Further studies on rabies virus isolated from healthy dogs in Nigeria

Veterinary Microbiology, 22 (1990) 17-22 Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands

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F u r t h e r Studies on Rabies Virus Isolated F r o m H e a l t h y D o g s in N i g e r i a H.O. AGHOMO and C.E. RUPPRECHT

The Wistar Institute o[ Anatomy and Biology, 3601 Spruce Street, Philadelphia, PA 19104 (U.S.A.) (Accepted 12 September 1989)

ABSTRACT Aghomo, H.O. and Rupprecht, C.E., 1990. Further studies on rabies virus isolated from healthy dogs in Nigeria. Vet. Microbiol., 22: 17-22. Rabies viruses isolated from healthy dogs, were passaged in mice and adapted to cell culture. After 5-7 passages, isolated viruses were subjected to monoclonal antibody (MAb) characterization with a panel of 36 anti-nucleocapsid (NC) and 40 anti-glycoprotein (G) MAbs. The four viruses showed positive fluorescence with all NC hybridomas except MAb 422-5, confirming them as true rabies virus isolates. The anti-G MAb reactivity pattern was the same in the four isolates indicating that they belong to the same antigenic group, but were antigenically distinct from the Flury LEP rabies vaccine virus which is widely used throughout Nigeria for canine vaccination, and from other previously characterized street lyssaviruses from Nigeria.

INTRODUCTION

Rabies virus was isolated from clinically healthy and previously unvaccinated dogs in Nigeria (Aghomo et al., 1989), contrary to the belief that rabies is uniformly fatal in all species of animals (Rife, 1968). The isolation confirms the work of Fekadu and Baer (1980) and Fekadu et al. (1981) who reported recovery of animals from clinical rabies with intermittent shedding of rabies virus in their saliva over the course of 1 year. The isolates from Nigerian dogs were uniformly fatal to mice but only one was fatal to puppies in experimental infection (Aghomo et al., 1989). While Fekadu et al. (1982) isolated rabies virus from a clinically healthy Ethiopian dog, this virus was fatal to some experimentally-infected adult dogs. Rabies diagnosis is based on immunofluorescence, histopathology, serology and animal inoculation (WHO, 1980) which do not differentiate between strains of rabies and related lyssaviruses. With the application of monoclonal antibody (MAb) analysis, it has been possible to differentiate between infec0378-1135/90/$03.50

© 1990 Elsevier Science Publishers B.V.

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H.O. AGHOMOAND C.E. RUPPRECHT

tions caused by strains of rabies and related viruses such as Mokola, Lagos bat, and Duvenhage (Flamand et al., 1980; Wiktor et al., 1980; Sureau et al., 1983; Wiktor et al., 1984; Dietzschold et al., 1988). The aim of this study was to differentiate these canine isolates from other local lyssavirus street strains and from the Flury LEP fixed rabies virus strain used for vaccine production in Nigeria. MATERIALS AND METHODS

Virus isolation The circumstances of isolation were described previously (Aghomo et al., 1989). Briefly, 1500 dogs presented for routine veterinary examination and antirabies vaccination were randomly screened over a period of 5 years from 4 states (Oyo, Lagos, Ondo, Ogun) in Southern Nigeria. Canine saliva was obtained by swabbing, and impressions were made on glass microscope slides from these salivary swabs. Individual swabs were placed in vials of transport medium consisting of Eagle's m i n i m u m essential medium (MEM) containing 10% fetal calf serum (MEM-10), 200 IU of penicillin, 200/~g of streptomycin, 250 m g / m l L-glutamine and 29.23 m g / m l of fungizone. Impressions were screened for rabies virus antigen by the fluorescent antibody test (Wiktor et al., 1980). Any positive salivary samples and 500 randomly selected others were inoculated intracerebrally into 24- to 48-h-old Swiss mice at a volume of 0.03 ml. Four samples were found positive by impression and mouse isolation during the 5-year survey. Brains were removed and homogenized separately in transport medium to give a 10% suspension. The brain suspension was clarified by centrifugation at 700 g for 10 min at 4 ° C. The virus-containing supernatant was decanted and placed in an ice bath during the procedure. After five mouse passages, approximately 0.25 ml of the clarified brain suspension supernatant was mixed with 2.0 ml of freshly trypsinized baby hamster kidney (BHK) cells (1.5× 106/ml) in a T25 flask (Falcon) and allowed to stand. After 5 min, 5 ml of MEM-10 was added; from this, 0.3 ml was transferred into a well of a 96-well plate (Falcon). Ten #l was transferred into each well in row of six wells in a Terasaki plate (Nunclon). The 96-well and Terasaki plates were incubated together with the T25 virus-containing flask at 37 ° C in a 5.5% CO.~ atmosphere for 3 days. After incubation, the Terasaki plate was washed 2 X with phosphate-buffered saline (PBS) (pH 7.0), rinsed in 80% cold acetone and fixed in another solution of 80% gold acetone for 30 rain. The plate was air-dried and stained with 5/~1 of rabies fluorescein isothiocyanate (FITC) conjugate (Centocor) and incubated for 30 min at 37 ° C. After incubation the plate was rinsed 2 × with distilled water and viewed while still moist under a fluorescent microscope (Leitz Dialux ) to asses the percentage of infected cells. The med;,lm in the infected T25 flask was collected and stored at - 85 ° C after

