clinical influenza virus and the embryonated hen's egg

Reviews in MEDICAL VIROLOGY VOL. 3: 97-106 (1993)

Clinical Influenza Virus and the Embryonated Hen's Egg J. S. Robertson National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, Herfs EN6 3QG, UK

INTRODUCTION

Virtually all our knowledge regarding the causative agent of influenza is based on studies of virus which has been grown in embryonated hens' eggs. These have been the most popular substrate for propagating influenza viruses for a considerable time because of their availability, their ease of use and yield of virus. However, for human influenza strains it was recognised 50 years ago by Bumet and Bull' that the freshly isolated clinical virus has to 'adapt' in order to grow in the allantoic cavity of the egg.

The extent to which past findings, which were based on egg-adapted virus, reflect the 'natural' virus present in original clinical material has been the subject of considerable investigation in this Institute since Schild ef ~ 1 . ~ described the host cell selection of antigenic variants of human influenza virus 10 years ago. They demonstrated that influenza B virus grown in embryonated hens' eggs is antigenically distinct from virus derived from the same clinical specimen but isolated and propagated exclusively on mammalian cell cultures.2

We now know that the adaptation process is a relatively sudden event occurring during the first passage of virus in the allantoic cavity of the egg and involves the selection of a variant with the required characteristics for successful growth in the cells of the allantois. This review will describe the molecular events associated with this initial egg adaptation of human influenza virus and also the implications of this selection process on the use of eggs for propagating human strains for epidemiological surveillance and vaccine production.

Virus structure Virions of influenza consist of a nucleocapsid core in which the single-stranded and segmented RNA genome is intimately associated with a nucleoprotein and with polymerase proteins. The cores are surrounded by a shell of matrix protein encapsulated within a lipid bilayer membrane derived from the host cell during maturation by budding. Protruding from the membrane are viral encoded glyco- proteins. For influenza A and B viruses the surface glyco- proteins are the haemagglutinin (HA) and neuraminidase (NA) whilst influenza C has a single haemagglutinin- esterase-fusion glycoprotein (see review3). The glyco- proteins function in attachment of virions to the host cell and in fusion of the viral membrane with host membranes to allow penetration of the nucleocapsid cores into the host cell and are implicated in maturation of the virion. The

ISSN 1052-9276/93/020097-10 $10.00 @ 1993 by John Wiley & Sons, Ltd.

haemagglutinin is also the major antigenic determinant of influenza virus and is responsible for inducing and binding neutralising antibodies.

Influenza viruses are classified serologically into types A, B and C dependent on the antigenicity of the nucleocapsid proteins. Influenza A viruses are further subdivided antigenically into subtypes dependent on the HA and NA surface antigens. Fourteen HA and nine NA subtypes of influenza A have been described in nature although only three of these have been associated with human disease, the HINl , the H2N2 (or Asian flu) and the H3N2 (or Hong Kong flu) subtype^.^ There are four types of influenza virus currently causing respiratory disease in man: the A(HIN1) and A(H3N2) subtypes and types B and C. Influenza A infections are a source of severe morbidity whose symptoms include fever, sore throat, myalgia and malaise. They can cause high mortality especially in the very young and the elderly. Influenza B infections are generally less severe whilst influenza C causes only mild infection. This review will focus on studies of influenza types A and B only.

SELECTION OF HOST CELL VARIANTS Isolation and propagation: cell culture versus eggs Studies on the selection of variants of influenza virus by the host cell have involved the comparison of pairs of viruses derived in the laboratory from the same clinical specimen on separate substrates, one in the allantoic cavity of eggs and the other on mammalian cell culture, typically Madin Darby canine kidney (MDCK) cells (Figure Such pairs of viruses were found to be biochemically, and often antigenically, distinct. In these studies insufficient virus was present within a clinical specimen to determine which isolate was most representative of the natural virus. However, since variants are selected when MDCK-isolated virus is passaged in eggs and egg-adapted virus can propagate successfully with no reversion in cells, this suggested that eggs provided the more restrictive system (Figure I). Additional serological evidence also suggested that the virus derived from cell culture is more like the natural virus: in a haemagglutination-inhibition (HI) assay the MDCK- derived virus detected antibodies in human post-infection sera more frequently and to higher titre than did the corresponding egg-adapted

