Characterisation of virus isolates from lesser whistling teals and channa species of fish

Abstract

Characterisation of virus isolates (T18 and T22) from lesser whistling teals (Dendrocygna javanica) and Channa species of fish (Fe and F12) was carried out to determine the similarities if any between the isolates and to identify the role of waterfowls in dissemination of these viruses. The virus isolates. preserved in the Department of Microbiology were revived by passaging through embryonated chicken! duck eggs through allantoic route. After the third passage, all the isolates were found to produce death of the embryos and the allantoic fluid collected agglutinated one percent chicken RBC. The isolates T18, Fs and F12 produced congestion of the embryo and CAM and the embryos showed sub-occipital and interdigital haemorrhages. Isolate T 22 also produced congestion of the embryo and CAM and the embryos were stunted. Liver of the embryos had yellowish brown patches. The EID50 of isolates were 3.2x10s, 5.6 x 105 , 1.65x 107 and 3.16 x 105 respectively for the isolates T18, T22. Fs and F12. The infectivity and haemagglutinating activity of all the isolates were retained at pH 7.2, but were completely lost at pH 3.2 and 9.0 and also by treatment at 56°C for 30 min. All the isolates were sensitive to chloroform indicating their enveloped nature. Pretreatment of chicken embryo fibroblast cultures with 1 OO~g! ml of luDR did not inhibit the multiplication of any of the isolates indicating they all had RNA genomes. All the isolates were resistant to treatment with brine indicating that they were capable of survlvinp at high salt concentration.

The isolates T18 and Fa produced marked CPE in chicken embryo fibroblast culture with rounding and clumping of cells and syncytia formation. Marked cytoplasmic vacuolation was also observed. Inclusion bodies could not be detected either in nucleus or cytoplasm. For isolates T22 and F12, CPE developed later only and was not as prominent as for T18 and Fa. Rounding of cells and their fusion forming syncytia was noticed by 72 h. Cytoplasmic vacuolation though present was much less marked. Inclusion bodies were absent. Large polykaryocytes were produced by the isolates T18, T 22 and F12 in BHK-21 cell line within 24h after inoculation. Between 48-72h large syncytia were formed. Intracytoplasmic inclusions could be observed by 24h after infection, which were quite prominent by 96h. The isolate Fa failed to produce any CPE in BHK-21 cell line. Pathogenecity tests in day old and six-week-old chicken and ducklings showed that all the four isolates were non-pathogenic when given by the oral Isubcutaneous route or by both. Neither clinical signs or mortality could be observed in the birds. Virus isolation was possible from the cloacal swabs of six-week-old chicken for the isolates T18 and T22 up to the 14th and th day respectively. Antigenic relationship between the isolates was tested by gel diffusion and haemagglutination inhibition tests, which showed that the isolate T18 did not have any similarity with any of the other three isolates. The isolate T 22 showed antigenic similarity by both the tests. Fa showed similarity to T18 by HI test but not by immunodiffusion test.

Isolate F12 was found to be distinct from the other three by HI test, but showed some similarity with them by immunodiffusion test. By sodium dodecyl sulphate polyacrylamide gel electrophoresis on 10 percent gels, 7-11 bands could be resolved for the different isolates. Three of the bands were common for all the four isolates and were having molecular weights similar to the three major proteins HN, NP and MP of avian paramyxoviruses, suggesting that the isolates belonged to the paramyxovirus group. Monoclonal antibody typing of the isolates T18 and T 22 at the Central Veterinary Laboratory, Surrey, England confirmed that both belonged to the paramyxovirus group with T18 belonging to group C (velogenic) and T 22 to group E (B1 and LaSota) viruses. The isolates Fe and F12 need to be further typed. It was concluded from the study that all the isolates were enveloped RNA viruses with T18 and T 22 being paramyxoviruses belonging to Group I. The properties of the isolates Fs and F12 resembled the paramyxoviruses and from the similarly in protein profile with the other two viruses can also be concluded to be paramyxoviruses.