Use of VP60 RT-PCR to overcome the limitation of haemagglutination inhibition diagnosis of rabbit viral haemorrhagic disease

M.A. Shakal, D.E. Khelfa, O.G.A. Salman, A.A. Yousif, E.-A.A Salwa


Rabbit viral haemorrhagic disease (RVHD) is a highly contagious, highly fatal, peracute and acute viral disease of both wild and domestic rabbits caused by rabbit haemorrhagic disease virus (RHDV). Testing for haemagglutination (HA) activity in processed liver samples is one of the cornerstones for rapid diagnosis of RHDV outbreaks in national rabbitries. However, RHDV isolates exhibiting no HA activity are increasingly being reported. The extent of deviation from classical HA activity patterns for RHDV strains in Egypt has not been investigated. This study compared the HA activity patterns of samples collected from 61 RHDV outbreaks that occurred between 1999 and 2005 to determine whether dependence on HA test (HAT) for diagnosis of RHDV outbreaks needs to be reviewed. All samples were confirmed RHDV positive using SDS-PAGE and western blotting. Using slide HAT, only 36.1% of samples were positive (22 samples). Plate HAT conducted at 4 0C detected an additional 16 positive samples bringing the total HA-positive samples to 38 (62.3%).  Plate HAT conducted at 22 0C failed to detect additional positive samples. The majority of samples detected after plate HA testing (62.5%) had HA titres comparable to those obtained from slide-HA-positive samples, indicating that the difference in HA activity is dependent on the nature of the HA antigen rather than its presence. Direct detection of HA activity failed in 37.7% of samples despite the presence of classical signs, pathology, and being RT-PCR positive for three different VP60 regions. Experimental infection of seronegative rabbits with 9 HA negative RHDV samples showed that 5 isolates were in-fact HA positive, while only 4 isolates remained HA negative. The increased detection of viruses lacking HA activity and the low HAT sensitivity mandates the use of molecular techniques for rapid confirmation of RHDV diagnosis in the Egyptian environment.


RHDV; Slide haemagglutination; Plate haemagglutination; RT-PCR; Non-haemagglutinating; Experimental infection

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Abd El-Mottelib T.Y. 1993. Role of hyperimmune serum in protection against viral haemorrhagic disease of rabbits. Assiut Vet. Med. J., 28: 297-307.

Abrantes J, Esteves PJ. 2010. Letter: Not-so-novel Michigan rabbit calicivirus. Emerg Infect Dis., 16:1331-13322.

Abrantes J, Esteves PJ, van der Loo W. 2008. Evidence for recombination in the major capsid gene VP60 of the rabbit haemorrhagic disease virus (RHDV). Arch Virol., 153: 329-35 doi: 10.1007/s00705-007-1084-0.

Barbieri I., Lavazza A., Brocchi E., Konig M., Capucci L. 1997. Morphological, structural and antigenic modifications of rabbit haemorrhagic disease virus in the course of the disease. In Proc.: 1st Symposium on Calicivirus of the European Society of Veterinary Virology (ESVV). Reading. UK. 15-17 September 1996, 182-193.

Bergin IL, Wise AG, Bolin SR, Mullaney TP, Kiupel M, Maes RK. 2009. Novel calicivirus identified in rabbits. Emerg Infect Dis., 15:1955-1962. doi: 10.3201/eid1512.090839

Biermann U., Herbst W., Balyer G. 1992. RHD comparative diagnostic studies: haemagglutination tests and electron microscopy. Berl. Munch. Tierarztl., 105: 86-87.

Capucci L., Scicluna M.T., Lavazza A., Brocchi E. 1990. Purification and characterization of the aetiological agent of viral haemorrhagic disease of rabbits. Selezione Veterinaria, 31: 301-312.

Capucci L., Scicluna M.T., Lavazza A. 1991. Diagnosis of viral haemorrhagic disease of rabbits and the European brown hare syndrome. Rev. Sci. Tech., 10: 347-370.