The breakthrough in determining the exact nature of the virus present in clinical material came with the develop- ment of the polymerase chain reaction (PCR) technique7

Accepted 12 January 1993

98 I. S. ROBERTSON

Clinical Specimen

0000 000 0000

MDCK 0000 ..a. 000 .a. 0000 ...a

Figure 1. Selection of influenza viruses. Virus derived from clinical spec mens on mammalian (MDCK) cell culture is representative of the naturi virus (open circles) whereas variants (filled circles) are selected when non-egg-adapted virus, either clinical material or MDCK-grown, is culti- vated in the allantoic cavity of embryonated hens' eggs. When the egg- adapted virus is propagated on MDCK cells, no reversion occurs, i.e. once egg-adapted, always egg-adapted.

and the ability to analyse directly the small amount of virus present in clinical samples without the need to propagate virus in the laboratory. Using PCR we have been able to establish that virus isolated and propagated in cell culture is representative of the natural virus present in clinical material, whereas the virus isolated in the allantoic cavity of eggs is a variant selected due to a restriction on the growth of the natural virus in eggs (Figure I).

The nature of the egg-adapted virus The nature of egg-adapted virus compared with virus derived in cell culture has been studied extensively by nucleotide sequencing. Data were obtained initially by direct RNA sequence analysis of purified egg-adapted and cell-derived virus pairs8-" Latterly,13-" sequencing of PCR amplimers derived from clinical material or laboratory derived virus has been performed both before and after cloning of the PCR products in M13. Since the observations on antigenic differences by Schild ef a1.' were made using anti-HA antibodies, the gene targetted for sequencing was the HA gene.

The HA molecule consists of two distinct domains, a stem structure protruding from the virion surface consist- ing of the HA2 and part of HA1 of the HA polypeptide,

and a globular head which is composed entirely of HAI. In early sequencing studies the entire HA coding region was analysed. Subsequently, after the initial data had been obtained, only the region coding for the HA1 portion of the molecule was analysed. Sequencing studies have identified, either by direct comparison with MDCK-derived virus or of PCR-amplified sequences, numerous amino acid residues within the globular head of the HA whose substitutions are directly associated with egg-adaptation. These residues are listed in Table I and their location on the three- dimensional structure of an H3 subtype HA is shown in Figure 2 . In most instances, the egg-adapted virus has a single base substitution compared either to the corresponding MDCK-isolate or to virus in the original clinical specimen, resulting in a single amino acid change. Occasionally, two substitutions have been observed and whether both or only one substitution are important for egg-adaptation cannot easily be assessed. For a few egg and cell-derived virus pairs, the neuraminidase and/or the matrix genes have been analysed but no differences have been detected (J. Katz, personal communication).

For influenza B virus the change generally associated with the egg-adapted virus is the loss of a specific glycosylation site from the tip of the HA molecule due to substitution of either the Am* or the Thr residue at positions 196-198 (187-189 in the H3 haemaggl~tinin).'"~ In addition, during the course of a study on the virulence of influenza B viruses derived from different sources (see below),16 an egg- adapted virus was isolated in which the HA retained this glycosylation site. Comparison with corresponding mammalian cell-derived material revealed an alternative substitution in this egg-adapted virus of Gly->Arg at residue 141 (138 in H3 numbering). More recently, some influenza B viruses isolated in eggs have retained the glycosylation sequence at 196-198 (reference 17 and unpublished observations). Unfortunately there are no equivalent cell culture or clinical viruses of these specific isolates available for analysis to determine if the HA has a substitution elsewhere associated with the ability to replicate in eggs. These isolates have Cly - 141 and so a change at this position would not appear to account for the ability of these viruses to grow in eggs.

Comparison of the HAS of egg-adapted and cell-derived influenza B virus by polyacrylamide gel electrophoresis suggests that the Asn-x-Thr (196-198) site in cell-derived virus is glycosylated.8 Also, endoglycosidase digestion of the HA of an egg-adapted virus which retained the Asn-x- Thr sequence suggests that this site is gly~osy1ated.l~ Since PCR sequencing of the HA of virus present in clinical material reveals the same sequence as the MDCK-derived virus, virus replicating in human epithelial cells presumably is also glycosylated at this site. There are of course many other potential glycosylation sites within the HA of influenza B which are common to both egg and cell-derived virus.

