extravascular tissue infection as noted in non-CNS organs such as the lung and

kidney also resembled the pathology seen in acute human NiV infection. The consistent

presence of viral antigen but not of viral genome in the covering epithelium of the renal

papilla suggests possible reabsorption of IHC-detectable viral proteins that had leaked into

the urine. The presence of viral antigen and RNA in the periarteriolar lymphoid cells of the

spleen suggests active viral replication confirming findings in human cases.

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Tissue localisation of virus by IHC and ISH was confirmed by virus isolation and/or

RT-PCR in all the solid organs tested. Overall, RT-PCR was more sensitive than virus

isolation as a confirmatory test for NiV infection in both IN and IP-infected animals. The

lower rate of virus isolation from IN-infected compared with IP-infected animals could be

related to the longer survival of the former, which presumably favoured effective immune

clearance of virus from solid organs.

RT-PCR was negative in sera of all 7 animals tested irrespective of survival

duration, suggesting that the immune system may be more efficient in clearing virus from

the circulation or that viraemia occurred early in the infection. In acute human NiV

infection, viraemia was thought to have occurred early based on the simultaneous

involvement of multiple organs and disseminated blood vessels, and the observation that

vascular lesions such as vasculitis, thrombosis and infarction occurred earlier than

extravascular parenchymal lesions. Viraemia appears to be corroborated by our data that

also showed simultaneous and widespread organ involvement in the hamster. Further

studies in the hamster model will be needed to confirm viraemia.

The presence of virus in urine as confirmed by RT-PCR and virus isolation

correlates well with kidney glomerular injury. Likewise, human glomerular injury could

explain virus excretion in patient’s urine, and could provide a possible means of viral

transmission to health care workers (Chua et al., 2001a). Interestingly, urine viral excretion

in hamsters by itself may not be sufficient to cause viral transmission as suggested by

observations in the experiment in which uninfected hamsters kept together with infected

animals for 30 days did not develop any positive serology.

Oral ingestion and/or aerosol inhalation of infected secretions is thought to be

responsible for pig-to-human viral transmission in Malaysia (Parashar et al., 2000) and

human-to-human transmission in Bangladesh and India (Gurley et al., 2007, Hsu et al.,

141

2004, Harit et al., 2006). The successful infection of hamsters by the IN route appears to

support this. The suspected primary viral replication sites in human NiV infection viz., lung

and lymph node, could not be confirmed in this study as there was no evidence of infection

in bronchial epithelium of the IN-infected animals nor any evidence of virus in the lymph

nodes. One possibility is that primary viral replication is an early event and therefore may

not be found in terminally ill animals.

The data strongly support the notion that NiV gains access into the human CNS via

vascular damage as infected neurons were often found in the vicinity of vasculitis. The

possibility of direct CNS infection via the olfactory bulb in this hamster model is not

supported by the data, which did not show a predilection for the olfactory bulb or structures

connected to it. There was also no evidence of human olfactory bulb involvement. In

experimentally-infected pigs, a recent report suggests that this route may be implicated

(Weingartl et al., 2005). Choroid plexus infection may allow virus to leak into the CSF and

thus spread the virus but we have not found strong evidence for this in our model or in

human subjects.

The limited published data on NiV-infected animals comprising observations on

field and experimentally-infected pigs, cats and guinea pigs, and field-infected dogs and

horse, showed that systemic vasculitis was the common feature in all these animals (Hooper

et al., 2001, Torres-Velez et al., 2008). However, it appears that in none of these animals,

was encephalitis and neuronal infection as convincingly demonstrated as in the hamsters in

our study. It was found that mice produced antibodies to NiV after repeated infection (Dr

Vincent Deubel, unpublished observations), but in our experiment they did not appear to

show clinical illness, and therefore were not studied further. Thus, present evidence

suggested that the pig, cat, dog, horse, mouse and guinea pig might not be good models for

the acute human disease, which is typified by severe illness, prominent vasculitis,

142

encephalitis and direct neuronal infection. More recent infection experiments on the guinea

pig showed involvement of the meninges and ependyma but changes in neural parenchyma

were less prominent, and lung involvement was reduced although systemic vasculitis was

found (Torres-Velez et al., 2008).


The pathological findings in hamsters with acute HeV infection paralleled the findings in

the single case of human infection, thus confirming the observations in the latter. In both

instances, the blood vessels and extravascular parenchymal cells in the CNS, lung, kidney

and other organs were involved. In the hamster, apart from vasculitis and vascular wall

immunolocalisation of antigens, characteristic endothelial syncytia were identified. This

suggests that in human HeV infection, although definite endothelial syncytia were not

identified, vasculitis followed direct vascular infection. The absence of endothelial syncytia

may be due to inadequate tissue sampling or related to the duration of the infection as

shown by acute NiV infection where syncytia is more likely to occur in the early stage of

infection (Figure 3.9).

