Studies on the pathogenicity of enterovirus-like viruses in chickens.

FP3 and 612 viruses are enterovirus-like viruses. Antibody to these viruses is widespread in chicken flocks, but nothing is known about their pathogenicity. Seven experiments were carried out to investigate the tissue tropism and associated pathology of these novel fowl enterovirus-like viruses and to compare these with the effects of the previously studied enterovirus-like viruses, ELV-1 and avian nephritis (ANV). ANV is now classified as an astrovirus. Preliminary experiments were carried out with FP3 virus, 612 virus and ELV-1 to determine the distribution of viral antigen. Each preliminary experiment was followed by a larger experiment that included more birds and in which a greater range of tissues was studied. It was shown that all four viruses studied replicated in the intestine and had differing abilities to spread to other tissues. Histological changes were present in most antigen-positive tissues but they were usually relatively mild. ELV-1 was associated with the most severe intestinal lesions, followed by FP3 virus. FP3 virus produced lesions in the kidney that were marginally more severe than those caused by the G-4260 strain of ANV. FP3 virus also caused pancreatic lesions. The 612 virus was found to be only mildly pathogenic in specific pathogen free chickens.

Effect of multiple cell culture passages on the biological behaviour of chicken anaemia virus.

The Cux-1 isolate of chicken anaemia virus (CAV) was passaged over 320 times in Marek's disease virus transformed chicken lymphoblastoid (MDCC-MSB1) cells. Comparison of the infectivity titres of virus pools derived from viruses that had received 0 (P0), 49 (P49), 170 (P170) and 320 (P320) passages in our laboratory indicated that the yields of infectious virus increased over 100-fold with passage number from P0 to P170. P320 exhibited unusual cell culture growth characteristics in that, unlike its lower passage counterparts, virus-specific immunofluorescence (IF) and cytopathic effect were detected at very low levels at 2 days post infection, with an additional passage of infected cells into fresh medium being required to produce high levels of infectious virus. Experimental infection of 1-day-old SPF chicks showed that P170 and P320 were substantially attenuated compared to P49 and a pathogenic, low-passage isolate used as control. When assessed by the indirect IF test, infection of 1-day-old chicks with the P49 and P170 isolates elicited similar levels of CAV-specific antibody to those elicited by infection with the pathogenic, low-passage virus and higher than those elicited by infection with the P320 isolate. Experimental infection of 5-week-old chicks indicated that the attenuated P170 and P320 isolates invoked similar CAV-specific antibody levels to those invoked by the pathogenic P49 and control isolates.

Molecular cloning of an attenuated chicken anaemia virus isolate following repeated cell culture passage.

The pathogenicity of the Cux-1 isolate of chicken anaemia virus (CAV) was substantially reduced following large numbers (50 to 173) of passages in MDCC-MSBl cells. Restriction endonuclease analysis of polymerase chain reaction (PCR)- amplified DNAs and recombinant plasmids containing DNA inserts specified by CAV that had been passaged 173 times, indicated that the population of high-passage virus was genetically diverse. A 210-base pair (bp) insertion, containing a 19-bp sequence identical to four repeated sequences that are located in the putative non-coding region of the genome was shown to have become established in the virus population by passage number 30. Individual virus isolates that were selected from the high-passage virus population using recombinant DNA cloning and transfection methodologies varied in their pathogenicities. One cloned virus isolate, designated number 10, produced virtually no anaemia and substantially reduced levels of aplasia of the bone marrow and thymus atrophy. The pathogenicity of this isolate was restored following 10 passages in young chicks.

Studies on a new enterovirus-like virus isolated from chickens.

During investigations into an outbreak of respiratory distress in broilers chicks, a small round virus was isolated following inoculation into chicken embryos. The isolate, designated 612, was identified as an enterovirus-like virus on the basis of its size and morphology, resistance to chloroform and to treatment at pH 3.0, and intracytoplasmic replication in cell culture. The virus produced a partial cytopathic effect following inoculation into chick embryo kidney cell cultures and viral antigens could be detected by immunostaining. The preferred culture method for 612 virus was by inoculation onto the CAM of chick embryos. Cross-immunofluorescence indicated that the virus is not antigenically related to five previously identified chicken enterovirus/enterovirus-like virus serogroups. Following experimental inoculation of 1-day-old male broilers a number of which had maternal antibody to 612, growth retardation ranging from 9.6 to 20.4% was detected. Serological studies demonstrated antibody to 612 virus was widespread in commercial chicken flocks in N. Ireland.

Development of a microtitre fluorescent antibody test for serological detection of adenovirus infection in birds.

The development of a microtitre fluorescent antibody test, for detection of adenovirus antibody is described. Chick embryo liver cells, infected with fowl adenovirus were found to be more suitable for the test than chick kidney, since a proportion of the chick kidney cells were found to contain non-viral cytoplasmic inclusions which were easily confused with specific viral fluorescence at the low power magnifications used in the test. It was demonstrated that a single positive serum would stain cultures infected with 11 different serotypes of fowl adenovirus, and two serotypes of turkey adenovirus with only minor differences in titre, thus confirming the presence of a common antigen in fowl and turkey adenoviruses. In a survey for adenovirus antibody a total of 595 avian sera (453 fowl and 142 turkey) were screened by double immuno-diffusion and microtitre fluorescence. A total of 52.6% of the sera was positive by double immuno-diffusion while 70.8% were positive by microtitre fluorescence.

Demonstration of anti-nuclear antibodies in fowl sera by immunofluorescence.

The occurrence of anti-nuclear antibodies in fowl sera is described. The anti-nuclear activity was demonstrated by immunofluorescence in a small percentage (3.9%) of the sera tested. The nuclei of cultured fowl and turkey cells were stained by anti-nuclear serum whereas the nuclei of several mammalian tissues were not. Staining of chicken kidney cell nuclei by anti-nuclear serum conjugated with fluorescein isothiocyanate was blocked by unconjugated anti-nuclear serum but not by serum without anti-nuclear activity indicating the specificity of the reaction. The possible confusion between anti-nuclear staining and specific viral staining is discussed, along with the possible association of anti-nuclear factors with disease in birds.