Indian Journal of Biotechnology Vol I, April 2002, pp 170-174

Antigenic Differentiation of Equine Herpes Virus-l (EHV -1) Isolates of Indian Origin using Monoclonal Antibodies

B K Singh i *, B R Gulati2, S C Tewari 2 and M P Yadav3

INational Research Centre on Equines, Sirsa Road, Hisar 125001, India

2Department of Veterinary Microbiology, CCS Haryana Agricultural University, Hisar 125004, India

' Indian Veterinary Research Institute, Izatnagar 2431 22, India

Received 5 November 200J ; revised 15 February 2002

A panel of seven monoclonal antibodies (Mabs) (5 non-neutralizing and 2 neutralizing) raised against 140 kDa polypeptide of EHV-l strain (Hisar-90-7) were used for antigenic characterization of five Indian isolates and com­pared with reference strain (592) of EHV-l. Jind-96 and Hisar-90-7 were antigenically indistinguishable from refer­ence strain in indirect enzyme-linked immunosorbent assay (ELISA) using 5 non-neutralizing Mabs. The antigenic differences were observed in three EHV-l isolates i.e. Tohana-96-2, Delhi-98 and Raj-98 as they failed to react with 5, 4, and 1 of five non-neutralizing Mabs, respectively. While all the six virus isolates were neutralized with Mab 9C4 in virus neutralization test (VNT), two (Tohana-96-2 and Raj-98) of the 6 viral isolates were not neutralized with one Mab, IH6. However, EHV -1 isolates could not be differentiated on western blot analysis as all the Mabs reacted with a 140 kDa protein in these isolates. The findings of the ELISA and VNT using 7 Mabs indicate that initial isolates (Hisar-90-7 and Jind-96) are antigenically closer to reference strain (592) and there is emergence of antigenically dif­ferent isolates (Tohana-96-2, Delhi-98 and Raj-98) subsequently. The study also establishes that more than one anti­genically different strains of EHV -1 are circulating in equines of northern India.

Keywords: equine herpes virus-I, monoclonal antibodies, antigenic differentiation, ELISA, virus neutralization, western blotting

Introduction Since the first report of equine herpes virus -1

(EHV-1) associated abortion in mares in 1965 (Sharma et al. 1965), the infection with this virus has been well established among horses in India. The virus has been isolated and characterized from the tissue of aborted foetus (Jain et ai, 1976; Singh et al. 1991), still birth and foal mortality (Jain & Ram, 1980), neurological disorders (Shankar & Yadav, 1986) and respiratory syndrome (Tewari & Prasad, 1983). During 1989, the National Research Centre on Equines (NRCE), Hisar, reported involvement of EHV -1 in an abortion storm among mares at an army stud in northern India. In this outbreak, among 360 pregnant mares, 54 abortions (15%) occurred (Uppal et ai, 1991). In a national assessment of EHV-l infection among equidae In India, the overall seropositivity to EHV-l was 13.5% (349/2573) (Singh et ai, 1998).

* Author for correspondence: Tel: 01662-76151 ; Fax: 01662-762 17 E-mail :nrcequine@ hubl.nic.in

The antigenic and genetic differences among two previous SUbtypes of equine herpes virus viz. EHV -1, SUbtype 1 and EHV-l subtype -2 led to their classifi­cation as separate types i.e. EHV type I and EHV type 4 (Thompson et ai, 1978; Allen et ai, 1983; Sa­bine et ai, 1981; Studdert et ai, 1981). Although all isolates of EHV type 1 (EHV -1), are antigenically closely related, minor variations in the neutralizing activity for different isolates with polyclonal sera re­vealed the existence of antigenic heterogeneity of EHV -lcirculating in the field (Thompson et ai, 1978). Conclusive evidence for the existence of antigenic diversity within EHV -1 was established by use of the panel of monoclonal antibodies(Mabs) (Yeargan et ai, 1985; Shimizu et aI, 1989). The antigenic variations within Indian EHV -1 isolates have not been studied due to non-availability of Mabs. The information about antigenjc differences among EHV -1 isolates has direct application for studying the pathogenesis and development of effective vaccine against EHV-l. The present study was aimed to detect antigenic differ­ences among EHV -1 virus isolates of Indian origin using Mabs developed at NRCE.