For the A(H1N1)9s14 and A(H3N2)10-'2~15 subtypes, the numerous substitutions in the HA1 which are directly associated with egg-adaptation are listed in Table 1 and

13

'See Reviews in Medical Virology Volume 1, Page 4 for amino acid codes.

CLINICAL INFLUENZA VIRUS 99

~~

100 J. S. ROBERTSON

r. b.s.

Figure 2. Location of HA1 amino acid substitutions associated with egg-adaptation. Changes in influenza B, A(HIN1) and A(H3NZ) viruses as listed in Table 1 are shown on the a-carbon backbone of the HA molecule. Generally only one of these substitutions is found in an individual egg-adapted virus. r.b.s., receptor binding site

their locations within the HA are shown in Figure 2. Typi- cally, only one of these substitutions is found in the HA of each egg-adapted virus and substitution generally involves a more basic residue relative to the equivalent residue in natural or cell-grown virus. This is especially so at, for example, position 189 in H l N l virus and at 156 in H3N2 virus where Glu- > Lys substitutions occurred. Most of the substitutions cluster around the receptor binding site (RBS) which is a shallow pocket on the surface of the globular head and implies involvement of the RBS in the selection process (Figure 2).18

A few of the identified changes involve the loss of a glycosylation sequence in the HA of egg-adapted virus, as occurs for influenza B, those at Asn-163 and Asn-129 for HI and at Asn-246 for H3. These positions are less intimately associated with the RBS than the other substitutions. How- ever, a carbohydrate structure at these locations on the natural or MDCK-grown virus could affect the specificity of the RBS in an adjacent monomer within the trimeric HA structure.

There is a single report in the literature of identity between the HA1 sequence of anallantoic-derived virus and the virus present in clinical material." This contradiction to the above studies remains unexplained. However, in that study, the PCR amplified DNA was sequenced directly without subcloning and if several different variants were present in the allantoic sample, any sequence differences may have gone undetected.

For each of the subtypes a variety of HA substitutions exist which are associated with egg-adaptation; for influenza B we have often observed sequence heterogeneity in egg-grown viruses at the Asn-x-Thr (196-198) site when performing direct RNA sequencing. We were interested therefore not only in the consensus sequence, which would be obtained by direct sequencing of PCR amplified DNA, but also on the extent of any heterogeneity of the virus population with respect to the residues associated with egg-adaptation, especially the presence of egg-adapted variants in clinical material.

We analysed the extent of heterogeneity in virus samples by cloning PCR amplified material into M13 and sequencing multiple clones for each sample. Typical results of such an analysis for the HA of an influenza B and for an A(HIN1) virus are shown in Table 2. First, these data indicated clearly, and for the first time, that the virus derived in cell culture is representative of the virus present in clinical material whilst egg-adapted virus, even after a single passage, is not. Additionally, it was also noted that virus derived from a single egg could contain several different ~a r i an t s . ' ~ , ' ~ Possibly, some variants will be more successful than others and with further passaging of a mixed population, the most successful variant should pre- dominate. In contrast to egg-adapted virus, the virus population in the original clinical sample and also that derived in cell culture was relatively homogenous. Occasionally, no substitution was observed in a clone derived from egg-

~


adapted virus. Such clones may derive from the original virus remaining in the inoculum or may indicate a very limited replication of the natural virus in the egg.

Influenza virus populations are described as quasispecies in that they consist of a complex mixture of microvariants due to the inherent error rate of the viral polymerase.” Studies” of this error rate and the frequency of selection of variants with single amino acid substitutions indicate that they are present at a frequency of approximately I in 10’. Such a frequency was observed for the infectivity of MDCK-derived influenza B virus directly in the allantoic cavity’ and also for an A(HIN1) virus.” Thus the level of egg-variants in clinical virus may be as low as or occasionally may be as high as IO-’ to IO-’ (see Table 2, A/NIB/49/88). I t is also feasible that egg-variants may arise naturally in the population due to antigenic drift although studies on the virulence of an egg-adapted influenza B virus16 (see below) suggest this is unlikely to occur. In the PCR studies many other base substitutions, unique to an individual M13 clone, were observed. These are most likely to be artefacts due to errors introduced by Taq polymerase or reverse transcriptase although some may represent a certain degree of natural HA microheterogeneity within the virus population.

