Laboratory Identification of Porcine Enteroviruses

Teschen disease was first described in Czechoslovakia by Trefny (1930). This severe clinical form of pig polioencephalomyelitis, with a mortality of up to 90%, was confined to central Europe until 1952, at which time it was confirmed in Madagascar (Pilet, 1952). In Denmark and England, a disease of pigs characterized by a mild nervous disorder was reported, and the names "poliomyelitis suum" and "Talfan" were used in the respective countries (Bendixen and Sjolte, 1955; Harding et al., 1957). These diseases were mild and there was almost no mortality. However, histological examination of the central nervous tissues revealed changes indistinguishable from those of the more severe Teschen disease. In 1958 it was established by reciprocal cross-neutralization tests in cell culture that Talfan virus was closely related to Teschen disease virus (Chaproniere et al., 1958). It was later shown by immunity tests in pigs that related viruses causing the mild disease can protect pigs against challenge with those viruses causing the severe disease (Huck, 1962; Mayr, 1961).

Porcine enteroviruses (PEV), other than Teschen disease virus, were first described in the late 1950s following the development of tissue culture techniques.

PEVs have been divided into three distinct groups (I, II and III) based on physicochemical properties, type of cytopathic effect (CPE) produced in pig kidney cells and different cell culture host ranges (Zoletto 1965; Zoletto et al., 1974; Knowles et al., 1979). They are also classified into 11 serologically distinct types (Dunne et al., 1971; Knowles et al., 1979), groupI consisting of serotypes 17 and 11, group II consisting of serotype 8 and group III consisting of serotypes 9 and 10. Teschen disease virus belongs to serotype 1. Swine vesicular disease (SVD) virus is not included as an additional PEV serotype since it is classified as a porcine variant of the human pathogen coxsackievirus B5 (Knowles et al., 1979).

PEVs are readily cultivated in the laboratory in cell cultures of porcine origin. Primary or secondary cultures of pig kidney or testes are generally used. However, continuous cell lines such as IB-RS-2, PK-15 or MVPK may be also be successfully employed. The type of CPE exhibited in porcine kidney cells can be recognised by experienced workers and used as a preliminary grouping method. The growth of PEV in cells derived from non-porcine sources varies between, and sometimes within, the three PEV groups (Knowles et al., 1979).

Depending on the reason for diagnosis a number of different tissues may be submitted. PEVs have been associated with nervous, respiratory, reproductive and digestive disorders. They are commonly found in faeces of both normal and sick pigs and isolation rates may be high (Knowles, 1983; Honda et al., 1990c). PEVs may also be isolated as incidental findings during the diagnosis of other diseases of pigs such as SVD or foot-and-mouth disease (FMD). In these cases, a test capable of differential diagnosis may be of vital importance to facilitate the rapid reporting of results (Knowles, 1988).

The ultimate typing test is still virus neutralization since the presence of multiple serotypes may be detected, however, where many tests are to be performed complement fixation is recommended (Knowles and Buckley, 1980; Knowles, 1983; Caracappa et al., 1985; Shin et al., 1987). Other techniques such as immunofluorescence (Watanabe, 1971), immunodiffusion (Sulochana and Derbyshire, 1978a), immunoperoxidase (Sulochana and Derbyshire, 1978b; Honda et al., 1990a) have been developed, however, none of these techniques have been employed for serotyping principally due to a lack of specificity. An enzyme-linked immunosorbent assay (ELISA) for PEV typing is currently being developed at Pirbright employing rabbit and guinea pig antisera raised against purified, inactivated viruses.

In addition to the 11 currently recognised serotypes, a number of PEVs have been described which are not neutralized by the prototype antisera (Knowles, 1983; Honda et al., 1990b; J. Auerbach & K.H. Witte, personal communication, 1993). Some of these have been characterized in more detail and have been proposed as additional serotypes (Table 1). However, since extensive antigenic variation may occur within some PEV serotypes (Dunne et al., 1971), a lack of neutralization may not necessarily indicate a new serotype and further studies are required.

Untyped isolates should be subjected to electron microscopy to estimate the particle size, acid stability treatment at pH 3.0, and exposure to an organic solvent to confirm the lack of essential lipids. The type of viral nucleic acid should also be determined. Some biological properties previously thought to be characteristic of PEVs, such as a lack of haemagglutination or failure to show CPE on bovine or ovine cells, may be less diagnostic since a PEV belonging to serotype 10 has been discovered which both agglutinates guinea pig red cells and grows in bovine and ovine cell cultures (Caracappa et al., 1985; Knowles et al., unpublished data).

Bendixen, H.C. and Sjolte, J.P. (1955). Undersogelser vedrorende optraeden af enzootisk (overforbar) griselammelse (poliomyelitis suum) i Danmark. Nord. Vet.-Med. 7: 97-140.