Capucci L, Frigoli G, Ronshold L, Lavazza A, Brocchi E, Rossi C. 1995. Antigenicity of the rabbit hemorrhagic disease virus studied by its reactivity with monoclonal antibodies. Virus Res., 37:221-238. doi: 10.1016/0168-1702(95)00033-M

Capucci L., Chasey D., Lavazza A., Westcott D. 1996a. Preliminary characterization of a non-haemagglutinating strain of rabbit haemorrhagic disease virus from the United Kingdom. J. Vet. Med. B, 43: 245-250. i:10.1111/j.1439-0450.1996.tb00311.x.

Capucci L., Fusi P., Lavazza A., Pacciarini M.L., Rossi C. 1996b. Detection and preliminary characterization of a new rabbit calicivirus related to rabbit hemorrhagic disease virus but nonpathogenic. J. Virol., 70: 8614-8623.

Chasey D., Lucas M.H., Westcott D.G., Sharp G., Kitching A., Hughes S.K. 1995. Development of diagnostic approach to the identification of rabbit haemorrhagic disease. Vet. Rec., 137: 158-160. doi:10.1136/vr.137.7.158

Cubitt W.D. 1987. The candidate caliciviruses. In Novel Diarrhoea Viruses, CIBA Foundation Symposium 128: 128-143.

Du N. 1990. Rabbit haemorrhagic disease (RHD)-a new disease and its viral etiology. Dtsch. Tierarztl. Wochenschr., 97: 114-116.

Erber M., Gerbermann H., Meiler H., Schindlmayer R. 1991. Rabbit haemorrhagic disease among domestic rabbits in Bavaria: epidemiological, pathological and virological investigations. Tierarztliche Umschau, 46: 146, 151-155.

Esteves P.J, Abrantes J, Carneiro M, Müller A, Thompson G, van der

Loo W. 2008. Detection of positive selection in the major capsid protein VP60 of the rabbit haemorrhagic disease virus (RHDV). Virus Res., 137: 253-256. doi: 10.1016/j.virusres.2008.07.025

Fitzner A., Kesy A., Chrobocinska M. 1992. Ability of human erythrocytes O, A, B and AB groups to agglutinate rabbit haemorrhagic disease virus. Med. Weter., 48: 89-90.

Forrester NL, Moss SR, Turner SL, Schirrmeier H, Gould EA. 2008. Recombination in rabbit haemorrhagic disease virus: possible impact on evolution and epidemiology. Virology, 376: 390-396. doi: 10.1016/j.virol.2008.03.023

Ghanem I.A., Ismail A.N. 1992. Occurrence of rabbit haemorrhagic disease in Sharkia province. Zagazig Veterinary Journal, 20: 491-502.

Granzow H., Weiland F., Strebelow H.-G., Liu C.M., Schirrmeier H. 1996. Rabbit haemorrhagic disease virus (RHDV): Ultrastructure and biochemical studies of typical and core-like particles present in liver homogenate. Virus Res., 41: 163-172. doi:10.1016/0168-1702(96)01285-3

Kesy A., Fitzner A., Niedbalski W., Paprocka G., Walkowiak B. 1996. A new variant of the viral haemorrhagic disease of rabbits virus. Rev Sci Tech., 15: 1029-1035.

Le Gall G., Arnauld C., Boilletot E., Morisse J.P., Rasschaert D. 1998. Molecular epidemiology of rabbit haemorrhagic disease virus outbreaks in France during 1988 to 1995. J. Gen. Virol., 79: 11-16.

Liebermann H., Bergmann H., Lange E., Schirrmeier H., Solisch P. 1992. Some physicochemical properties of the virus of rabbit haemorrhagic disease. J. Vet. Med., Series B, 39: 317-329. doi: 10.1111/j.1439-0450.1992.tb01176.x

Liu S.J., Xue H.P., Pu B.Q., Qian N.H. 1984. [New viral disease in rabbits]. Animal Husbandry and Veterinary Medicine, 16: 253-255.

McIntosh M.T., Behan S.C., Mohamed F.M., M.; Lu Z., Moran K.E. Burrage T. G., Neilan J.G., Ward G.B., Botti G., Capucci L., Metwally S.A. 2007. A pandemic strain of calicivirus threatens rabbit industries in the Americas. J. Virol., 4: 96-108. doi: 10.1186/1743-422X-4-96.