SINGH el af: DIFFERENTIATION OF EHV - I USING MONOCLONAL ANTIBODIES 171

Materials and Methods

Cells

Equine embryonic dermis (EED) cell line obtained from National Centre for Cell Science, Pune was used for virus cultivation, titration and neutralization ex­periments . The cell line was cultivated as per standard technique.

EHV-J Isolates

Five virus isolates were recovered from samples of aborted mares and foal mortality collected from different outbreaks, places and point of times. In addition, a reference strain of EHV -1 (592) was also included in the study (Table 1). All virus isolates were propagated from initial passage level frozen aliquots (-70°C) samples by inoculating EED at a 0.001 multiplicity of infection (m.o.i .) and infected cells were maintained in minimum essential medium supplemented with 2% foetal calf serum at 37° C in 5% CO2 incubator till 80% infected cells showed cytopathic effect (CPE). The virus was purified by the discontinuous sucrose gradient method (Sinclair et ai, 1989).

Mabs

Mabs used for antigenic differentiation of EHV-1 isolates in the present study were developed against abortigenic EHV -1 strain (Hisar-90-7) (Singh et ai, 2001). Properly cloned EHV-1 antibody secretary hybridoma against Hisar-90-7 was inoculated intra­peritoneally in BALB/c for development of ascites fluid . This fluid was purified by ammonium sulphate and used as Mabs (Sinclair et ai, 1989). Seven Mabs (lH6, 9C4, 6A1, 6A2, 6B1, 6D1 and 9D4), all di­rected against 140 kDa polypeptide, were used.

ELISA

The indirect ELISA (Allen et ai, 1988) was per­formed using diluted stock of immunoglobulins puri­fied from ascitic fluid (1 :500) with purified EHV-l antigen. Briefly, 96 well flat bottom microassay plate (Falcon, Bectson Dickinson Labware, Oxnard, CA) was coated with approximately 1 j.l.g/well of purified intact virus overnight at 4°C in coating buffer (0.05 M NaHCOiNa2 C03, pH 9.6). After washing 3 times with phosphate buffer saline (PH 7.2) containing 0.05% Tween 20 (PBS-T), the test Mabs (1 :500) were added to the wells and incubated for 90 min. EHV-1 antigen coated wells in which purified ascites of non-

-secretary clone (6B6) was added, served as negative control. This was followed by successive addition of an optimum dilution of rabbit anti mouse horse radish peroxidase (HRPO) IgG-conjugate diluted 1: 1200 in 0.5 mg/ml bovine serum albumin. After washing 5 times, O-phenylenediamine substrate (OPD) was added. All assays were carried out in triplicate wells and mean OD492nm value of two separate experiments were recorded. Mabs were considered to be positive when the mean of the positive/negative ratio (P/N) i.e. OD492 nm for test Mab divided by OD492 nm negative control was?: 2.0.

Micro Virus Neutralization Test (VNT)

In EED cells, VNT was done to detect virus neu­tralization ability of Mabs by constant virus-variable serum method (Singh et ai, 1995). Each test was done in the presence of 100 TCIDso (50% tissue culture infective dose) of virus isolate in 25 j.l.1 quantity in quadruplicate wells containing doubling dilution of Mab and in the presence of 16 units of guinea-pig complement (GPC'). The mixture was then incubated at 37°C for 2 hrs. EED cells (l.7xl0s cells/ml) were added in minimum essential medium supplemented with 15% FCS. VNT was also performed without GPC'. Each assay consisted of controls for cells, Mabs, anti-EHV-1 positive rabbit and negative con­trol sera. Simultaneously, a virus titration was also performed during each test to ensure a challenge dose of 102

±O.3 TCIDsolwell. Anti-EHV -1 poly clonal antise­rum raised in rabbit was also tested similarly in VNT against all the viral isolates. The plates were then transferred to a 5% CO2 incubator and incubated at 37°C for 5 days. Finally, neutralizing antibody titre of Mabs was expressed as reciprocal of the dilution of Mab that caused 50% reduction in CPE (Reed & Muench, 1938).

