Characterisation of fowl adenoviruses from chickens affected with infectious hydropericardium during 1994-1998 in India.

In the present study characterisation has been done for six group I fowl adenoviruses (FAV) isolated from outbreaks of infectious hydropericardium (IHP) of chickens that occurred in different states/regions of India during the years 1994-98. These six viruses were identified as FAV serotype 4 by virus neutralisation and restriction endonuclease analyses. Antigenic analyses of the viruses revealed close relationship (R-values 0.93-0.96). Under the experimental conditions, we have been able to induce IHP using FAV serotype 4 isolate AD: 411 and were also able detect FAV antigens in myocardial tissues by immunofluorescence assay (a new observation), an indication that IHP causing FAV serotype 4 strain replicate in myocardial tissue. Restriction endonuclease analysis of the viral genomes (approximately 46 Kb), using Hind III, Sma I, Xba I, Bam HI, Pst I and Dra I produced identical genetic profiles. Pst I and Bam HI profiles for these six vitus isolates were identical to those published earlier for an IHP causing Pakistani FAV serotype 4 isolate KR31. The identical genetic profiles of viruses, chronology of the outbreaks of IHP in Pakistan during 1989 onward and later in Jammu and Kashmir, India (1994), suggest that FAV serotype 4 isolates involved in outbreaks of IHP in India had probably spread from Pakistan. In order to prevent further spread and economic losses due to IHP in India, based on the antigenic relatedness data in this paper, any one of the six studied FAV serotype 4 isolates can be used as a candidate for mass production of CEH culture based killed vaccine.

Purification and differentiation of three avian adenoviruses by restriction endonuclease analysis.

A rapid method of ultracentrifugation pelleting of avian adenovirus (AAV) from small volume of chloroform treated infected cell culture fluid or allantoic fluid was adapted for isolation of adenoviral DNA. The viral DNA extracted from semipurified viruses was found to be intact on agarose gel and pure enough (A260/280 = 1.85-1.92) for restriction enzyme analysis. Restriction endonuclease analysis of Indian strain of AAV serotype 1, AAV serotype 4 (group I AAVs) and egg drop syndrome-76 (EDS-76) virus genomes (group III AAV) with Hind III enzyme differentiated these viruses. The AAV serotype 1 and serotype 4 strain exhibited identical Hind III profile to European viral strains belonging to same serotypes however, the EDS-76 virus gave similar but not identical profile. The calculated genomic lengths for AAV serotype 1 and EDS-76 virus were approximately found to be 33.9 and 44.4 Kb, respectively.

Monoclonal antibody based dot-enzyme linked immunosorbent assay (Dot-ELISA) and agar gel precipitation test (AGPT) for identification of Newcastle disease virus (NDV).

Two mouse monoclonal antibodies (MAbs), viz. 2B7 and 2 D10 raised against haemagglutinin-neuraminidase glycoprotein of Newcastle disease virus (NDV) were used to identify several other field isolates and vaccine strains of NDV. These MAbs reacted specifically with all the NDV strains/isolates in Dot-ELISA whereas, only MAb 2D10 reacted with all the NDV strains/isolates in agar gel precipitation test. These two tests employing the MAbs were standardised for rapid diagnosis and identification of NDV.

Immunogenic activity of viral polypeptides of an Indian isolate of infectious bursal disease virus.

An Indian isolate of infectious bursal disease virus, i.e. IBDV-P/AD/81, was analysed for immunogenic activity of its structural polypeptides. Virus was purified from infected bursal homogenate by sucrose density gradient centrifugation. It showed five different structural polypeptides of 75.8, 45, 40.7, 33.1 and 27 kDa molecular weights in sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Anti infectious bursal disease virus (IBDV) antibodies were tested using enzyme-linked immunosorbent assay (ELISA) and neutralization test (NT). Polypeptide 40.7 kDa (VP2) was known to have neutralizing epitopes. However, polyclonal anti VP2 failed to neutralize the virus. It was interpreted that VP2 had labile neutralizing epitopes which get altered confirmationally by SDS. Surprisingly, polyclonal anti 33.1 kDa (VP3) had mild neutralizing activity.

Ultrastructural studies on interaction of infectious bursal disease virus (IBDV) and aflatoxin B1 on chick embryo fibroblast cell culture.

Light and electron microscopic evaluation of chick embryo fibroblast (CEF) cell culture inoculated with graded doses (0.25, 2.5 and 25 micrograms/ml medium) of aflatoxin B1 with and without infectious bursal disease virus (IBDV) was undertaken. The light microscopy revealed degeneration, detachment and necrosis of fibroblasts and multiple plaques formation in IBDV infected group without and with (0.25, 2.5 micrograms) aflatoxin B1. The cultures infected with virus, with or without 25 micrograms aflatoxin B1 showed complete detachment from glass surface. Electron microscopy of these cultures showed marked pyknotic or bizarre shaped nuclei, pronounced degenerative changes in the rough endoplasmic reticulum (RER), mitochondria and the presence of multiple vacuoles in the cytoplasm. The viruses were spherical, arrayed, complete, generally closer to nuclei and RER and indistinctly membrane bound. The viruses were either localised or scattered in the cytoplasm. Cultures containing 25 micrograms aflatoxin B1 without or infected with virus showed marked necrosis of cells. In latter group only a few viruses were seen either in infected cells or free in culture. Control cultures failed to show cytopathic changes as observed in the other three groups.