Moss S.R., Turner S.L., Trout R.C., White P.J., Hudson P.J., Desai A., Armesto M., Forrester N.L., Gould E.A. 2002. Molecular epidemiology of Rabbit haemorrhagic disease virus. J. Gen. Virol., 83: 2461-2467.

Motha M.X., Kittelberger R. 1998. Evaluation of three tests for the detection of rabbit haemorrhagic disease virus in wild rabbits. Vet. Rec., 143: 627-629.

Nardelli S., Agnoletti F., Costantini F., Parpajola R., 1996. Diagnosis of rabbit haemorrhagic disease (RHD) and European brown hare syndrome (EBHS) by indirect sandwich polyclonal ELISA. Zentralbl. Veterinarmed. B, 43: 393-400.

Nowotny N., Leidinger J., Fushs A., Vlasak R., Schwendenwein I., Schilcher F., Loupal G. 1993. RHD: Experimental infections of domestic rabbits with regard to clinical, haematological, chemical, virological, serological and pathomorphological features. Wien.Tierarztl Monat., 80: 65-74.

Ohlinger V.F., Haas B., Meyers G., Weiland F., Thiel H.J. 1990. Identification and characterization of the virus causing RHD. J. Virol., 64: 3331-3336.

OIE. World Organisation for Animal Health. 2008. Rabbit haemorrhagic disease.In Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. 2.6.2.: 947-961.

Park J.H., Kida H., Ueda K., Goryo M., Itakura C. 1991. Etiology of RHDV spontaneously in Korea. J. Vet. Med., Series B, 38: 749-754.doi: 10.1111/j.1439-0450.1991.tb00939.x.

Parra F., Prieto M. 1990. Purification and characterization of a calicivirus as the causative agent of a lethal haemorrhagic disease in rabbits. J. Virol., 64: 4013-4015.

Peeters J.E., Broes A., Charlier G. 1990. First observations of viral haemorrhagic disease of rabbits (RHD) in Belgium. Ann. Med. Vet., 134: 567-571.

Prieto J.M., Martin J.M., Espi A., Parra F. 2000. A new non-haemagglutinating strain of RHDV. In Proc.: 5th international congress of the European society for veterinary virology. Brescia, Italy. 27-30 August 2000. 204-205.

Salman, O.G.A. 1999. Studies on haemorrhagic viral disease in rabbits. In M. V. Sci. Thesis, Faculty of Veterinary Medicine, Cairo University.

Sambrook J., Fritsch F.E., Manitis T. 1989. Molecular cloning. In A laboratory manual. 2nd ed., Cold spring Harber Laboratory Press, Plain View, N,Y, USA, : 18047-18059.

Schirrmeier H., Reimann I., Kollner B., Granzow H. 1999. Pathogenic, antigenic and molecular properties of rabbit haemorrhagic disease virus (RHDV) isolated from vaccinated rabbits: detection and characterization of antigenic variants. Arch. Virol., 144:719-735.doi: 10.1007/s007050050538.

Sharawi S.S.A. 1992. Studies on the virus causing haemorrhagic septicaemia in rabbits. In M.V.Sc. Thesis, Fac. Vet. Med., Zagazig Univ., Benha Branch.

Strive T., Wright J.D., Robinson A.J. 2009. Identification and partial characterization of a new Lagovirus in Australian wild rabbits. Virology, 384:97-105. doi: 10.1016/j.virol.2008.11.004.

Studdert M.J. 1978. Caliciviruses. Brief Review. Arch. Virol., 58: 157-191. doi: 10.1007/BF01317600.

White P.J., Norman R.A., Hudson P. J. 2002. Epidemiological consequences of a pathogen having both virulent and avirulent modes of transmission: the case of rabbit haemorrhagic disease virus. Epidemiol. Infect., 129: 665-677. doi: 10.1017/S095026880200777X

Wirblich C, Meyers G, Ohlinger VF, Capucci L, Eskens U, Haas B, Thiel H.J. 1994. European brown hare syndrome virus: relationship to rabbit hemorrhagic disease virus and other caliciviruses. J. Virol., 68: 5164-5173.

Wirblich C., Thiel H.J., Meyers G. 1996. Genetic map of the calicivirus rabbit hemorrhagic disease virus as deduced from in vitrotranslation studies. J. Virol., 70: 7974-7983

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 Universitat Politècnica de València


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