HeV antigens and/or RNA were also demonstrated in neurons of the hamster CNS,

confirming findings from human studies that HeV is neuronotropic. Ependyma was also

susceptible but on the whole glial cells were far less susceptible to infection. In a guinea pig

model for HeV infection (Williamson et al., 2001), choroid plexus involvement was

reported but we found no evidence for this in the hamster. Significant pathological changes

in most organs appeared by 3-4 days of infection, consistent with the short incubation

period of the case of human acute HeV infection (Case 1, Table 3.7)

143

CHAPTER 5: CONCLUSIONS

It is apparent that NiV and HeV, both from the same Henipavirus genus, share many

common clinicopathological features suggesting that the pathogenesis of the human

infectious diseases, respectively, is essentially the same. Both caused acute infections with

predominant CNS disease, although the infections were systemic and characterised by

disseminated vasculitis and extravascular parenchymal cell involvement. In most tissues,

except perhaps the CNS white matter, tissue damage appears to be due to a unique dual

pathomechanisms of vasculitis-induced thrombosis, ischaemia and microinfarction, and

direct viral infection of parenchymal cells. Although only 1 acute human HeV infection was

studied, the findings appeared to be confirmed by findings in the hamster infected by HeV

and NiV, respectively. In the acute infection, viral spread into the CNS is almost certainly

by haematogenous route and breach of the blood-brain-barrier as a result of vasculitis that

allows the virus to enter the parenchyma. Ependymal lining and meningeal vascular

infection in both humans and the hamster model could possibly allow virus to enter the

CSF directly as well and thus spread the virus more rapidly.

The relapse/late-onset encephalitis caused by henipaviruses is a de novo, recurrent

viral infection that appears to be unique amongst viral encephalitides, although there may

be some resemblance to SSPE and measles inclusion body encephalitis. The pathogenesis

appears to be related to neuronal infection following neuron-to-neuron spread from

surviving virus foci that were disseminated to the CNS during the acute infection.

Vasculitis-induced thrombosis, ischaemia and microinfarction do not appear to play a role

in the pathogenesis in contrast to acute henipavirus encephalitis. The factors that lead to

this complication are mostly unexplored.

144

Certain pathological features in acute and relapsed/late-onset henipavirus infection

may be distinctive enough to suggest the diagnosis, particularly if this can be confirmed by

IHC, ISH, antibody assays, RT-PCR and virus culture. In acute infection, the combination

of disseminated vasculitis associated with endothelial syncytia and parenchymal cell

infection appears unique. Likewise, in relapsed/late-onset, concentric vacuolated necrotic

lesions are remarkable.

It is perhaps not surprising that henipaviruses cause similar infectious disease

pathology as it has been shown very recently that they share the same virus entry receptor.

The main receptor has been identified as ephrin B2 (Negrete et al., 2005, Bonaparte et al.,

2005) and the alternative receptor, ephrin B3 (Negrete et al., 2006). These receptors are

ubiquitous on the plasma membranes of many mammalian cells, particularly in the blood

vessels and CNS, thus accounting for the prominent clinico-pathological features of

vasculitis and CNS involvement.

Because pteropid bats are the natural hosts of henipaviruses (Chua et al., 2002,

Epstein et al., 2008) and are found in many parts of the world, future henipavirus outbreaks

ought to be anticipated. Further understanding of the pathology and pathogenesis of this

important group of emerging infection should continue to contribute significantly to the

development of therapeutic strategies and vaccines as several recent efforts have

demonstrated (Guillaume et al., 2004, McEachern et al., 2008, Zhu et al., 2006).

145

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Appendix

Appendix A (Hybridisation Solution for ISH)

The hybridisation solution is prepared by mixing the following component and prepared

fresh before use.