Western Blotting

The proteins of the purified EHV -1 isolates were resolved by SDS-PAGE (Crabb & Studdert, 1990) and were electrophoretic ally transferred from gel to a 0.45-j.l.m pore size PVD membrane (Millipore) (Tow­bin et ai, 1979) using semi-dry electrophoresis appa­ratus. Following transfer, the membrane was blocked in 5% bovine serum albumin (BSA), washed 5 times in PBST and incubated with Mabs at optimum dilu­tion at 37°C for 90 min. After washing the membrane, it was incubated with anti-mouse peroxidase­conjugated rabbit immunoglobulin for 90 min at

172 INDIAN 1 8IOTECHNOL, APRIL 2002

37°C. After washing 3 times, the membrane was de­veloped with freshly prepared diaminobenzidine (DAB). Washing in distilled water stopped the reac­tion and air-drying preserved the membrane.

Results All the 7 Mabs were directed against a 140 kDa

polypeptide of Hisar-90-7 isolate of EHV -1. All the Mabs reacted in indirect ELISA with the homologous viral strain, however, only 2 Mabs (lH6 & 9C4) were able to neutralize the Hisar-90-7 strain in the presence of GPC' (Table 1). When different EHV -1 isolates were tested with two neutralizing Mabs in VNT, Mab 9C4 was able to neutralize all the isolates tested giv­ing a neutralizing titre ranging between 8 and 64 (Table 1). However, Mab IH6 failed to neutralize To­hana-96-2 and Raj-98 isolates indicating that these two isolates lack the neutralizing epitope for Mab IH6.

A polyclonal anti -EHV-l rabbit serum could neu­tralize all the six isolates both in presence or absence of GPc. None of the Mabs neutralized any isolate in absence of GPc. The purified protein of all the six EHV -1 isolates reacted in western blots analysis with all the 7 Mabs . No differences could be detected by western blot analysis as all Mabs reacted equally with 140 kDa protein of different isolates (Fig. 1).

The Hisar-90-7, Jind-96 and reference strain 592 were antigenically indistinguishable in indirect ELISA as all the 7 Mabs reacted with them giving PIN ratio 2: 2 (Table 2). Delhi-98 could be differenti­ated from other EHV -1 isolates as it fai led to react with 4 of 7 Mabs (6Al, 6A2, 6BI and 9D4) while Raj-98 did not react with 2 Mabs (1 H6 and 9D4) in indirect ELISA (Table 2). Interestingly, Tohana-96-2 isolate could not be identified by any of the Mabs in indirect ELISA, although it was neutralized by one of the neutralizing Mabs in VNT (Table 1 & 2) . The re-

Table I - Complement-dependent neutralization of different EHV- I isolates using Mabs

S. Virus isolates Place of Year of Clinical mani- Reciprocal of 10g IO neutralizing titre No. (designation) ori gin isolation festations

Mabs* lH6(IgG2a) 9C4(IgG2b)

I Hisar-90-7 Hi sar 1990 Abortion 32 64 2 Tohana-96-2 Tohana 1996 Abortion 32 3 Raj -98 Rajasthan 1998 Abortion 8 4 Delhi-98 Delhi 1998 Abortion 8 20 5 lind-96 lind 1996 Foal mortality 32 32 6 592 UK Foal mortality 32 12

(Reference strain)

- = negative results ; 'Other Mabs did not neutralize any of EHV-I isolate either in presence or absence of GPC' .

Table 2 - Antigenic differentiation of 6 EHV -I isolates using Mabs by indirect ELISA

Mabs ELISA positive/negative ratio l with different EHV-I isolates Hi sar-90-7 Tohana-96-2 Raj-98 Delhi-98 lind-96

IH6" 5.92 0.12* 1.46* 5.52 2.00 9C4" 5.85 0.75* 2.95 2.55 2.13 6AI 7.42 0.96* 2.09 0.23* 2.84 6A2 6.55 1.52* 2.38 0.46* 2.75

681 5.79 0.01 * 2.36 0.69* 2.00

6Dl 9.97 0.71 * 12.63 4.15 2.60

9D4 4.79 0. 12* 1.64* 0.641 * 2.81

# = Neutralizing Mabs; * = negative results; fi gures in bold letters indicate negative results in VNT also.

Polyclonal anti-EHV-I

rabbit serum

32 16 16 16 32 32

592 (reference

strain)

2.53 3.91 4.64 6. 11

2.63

3.83

3.94

IThe result was considered positive if the mean of the OD492 nm value of the test sample/negative control OD492nm was >2.