RABIES VIRUS ISOLATED FROM HEALTHY DOGS

19

clarification. The infected cells were trypsinized and 50% were discarded at each passage, and 5 ml of MEM-10 was added to the remaining 50%. The above procedure was repeated at each passage. Fresh BHK cells (50%) were added to the culture at the third passage as more cells became infected. The percentage of infectivity was monitored as described at each passage until 100% cell infectivity was obtained. At 100% cell infectivity the virus-containing supernatant was titrated. In a 96-well plate five-fold serial dilutions were made and to each well 30/~l of BHK cells ( 1.0 X 106 ) was added. The contents of the wells were thoroughly mixed, and 10/tl of virus-cell suspension was transferred into each well of a Terasaki plate in duplicate. The 96-well and Terasaki plates were incubated together for 4 days at 37 °C in 5.5% CO2 incubator. After incubation the Terasaki plate was fixed for fluorescent microscopy.

Anti-nucleocapsid (NC) characterization Ten/zl of 50% infected cells (1.0 X 106/ml) was distributed into 36 wells of a Terasaki plate and incubated for 24 h in a 37°C plus 5.5% CO2 incubator, as described (Wiktor et al., 1984). The plate was washed 2X in PBS, drained, rinsed in 80% cold acetone and finally fixed in another solution of 80% cold acetone for 30 min. The plate was dried for 10 min in an incubator (37°C) before each well was stained with 5/11 of each of the panel of 36 anti-NC MAbs. The plate was incubated for 30 min at 37°C before washing 2X with PBS, drained and counterstained with 5/~l of goat anti-mouse rabies gamma globulin FITC-labeled conjugate (Cappel) and incubated for 30 rain at 37 oC. The plate was washed 2 X with distilled water and viewed while moist under fluorescent microscopy at 200 X magnification.

Anti-glycoprotein (G) characterization The virus neutralizing index of each of the four isolates was determined with a panel of 40 anti-G MAbs as described (Wiktor et al., 1984). Briefly, 30/tl of each anti-G MAb (of predetermined concentration) was placed in six wells of 96 plates followed by 30/~l of six serial three-fold dilutions of virus. The contents of the wells were thoroughly mixed and incubated at 37 °C for 1 h in a 5.5 % CO2 incubator. Thirty/zl of BHK cells (1.0 X 106/ml) was added to each well and mixed thoroughly. Ten #l from each well was transformed to a corresponding well in a Terasaki plate. The 96-well and Terasaki plates were incubated for 4 days at 37°C in a 5.5% CO2 incubator. The Terasaki plate was washed 2 X with PBS (pH 7.0) drained, rinsed in 80% cold acetone and fixed in another 80% cold acetone for 30 rain. The plate was dried and stained with 5/tl of rabies FITC-labeled conjugate and incubated for 30 min at 37°C. The plate was rinsed in distilled water and viewed while moist by fluorescent mi-

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H.O. AGHOMOAND C.E. RUPPRECHT

croscopy at 200 X magnification. A reduction in virus titer of 100 or more infective units in the presence of MAb was considered as positive neutralization. RESULTS

The four isolates were identified as true rabies viruses on the basis of their reactivity with anti-NC MAbs 502-2 and 422-5. The four isolates showed positive staining by bright fluorescence with 35 of the anti-NC MAbs, including 502-2. There was no fluorescence with anti-NC MAb 422-5. The four isolates showed similarities in their patterns of reactivity with the 40 anti-G MAbs. They were neutralized by anti-G MAbs 509-6, 231-2, 220-8, 240-3,719-3, 1117-8, 226-1,194-2,248-8, 507-1,718-4, 127-5,904-4,523-11 and 1113-1 (Fig. 1 ). These patterns of reactivity place the 4 isolates in the same antigenic group (Fig. 1 ), but indicate that they are quite distinct antigenically from the Flury LEP vaccine virus or previously characterized related lyssaviruses (Dietzschold et al., 1988). DISCUSSION

Four isolates of rabies virus were obtained from clinically healthy but unvaccinated dogs. These viruses were all pathogenic to mice but only one was pathogenic to puppies in experimental infections (Aghomo et al., 1989). The Flury LEP strain is used exclusively for vaccine production in Nigeria (Ezeokoli and Umoh, 1986; Okoh et al., 1988). These dogs were not vaccinated, and the isolates were unlikely to have originated directly from vaccine virus. The analysis by anti-NC MAbs confirm these isolates as true rabies rather then related lyssavirus strains by their reactivity patterns with MAbs 502-2 and 422-5. The viruses showed positive staining with MAb 502-2 and negative with 422-5. Anti-NC MAb 502-2 recognizes all known lyssaviruses, while antiNC MAb 422-5 recognizes related African lyssaviruses only(Wiktor et al., 1984). The anti-G MAb characterization demonstrated that these isolates were true rabies viruses because they were neutralized by anti-G MAbs 509-6 (Site I) and 231-2 (Site IIA) and were not neutralized by the unclassified MAbs 5041,508-9, 419-2 and 422-2 which recognize related lyssaviruses (Wiktor et al., 1980). The isolated viruses were also distinct from the Nigerian vaccine strain (Flury LEP) because LEP virus is not neutralized by MAbs 220-8, 1117-8, 240-3, 719-3, 226-1 and 127-5. The isolates were also different antigenically from the field strains characterized by Okoh et al. (1988). Loss of virulence by the isolates in dogs may be due to a number of factors, such as multiple natural passage from dog to dog giving rise to an adaptive tolerance on the part of the canine. Conversely, loss of virulence may have resulted Crom selective mutation in the virus; for example, Wunner and