20 x SSC, 0.3ml (NaCl 175.3 g, Sodium citrate 88.2 g, pure

H2

O to a final volume of 1 litre, pH 7)

50 x Denhardts solution, 0.1ml (Ficoll 0.1 g, polyvinylpyrolidone 0.1 g,

pure H2

O to a final volume of 10 ml)

10% dextran sulphate, 0.5 ml (Dextran sulphate 0.50 g, Formamide to a

final volume of 5 ml)

Denatured salmon sperm DNA, 0.01 ml

Pure H2 O, 0.09 ml

Appendix B (Anti-Nipah Antibody Assay in Human Samples)

The IgM assay was performed in a Mu-capture format and utilized Hendra antigen grown

in Vero E6 cells and inactivated by gamma irradiation and a hyperimmune anti-Hendra

mouse ascitic fluid as the detection system for bound antigen. The IgG assay used a

detergent-extracted Hendra antigen grown in Vero E6 cells and inactivated by gamma

irradiation; antigen was adsorbed directly onto microtitre plates. IgG and IgM assays were

also performed as above with control antigens from mock-infected Vero E6 cells.

Sera were tested in a 4-fold dilution series from 1:100 to 1:6400, and CSF were

similarly tested in a 4-fold series from 1:20 to 1:2560. A sample of negative donors was

161

used to validate the cut-off values for the assays. Samples were considered positive for the

IgM assay if the sum of the adjusted optical densities from all of the dilutions (infected

antigen less the mock-infected antigen) was >0.45 through the entire dilution series and the

titre was 1:400 (1:16 for CSF). Samples were likewise considered positive in the IgG assay

if the sum for the adjusted optical densities from all of the dilutions (infected antigen less

the mock-infected antigen) was >0.90 through the entire dilution series and the titre was

1:400 (1:16 for CSF).

The IgM capture assay used goat anti-human Mu to capture IgM (Biosource,

Camarilla, CA) and a horseradish peroxidase-conjugated goat anti-mouse IgG from

Biosource in the Hendra antigen detection system. The IgG assay used a horseradish

peroxidase-conjugated mouse anti-human γ chain-specific antibody (Accurate Chemical,

Westbury, NY) to detect bound IgG.

Appendix C, (Nipah Virus Culture and Identification in Human CSF)

In brief, CSF was collected aseptically by lumbar puncture into a sterile container and

immediately transported in wet ice to the laboratory. One hundred microliter-aliquots of

CSF were transferred into a 24-well culture plate (Costar, Cambridge, MA) that previously

had been seeded with 105 Vero cells (CCL-81; American Type Culture Collection,

Rockville, MD) in 1 ml of Eagle minimal essential growth medium containing 10% fetal

calf serum (Flowlab, Sydney, Australia) for each well. The culture plate was sealed,

incubated at 370C, and examined daily for cytopathic effects. Positive identification of virus

was made by immunofluorescence using anti-Hendra hyperimmune mouse ascitic fluid and

162

goat anti-mouse IgG fluorescein isothiocyanate conjugate (Sigma, St Louis, MO) as

secondary antibody as previously described.

Appendix D (Nipah Virus Stock and Titration)

After 1-2 days when the infected Vero cells showed fusion and syncytia formation, the

supernatant was harvested for virus. Virus stock was titrated in 6-well plates by incubating

200 µl of serial 10 times dilution of supernatant in each well (containing 106

Vero cells per

well) for 1 hr at 37°C. The cells in each well were washed twice with Dulbecco’s minimum

essential medium (DMEM), and 2 ml of 1.6% carboxymethylcellulose in DMEM

containing 2% fetal calf serum were added to each well. After incubation for 5 days at

37°C, and the wells were washed with phosphate buffer pH 7.4 (PBS), fixed with 10%

formalin for 20 min, washed and stained with methylene blue.

Appendix E (Anti-Nipah Antibody Assay in Hamster Samples)

Crude extracts of NiV antigens were prepared from infected Vero cells at a multiplicity of

infection of 0.01 pfu/cell for 24 hours. The cells were washed with PBS and lysed in PBS

containing 1% Triton X 100 (107 cells/ml) at 4 0C for 10 min. The cell lysate was sonicated

twice for 30 seconds each to full cell destruction and centrifuged at 5000 rpm at 4 0C for 10

min. The supernatant was frozen at –80 0

C. Non-infected Vero cells were similarly treated

to prepare an antigen control. Cross-titration of the NiV antigens was performed with serum

from a convalescent, NiV-infected patient to determine the antigen titre corresponding to

the dilution showing the highest O.D. reading.

163

Appendix F (List of Publications)

Journal articles

1) Chua KB, Goh KJ, Wong KT, Kamarulzaman A, Tan PSK, Ksiazek TG, Zaki SR, Paul G, Lam SK, Tan CT. Fatal encephalitis due to Nipah virus among pig farmers in Malaysia. Lancet 1999; 354: 1257-9.