Does the amnion select? A growth restriction of the natural virus in the allantoic cavity is the basis for the observed difficulty in isolating influenza virus from clinical material in eggs. However, the allantoic compartment is not the only site within the embryonated egg in which replication of human influenza virus can O C C U ~ . ’ ~ It has long been re~ognised’~ that initial

Routes of Inoculation

AMNIOTIC ALLANTOIC

Figure 3. Cross-section of the embryonated hen’s egg showing inoculation into the amniotic cavity and the allantoic cavity.

passage of clinical material in the amniotic cavity prior to allantoic propagation is a more efficient route for obtaining an egg-isolate (Figure 3). To investigate the extent to which variants are selected by amniotic passage, samples of clinical material passaged once in the amnion have been subjected to PCR amplification, M13 cloning and sequencing.

Virus present in the allantoic fluid, harvested from an egg which was inoculated amniotically, contained a similar

102 J. S. ROBERTSON

spectrum of natural and variant virus compared to that present in the amniotic fluid. This implies that the two harvested fluids are identical. Inoculation into the amnion requires injection through the allantoic cavity (Figure 3). During incubation of the egg, transfer of fluid between the amniotic and allantoic compartments, due to movement of the embryo and diffusion, will inevitably occur through the hole caused by the inoculating needle. The sequencing data (unpublished) suggest that the natural virus can replicate without restriction within the amnion but that exposure of the inoculum, or of de ~ O D O virus derived in the amnion, to the allantois during the incubation period, allows for the possibility of an appropriate variant successfully establishing itself in the allantois. Such virus will then also equili- brate with the amniotic cavity where it may replicate further. This was observed to occur to varying degrees and reveals why prior passage in the amnion increases the probability of a variant successfully establishing itself during a subsequent allantoic passage. In this study, a further single allantoic passage of each amniotic fluid resulted in the complete selection of a variant in each case.

The above hypothesis presumably applies to the observations made by Bumet and Bull' 50 years ago who noted that the agglutination properties of A(H1NI) virus changed from 0 (original) to D (derived) when amniotic virus was passaged in the allantois-the O/D variation.

Virus derived on other mammalian cell cultures, e.g. LLC-MK, and MRC-5 cells, and also on primary chick kidney cells, are, like that derived on MDCKs, representative of the virus present in clinical material.25 Since the natural virus can replicate without restriction in the amniotic cavity (where the virus has access to the respiratory and intestinal tracts of the embryo), it appears to be uniquely the cells lining the allantoic cavity, the complex layer known as the allantois, which pose this restriction to growth. Once the restriction is overcome by the successful replication of a variant with the appropriate HA1 amino acid substitution, the allantoic cavity presents an efficient, high yielding, easily utilised and inexpensive substrate for virus propragation.

The biological basis for selection What is the basis for this severe restriction to the growth of the natural virus in the allantoic cavity? The substitutions found in the HA of variants selected in the egg cluster around the RBS in the globular head of the HA (Figure 2) and this implies the involvement of the RBS in the selection process. The receptor for influenza virus is sialic acid" and the predominant type found in humans, N-acetyl neuraminic acid, is also present in the allantois. Whilst there are limited data relating different HA sequences to the nature of the sialic acid species26 and to the nature of the linkage between the sialic acid and its adjacent sugar

there is a paucity of information regarding the type of macromolecule which acts as an effective receptor, i.e. whether it is a glycoprotein, a glycolipid, or both, and whether a specific glycoproteinAipid is involved.

Agglutination studies with red blood cells have indicated differential receptor binding activities between egg- adapted and non-egg-adapted viruses.2z11 However, direct

evidence for the biological basis of the restriction of non- egg-adapted virus to growth in the allantois was required. We therefore investigated this using radioactively labelled virus in binding studies with viable portions of the chorio- allantoic membrane (CAM) of embryonated eggs.22 Non- egg-adapted virus (i.e. virus grown solely on mammalian cell cultures) bound to the sialic acid on allantois cells of the CAM to significant levels. However, since the virus could be stripped off by treatment with neuraminidase, this indicated that it failed to be internalised. In contrast, egg-adapted virus both bound to and was internalised efficiently by CAMS since it attained a high level of neuraminidase resist- ance. Both virus types efficiently bound to and were internalised by MDCK cells. Thus, the subtle differences in the primary structure of the HA molecule of variants in the vicinity of the RBS appear to be responsible for the ability of egg-adapted virus not just to attach to, but also to trigger effective receptors on the surface of allantois cells.