Caracappa, S., Vesco, G., Iannizzotto, G., Guercio, V. and Knowles, N.J. (1985). Isolation and identification of porcine enteroviruses in Sicily. (Isolamento ed identificazione di enterovirus suini in Sicilia). Archivio Veterinario Italiano 36: 167-170.

Chaproniere, D.M., Done, J.T. and Andrewes, C.H. (1958). Comparative serological studies on Talfan and Teschen diseases and similar conditions. British Journal Experimental Pathology 39: 74-77.

Dunne, H.W., Wang, J.T., Ammerman, E.H. (1971). Further studies on the classification of North American porcine enteroviruses: A comparison with European and Japanese strains. Infection and Immunity 4: 619-631.

Harding, J.D.J., Done, J.T. and Kershaw, G.F. (1957). A transmissible polioencephalomyelitis of pigs (Talfan disease). Veterinary Record 69: 824-832.

Honda, E., Watanabe, I., Okazaki, K. and Kumagai, T. (1990a). Relation of serological and CPE classification of porcine enteroviruses to the classification by immunoperoxidase (IP) staining, and observation of CPE by IP staining method. Japanese Journal of Veterinary Science 52: 795-800.

Honda, E., Kimata, A., Hattori, I., Kumagai, T., Tsuda, T. and Tokui, T. (1990b). A serological comparison of 4 Japanese isolates of porcine enteroviruses with the international reference strains. Japanese Journal of Veterinary Science 52: 49-54.

Honda, E., Hattori, I., Oohara, Y., Taniguchi, T., Ariyama, K., Kimata, A., Nagamine, N. and Kumagai, T. (1990c). Sero and CPEtypes of porcine enteroviruses isolated from healthy and diarrheal pigs: possible association of CPE type II with diarrhea. Japanese Journal of Veterinary Science 52: 85-90.

Huck, R.A. (1962). Encephalomyelitis of pigs - studies of the relationships of the Teschen group of viruses and the identification of a third subtype F.S. 55. VIII International Congress of Microbiology 101.

Kadoi, K., Kobori, S., Morimoto, T. (1970). Studies on swine enteroviruses: Japanese 6th serotype and relationship between heat susceptibility and cytopathic effects. Japanese Journal of Microbiology 14: 111-121.

Knowles, N.J. (1983). Isolation and identification of porcine enteroviruses in Great Britain, 1979 to 1980. British Veterinary Journal 139: 19-22.

Knowles, N.J. (1988). The association of group III porcine enteroviruses with epithelial tissue. Veterinary Record 122: 441-442.

Knowles, N.J. and Buckley, L.S. (1980). Differentiation of porcine enterovirus serotypes by complement fixation. Research in Veterinary Science 29: 113-115.

Knowles, N.J., Buckley, L.S. and Pereira, H.G. (1979). Classification of porcine enteroviruses by antigenic analysis and cytopathic effects in tissue culture: description of 3 new serotypes. Archives of Virology 62: 201-208.

Mayr, A. (1961). Degrees of variation of the virus of Teschen disease and relationships to other enteroviruses of swine. Bull. Off. int. Epiz. 56: 106-110.

Morimoto, T., Dunne, H.W. and Wang, J.T. (1968). Serologic comparison of North American and Japanese porcine picornaviruses. American Journal of Veterinary Research 29: 2275-2280.

Pilet, E. (1952). La meningo-encephalomyelite enzootique du porc a Madagascar. Bull. Off. int. Epiz. 38: 61-105.

Shin, T.K., Lee, C.S., Kwon, H.J. and Knowles, N.J. (1987). Serological classification of porcine enteroviruses isolated in Korea. Korean Journal of Veterinary Research 27: 223-226.

Sulochana, S. and Derbyshire, J.B. (1978a). Immunodiffusion reactions among porcine enteroviruses and other picornaviruses. Veterinary Microbiology 2: 205-212.

Sulochana, S. and Derbyshire, J.B. (1978b). Use of indirect immunoperoxidase test for detection of porcine enteroviral antigens in infected PK15 cell cultures. Kerala Journal of Veterinary Science 9: 111-119.

Trefny, L. (1930). Massive illness of swine in Teschen area. Zverolek. Obz. 23: 235-236.

Watanabe, H. (1971). Fluorescent antibody technique in cultured cells infected with porcine enteroviruses. Japanese Journal of Veterinary Research 19: 1-5.

Zoletto, R. (1965). Differential characteristics of swine enteroviruses. Veterinaria Italiana 16: 13-20.

Zoletto, R., Kadoi, K., Turilli, C., Cancelloti, F., Stilas, B. (1974). Cytopathic effect and physicochemical characteristics of swine vesicular disease virus and its relationship with other swine enteroviruses. 3rd International Congress (IPVS), Lyon, France, June 12-14.

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