2) Chew NK, Goh KJ, Tan CT, Sarji SA, Wong KT. Electroencephalography in

Nipah encephalitis. Neurological Journal of South East Asia 1999; 4: 45-51. 3) Wong KT. Emerging and re-emerging epidemic encephalitides: a tale of two

viruses. Journal of Neuropathology and Applied Neurobiology 2000; 26: 313-8 4) Sarji SA, Abdullah BJ, Goh KJ, Tan CT, Wong KT. MR imaging features of

Nipah encephalitis. American Journal of Roentgenology 2000; 175: 437-42 5) Goh KJ, Tan CT, Chew NK, Tan PSK, Kamarulzaman A, Sarji SA, Wong KT,

Abdullah BSJ, Chua KB, Lam SK. Clinical features of Nipah virus infection, a new viral encephalitis among pig farmers in Malaysia. New England Journal of Medicine 2000; 342: 1229-35

6) Chua KB, Lam SK, Tan CT, Hooi PS, Goh KJ, Chew NK, Tan KS,

Kamarulzaman A, Wong KT. High mortality in Nipah encephalitis is associated with presence of virus in cerebrospinal fluid. Annals of Neurology 2000; 48: 802-5

7) Chong HT, Tan CT, Karim N, Wong KT, Kumar S, Abdullah W, Chua KB, Lam

SK, Goh KJ, Chew NK, Petharunam V, Kunjapan SR, Thayaparan T. Outbreak of Nipah encephalitis among pig farm workers in Malaysia in 1998/1999: Was there any role for Japanese encephalitis? Neurol J Southeast Asia 2001; 6: 129-134

8) Tan CT, Goh KJ, Wong KT, Sarji SA, Chua KB, Chew NK, Murugasu P, Loh YL,

Chong HT, Tan KS, Thayaparan T, Kumar S, Jusoh MR. Relapse and late-onset Nipah encephalitis. Annals of Neurology 2002; 51:703-8

9) Wong KT, Shieh WJ, Kumar S, Norain K, Abdullah W, Guarner J, Goldsmith CS,

Chua KB, Lam SK, Tan CT, Goh KJ, Chong HT, Jusoh R, Rollin PE, Ksiazek TG, Zaki SR and the Nipah Virus Pathology Working Group. Nipah virus infection: Pathology and pathogenesis of an emerging paramyxoviral zoonosis. American Journal of Pathology, 2002; 161: 2153-2167

10) Wong KT, Shieh WJ, Zaki SR, Tan CT. Nipah virus infection, an emerging

paramyxoviral zoonosis. Springer Seminars in Immunopathology, 2002; 24: 215-228.

11) Goldsmith CS, Whistler T, Rollin RE, Ksiazek TG, Rota PA, Bellini WJ, Daszek

P, Wong KT, Shieh WJ, Zaki SR. Elucidating Nipah virus morphogenesis and replication using ultrastructural and molecular approaches. Virus Research 2003; 92: 89-98

164

12) Tan CT, Wong KT. Nipah encephalitis outbreak in Malaysia. Annals of the

Academy of Medicine Singapore 2003; 32; 112-117 13) Wong KT, Grosjean I, Brisson C, Blanquier B, Fevre-Montange M, Bernard A,

Loth P, Georges-Courbot MC, Chevallier M, Akaoka H, Marianneau P,

Lam SK, Wild TF, Deubel V. A golden hamster model for human acute Nipah virus infection. American Journal of Pathology 2003; 163: 2127-37

14) Wong KT, Robertson T, Ong BB, Chong JW, Yaiw KC, Wang LF, Ansford, AJ, Tannenberg A. Human Hendra virus infection causes acute and relapsing encephalitis. Neuropathology and Applied Neurobiology 2009; 35: 296-305

15) Guillaume V, Wong KT, Looi RY, Georges-Courbot MC, Barrot L, Buckland R,

Wild TF, Horvat B. Acute Hendra virus infection: Analysis of the pathogenesis and passive antibody protection in the hamster model. Virology 2009; 387: 459-65

Book Chapters

1) Tan CT, Wong KT. Nipah encephalitis. In: Clinical Neurovirology, Chapter 23, Marcel Dekker Inc, New York, 2003

2) Tan CT, Wong KT, Chua KB, Nipah encephalitis. In: Emerging Neurological Infections. Eds Power C & Johnson R, Marcel Dekker Inc, New York, 2005.

3) Goh KJ, Wong KT, Tan CT. Nipah and Hendra viruses encephalitis. In: New and

Evolving Infections of the 21st

Century. Eds. Fong IW Alibek K, Springer. New York 2007, pp 279-293

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