Variants of a laboratory strain of influenza virus have been observed to emerge upon sequential passage of virus in cell culture.29 We have observed on two occasions the emergence of egg-adapted type variants during propagation of MDCK-derived virus on MDCK cells.22 This prompted us to spike MDCK-derived virus with very small quantities of egg-adapted variants and subject the population to sequential passage on MDCK cells. The egg- adapted variants often became dominant in the population. These observations correlate with increased levels of binding of egg-adapted virus to MDCK cells compared to the MDCK-derived virus. It appears that the natural virus, whilst quite capable of replication on MDCK cells, is not the 'best fit' virus on this substrate, and the virus population may evolve to provide better growth characteristics by substitutions around the RBS to provide a more efficient receptor binding site. Curiously these substitutions are the same as those found in egg-adapted virus.

IMPLICATIONS OF HOST CELL SELECTION The HA is the major antigenic determinant of influenza virus, inducing and binding neutralising antibodies. Conse- quently it is the major component of influenza vaccine. Due to antigenic drift of human influenza virus it is necessary to monitor the antigenicity of epidemiologically significant viruses to ensure that the most appropriate strain is incorporated into vaccine. Since surveillance studies are performed with and vaccine prepared from egg-adapted viruses, the selection of egg-adapted viruses with variant HAS has potentially serious implications for the choice of the vaccine strain. Several studies have investigated the exent to which the single amino acid substitutions observed affect the antigenic and immunogenic properties of the variants and the effects are not insignificant.

Antigenicity Specific antigenic differences between MDCK-derived and egg-adapted viruses were first observed for influenza B, using monoclonal antibodies (mAbs) in HI These differences could also be observed with ferret and human


polyclonal sera and in enzyme-immunoassay (EIA) and virus neutralisation (VN) assays using either monoclonal or human polyclonal antibodies.',' Although there is cir- cumstantial evidence to suggest that the specific glycosylation site associated with egg-adaptation of influenza B is glycosylated in MDCK-derived virus, the extent to which the difference in antigenicity observed between egg- adapted and cell-derived virus is due to glycosylation or to the observed difference in primary amino acid sequence is unknown.

For the A(HlN1)"' and A(H3N2)10-12,30 subtypes of the virus, many of the substitutions which accompany egg- adaptation are antigenically significant and the various egg-adapted viruses can be placed into several distinct antigenic groupings based on HI assay with mAbs. Generally, mAbs which react exclusively with egg-adapted virus will have been raised against an egg-adapted virus and vice-versa. For each of the two subtypes one of these antigenic groups was indistinguishable from the corresponding MDCK-derived virus (termed 'cell-like' virus). This group nonetheless was genetically distinct, not all of the observed amino acid substitutions exerting an antigenic effect. In contrast, within an individual study, viruses isolated on cells were antigenically homogeneous and by extrapolation the natural virus also is presumably homogeneous.

Significantly, some of the antigenic groupings defined by mAbs can also be distinguished with ferret polyclonal antiserum in HI assay, which is the assay currently in use extensively to characterise epidemiologically significant viruses on a worldwide basis. Also, antigenic differences have been observed in many instances with EIA and VN studies and with polyclonal ferret, hamster, guinea-pig and human sera.

Occasionally there have been discrepancies in that antigenic differences have been observed for A(H3N2) virus pairs whose HA sequences are distinct but the same as another virus pair for which no antigenic difference has been observed (unpublished). Possibly these observations are due to the use of polyclonal sera in an HI assay, the presence of virus mixtures, and/or laboratory sequencing artefacts.

Surveillance Reference strains of human influenza virus chosen by the World Health Organization (WHO) to represent antigenic phenotypes circulating in a given influenza season are currently grown in eggs. To achieve this, public health laboratories on a worldwide basis isolate influenza viruses for full antigenic characterisation by reaction with sera from animals and with human sera from clinical trials. Such data are reviewed annually by the WHO to determine which virus strains should be incorporated into vaccine.