SINGH et al: DIFFERENTIATION OF EHV-I USING MONOCLONAL ANTIBODIES 173

kDa 2 3 4 5 6

200 ii&Ml:~~!J1

97

Fig. I- Western blot analysis of equine herpes virus (EHV-J) strains with anti-EHV-I monoclonal antibody, 9C4. Lane I : Mo­lecul ar weight marker (Bangalore Genei); lane 2-6: EHV- I strains: Hisar-90-7, Tohana-96-2, Raj-98, Delhi-98 and Jind-96.

sults of indirect ELISA indicate antigenic differences in EHV -1 isolates Tohana-96-2, Delhi-98 and Raj-98 as they lacked 7, 4 and 2 ELISA epitopes, respec­tively.

Discussion In the present study, all the Mabs reacted with a

single band of 140 kDa EHV -1 polypeptide of differ­ent isolates in western blotting. A target antigen of 140 kDa polypeptide under non-reducing condition also reacted with various Mabs of different properties (Allen et ai, 1991). Further, it is quite possible that several epitopes may be present in any large protein molecule and Mabs in this study are directed against different epitopes on the same 140 kDa protein.

The virus neutralizing activity of 2 Mabs (lH6 & 9C4) was not observed in absence of guinea-pig com­plement (GPC') . GPC' -dependent and GPC'­independent virus neutralization has been previously documented with EHV -1 Mabs (Sinclair et ai, 1989; Shimizu et ai, 1989). Since some of the Mabs directed against the 140 kDa protein are neutralizing, it is likely that these Mabs are directed against the surface glycoprotein (envelope protein) of EHV-l. However, nature of the specific protein against which these Mabs are directed, is being confirmed.

Mab-based ELISA is considered sensitive diagnos­tic tool for antigenic differentiation of herpes viruses (Yeargan et ai, 1985; Balchandran et ai, 1982; Peter­son et ai, 1983). However, Tohana-96-2 isolate was not detected by any of the Mabs in ELISA. Mab (9C4) reacted in VNT but not in ELISA with the puri­fied anticrens of Tohana-96-2 . It is not unusual to ob-o

serve variability in the reaction of the same Mab in different test systems . Such variations in the reaction of Mab with different isolates in ELISA and VNT have also been documented against bovine herpes

virus-l (Collins et ai, 1984). Mabs glVlng positive immunofluorescence staining and VNT against EHV-1 gave positive results by FAT against EHV -4 but gave negative VNT (Sinclair et ai, 1989). In this study , non-reactivity of Tohana-96-2 isolate in ELISA and VNT with 1 H6 suggests its antigenic differences from other EHV -1 isolates. Similarly, Raj-98 isolate did not react with 1 H6 neutralizing Mab both in ELISA and VNT indicating that Raj-98 is also anti­genically different from other isolates .

Since quantitative serological differences by poly­clonal antibodies is difficult to detect and because of the close serological relationship which exists among EHVs, the panel of Mabs to EHV-1 reported here has potential as diagnostic tool to facilitate differentiation among Indian EHV -1 strains. Mabs raised against EHV -1 in Europe and USA showed antigenic differ­entiation among EHV -1 strains prevalent in these countries (Yeargan et ai, 1985; Allen & Bryans, 1986; Edington et ai, 1987).

It may be assumed as discussed above that some Indian strains of EHV -1 used in this study also differ at more than one antigenic sites. This study suggests that initial strains (Hisar-90-7 & Jind-96) of EHV-1 are antigenically closer to reference strain (592) while there is emergence of antigenic heterogeneity amongst subsequent EHV -1 strains from northern In­dia (Tohana-96-2, Delhi-98 & Raj-98) . It also indi­cates the prevalence of antigenic heterogeneity among Indian strains.

Acknowledgement The authors thank Dr P K Uppal and Dr S K Sri­

vastava for guidance and encouragement, and Shri D D Pandey for technical help during the research work. B K Singh is grateful to the Indian Council of Agri­cultural Research, New Delhi, for grant of senior fel­lowship.

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