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Fig. 1. Pattern of neutralization reactivity of the four rabies isolates from clinically healthy and unvaccinated dogs with a panel of 40 anti-G MAbs by antigenic (AG) site or unclassified (UNC). Clear boxes show neutralization (and hence no positive staining). Striped bars indicate no virus neutralization (and hence immunofluorescence).

Dietzschold (1987) reported alterations from pathogenic parent to nonpathogenic variant viruses due to substitution of amino acid arginine at position 333 for another amino acid, depending on the rabies virus strain under neutralizing MAb selection pressure. This is yet to be investigated in these particular Nigerian isolates.

22

H.O.AGHOMOANDC.E.RUPPRECHT

These isolates were antigenically different from the vaccine strain (Flury LEP) (Fig. 1 ), and the strains identified by Okoh et al. (1988). This suggests that there are many different local strains of rabies virus of unknown public health significance and control of all strains will require considerable research on the epidemiology and pathogenesis of lyssaviruses in Nigeria. ACKNOWLEDGEMENTS

This work was supported in part by Public Health Service Grant AI-0970616 from the National Institute of Allergy and Infectious Diseases.

REFERENCES Aghomo, H.O., Oduye, 0.0., Tomori, O. and Ibe, M., 1989. Isolation of rabies virus from clinically healthy and previously unvaccinated dogs. Bull. Anim. Health Prod. Afr., in press. Dietzschold, B., Rupprecht, C.E., Tollis, M., Lafon, M., Mattei, J., Wiktor, T.J. and Koprowski, H., 1988. Antigenic diversity of the glycoprotein and nucleocapsid proteins of rabies and rabiesrelated viruses: implications for epidemiology and control of rabies. Rev. Infect. Dis., 10 (Suppl. 4): $785-798. Ezeokoli, C.D. and Umoh, J.U., 1986. Rabies. Zariya Veterinarian, 1: 100-114. Fekadu, M. and Baer, G.M., 1980. Recovery from clinical rabies of 2 dogs inoculated with a rabies virus strain from Ethiopia. Am. J. Vet. Res., 41: 1632-1634. Fekadu, M., Shaddock, J.H. and Baer, G.M., 1981. Intermittent excretion of rabies virus in the saliva of a dog two and six months after it had recovered from experimental rabies. Am. J. Trop. Med. Hyg., 30: 1113-1115. Fekadu, M., Chandler, F.W. and Harrison, A.K., 1982. Pathogenesis of rabies in dogs inoculated with an Ethiopian rabies virus strain: immunofluorescence, histologic and ultrastructural studies of the central nervous system. Arch. Virol., 71: 109-126. Flamand, A., Wiktor, T.J. and Koprowski, H., 1980. Use of hybridoma monoclonal antibodies in detection of antigenic differences between rabies and rabies-related virus protein. II. The glycoprotein. J. Gen. Virol., 48: 105-109. Okoh, A.E.J., Umoh, J.U., Ezeokoli, C.D. and Addo, P.B., 1989. Vaccination challenge studies with variants of street rabies virus isolated in Nigeria. Vaccine, 6: 19-24. Rife, C.C., 1968. Rabies. In: Canine Medicine. Am. Vet. Pub. Inc., Philadelphia, PA, pp. 133-144. Sureau, P., Rollin, P. and Wiktor, T.J., 1983. Epidemiologic analysis of antigenic variants of street rabies virus. Detection by monoclonal antibodies. Am. J. Epidemiol., 117: 605-609. World Health Organization, 1980. Report on consultation on rabies prevention and control. 1012 March, Lyon, France. Wiktor, T.J., Flamand, A. and Koprowski, H., 1980. Use of monoclonal antibodies in diagnosis of rabies virus infection and differentiation of rabies and rabies-related viruses. J. Virol. Methods, 1: 33-36. Wiktor, T.J., Macfarlan, R.I., Foggin, C.M. and Koprowski, H., 1984. Antigenic analysis of rabies and Mokola virus from Zimbabwe using monoclonal antibodies. Dev. Biol. Stand., 57: 199211. Wunner, W.H. and Dietzschold, B., 1987. Rabies virus infection: genetic mutations and the impact on viral pathogenicity and immunity. Contrib. Microbiol. Immunol., 8: 103-124.