Several studies have shown that MDCK-grown viruses are more sensitive at detecting antibodies in postinfection human sera than is egg-grown antigen.2r6,31-33 Also, the impact of the previous exclusive use of egg-adapted viruses in assessing the antigenic nature of epidemiologically significant strains has recently been in~es t iga t ed .~~ It was noted that A(H3N2) viruses isolated from around the world over several years exhibit less antigenic drift if

isolated on cells than if isolated in eggs. This suggests there has been an overestimation of the extent of antigenic diversity amongst human influenza viruses. Overall, the studies indicate that considerable care must be taken in the selection of reference and vaccine strains.

Immunogenicity A few studies have investigated the immunogenic characteristics and protective efficacy of MDCK-derived vaccine versus egg-adapted vaccine in a variety of animal models. Whilst there has been no single definitive conclusion, generally mammalian cell-derived virus vaccine induced a more cross-reactive antibody response and gave better protection than the corresponding egg-adapted virus vaccine.

In a study by Wood et aL3' inactivated vaccine was prepared from a pair of cell-derived and egg-grown A(HlN1) viruses obtained from the same clinical specimen and differing in their HA by two amino acid residues. Their immunogenicity and protective efficacy were compared in hamsters. The egg-adapted virus vaccine induced antibodies which were significantly more strain specific than those produced by the cell-derived virus vaccine which induced a more cross reactive response. The protective efficacy of the egg-vaccine in this study was poor, reaching only 20% for a homologous challenge despite a significant (geometric mean HI titre 2 40) postvaccine antibody response, and gave no protection against a heterologous challenge with the cell-derived virus. In contrast, the cell- derived vaccine provided > 50% homologous protection and 20% protection against egg-virus challenge.

Similar results were obtained by Katz and W e b ~ t e r ~ ~ in a study on a ferret model using an A(H3N2) pair of egg and cell derived virus vaccines. These two viruses differed by a single amino acid residue in the HA and were antigenically distinguishable. Again, the cell-derived vaccine provided greater protection against both virus types than did the egg-derived vaccine. The results of the above two studies with inactivated vaccines are in contrast to a study in which mice were immunised with live vaccinia recombinants expressing the HA derived from a pair of influenza B viruses isolated either on cells or in eggs.37r38 In these studies, good cross-protection was achieved by both vaccines.

In the above three studies, different (sub)types of virus were employed, each study used a different animal model, and in two of the studies vaccine was presented in inactivated form whilst the third study used a live vaccinia vec- tor expressing the HA molecule. Thus it is difficult to make meaningful comparisons. Ultimately, none of them is the ideal model for assessing the comparative immunogenicity and protective efficacy that can be achieved in humans although current vaccine for human use is based on inactivated virus rather than a live vaccinia recombinant. Neither should these studies be taken to indicate that the egg- derived influenza vaccine currently in use is likely to be irrelevant in providing protection in humans. Rather, they were designed to determine if HAS which differ by only one or two amino acids residues would invoke a differential immunogenic response and protective efficacy. In so

J. S. ROBERTSON 104

doing, virus pairs were chosen which had previously been well characterised and which could be distinguished antigenically in uitro.

A study has been conducted in human volunteers of the immunogenicity of inactivated vaccines based on two distinct egg-grown A(HIN1) viruses derived from the same clinical specimen.39 The two variants differed in their HA by three amino acid residues and whilst one of the variants was antigenically distinct from virus isolated in cell culture, the other was antigenically indistinguishable (cell-like). Both vaccines induced high levels of cross-reactive antibody capable of reacting with both strains, however, only the vaccine which was antigenically cell-like induced significant levels of strain specific antibody. Previous natural infection with strains antigenically related to the exper- imental vaccines, especially to the cell-like vaccine, apparently resulted in the vaccinees inducing mainly a secondary immunological response. This emphasises the difficulty in performing vaccine trials in humans who have had previous exposure to virus.

Attenuation An interesting study on the virulence of egg-adapted virus versus mammalian cell derived virus was conducted at the MRC Common Cold Unit, Salisbury.16 Four groups of volunteers were infected with influenza B virus; one with untreated clinical material (nasal wash) from a patient with a documented influenza B infection, one with virus from the clinical material isolated and grown on human embryonic tracheal cultures, one with an egg-adapted virus which had lost the glycosylation site at HA1 residues 196-198, and one with an alternative egg-adapted virus which retained the glycosylation site but instead had the substitution Gly- >Arg at residue 141. Only the group infected with the egg-adapted virus which had lost the glycosylation site at 196-198 failed to become significantly ill. The level of symptoms and the extent of antibody rises and of virus isolation from members of this last group were extremely low whilst there were significant symptoms of disease, of rise in antibody titre and of virus isolation in the volunteers infected with any of the other three virus samples. Apparently the loss of the 196-198 glycosylation site from the HA had considerably attenuated the virulence and infectivity of this egg-adapted virus. This may be why influenza B virus lacking this site has not occurred naturally in the human population through antigenic drift. Similarly, for influenza A virus, adaptation continues to be a require- ment for wild type virus to grow in eggs and a virus which is able to do so naturally does not appear to have evolved in the human population to date.

OTHER VIRUSES

The role that the host cell may play in the selection of variants has been investigated for other virus systems. Aytay and Schulze4' have reported than an influenza variant of the (egg-adapted) WSN strain selected by MDBK (bovine) cells has a single amino acid substitution affecting the glycosylation of the HA. Equine influenza vaccine,

similarly to the human vaccine, is prepared from egg- grown virus and the effect of egg-adaptation is under investigation at this Institute. For equine influenza, the situation is more complex; the natural virus appears to grow unrestricted in the allantoic cavity whilst variants may be selected on MDCK cells. In this respect it is interesting that avian and equine A(H3N2) viruses have a receptor specificity for sialic acid linked a,2-3 to galactose in contrast to human A(H3N2)41 and A(H1Nl)27 viruses which have a,2-6 specificity. However, there is no con- sistent difference in the receptor specificity, with respect to the sialic acid-galactose linkage, between egg-adapted and cell-derived A(H3N2) (J. Katz, personal communication) and A(HIN1) (G. Rogers, personal communication) viruses, which remain a,2-6 specific. For influenza C virus, a recent report similarly indicates that cell cultures may select antigenically distinct variants with altered receptor binding abilities compared to virus isolated and propagated in eggs.42

Selection of variants of Sendai virus, a member of the family Pararnyxouiridae, in the allantoic cavity of embryonated eggs has been reported.43 Virus derived from a field isolate in LLC-MK, cells differed antigenically, in haemagglutinating activity and in gene sequence from corresponding virus derived in eggs. The egg-derived virus replicated in the LLC-MK, cells without reversion whilst variants were selected when the cell culture isolate was passaged in eggs. Thus variants of Sendai virus are apparently selected in the egg in a manner analogous to that for human influenza A and B.

CONCLUSIONS

There is now a consensus of opinion that great care should be taken in the use of egg-grown viruses for human influenza surveillance and vaccine. Although all the evidence indicates that the cell-isolated virus is more like, if not identical to, the natural virus, there is not an immediate need to suggest the exclusive use of cell culture for vaccine production. Most egg-adapted influenza A isolates are indistinguishable antigenically from their cell-derived counterparts even though an amino acid within the HA has been substituted and we have suggested that it would be prudent to target such egg-derived viruses for vaccine m a n u f a c t ~ r e ~ ~ even though little data are available con- cerning the comparative protective efficacy which can be obtained with a cell-like egg-adapted vaccine against natural infection in man compared with a cell-derived vaccine. Such cell-like viruses can be identified by carrying out parallel surveillance with cell-isolated and egg-adapted viruses. Furthermore, the WHO has recently stated that it has no objection to the use of cells for the isolation and limited propagation of human influenza virus although it did not specifically sanction the use of cells for vaccine p r o d ~ c t i o n . ~ ~ It is now recognised by WHO surveillance laboratories that the effects of egg-adaptation on the HA must be carefully monitored to avoid the diagnosis of a new influenza strain as epidemiologically significant when it is in fact a laboratory artefact.


ACKNOWLEDGEMENTS I wish to acknowledge the considerable contribution of my colleagues Drs J. S. Oxford, G. C. Schild and J. M. Wood at NIBSC and Drs J. M. Katz and R. G. Webster at St Jude Children’s Research Hospital, Memphis, USA, to the work contained in this review, and to Dr Sue Williams whose studies on receptor binding formed part of her PhD thesis. I also wish to thank Carolyn Nicolson and Janet Bootman for their immense contributions to the sequencing data reported.

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Note Added in Proof Evidence is accruing that recent influenza B/Panama/45/9c)-like isolates are apparently able to replicate in the allantoic cavity without the need to adapt.

clinical influenza virus and the embryonated hen's egg

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