Polymerase chain reaction combined with restriction enzyme analysis for detection and differentiation of fowl adenoviruses.

A polymerase chain reaction combined with restriction enzyme analysis was developed for detection and differentiation of all 12 fowl adenovirus (FAdV) serotypes representing the five fowl adenovirus (A to E) species. For primer design, the published sequences of the hexon proteins of FAdV1, FAdV8 and FAdV9 were aligned and conserved regions in the two pedestal regions adjacent to the L1 loop region were determined. A primer pair (hexon A/hexon B) was constructed and was shown to amplify approximately 900 bp of the hexon gene including the L1 loop region. An amplification product was detected using supernatant of infected cell cultures from all FAdV1 to FAdV12 reference strains used in our study. The sequence and the restriction patterns of the hexon A/B fragments of the 12 FAdV strains were determined and compared. The successive use of four different endonucleases allowed the complete differentiation of the reference FAdV strains. Twenty-six fowl adenoviruses isolated during our routine virological diagnosis activities could all be amplified using hexon A/hexon B primers. Restriction analysis results showed that 8/26 adenovirus strains contained two different FAdV types. FAdV4, FAdV12, FAdV1, FAdV5 and FAdV6 were the most frequently isolated.

Epidemiology of infectious bronchitis virus in Belgian broilers: a retrospective study, 1986 to 1995.

Infectious bronchitis virus (IBV) was isolated from each of 236 broiler flocks that had respiratory infection (86%), impaired growth, enteritis and/or nephritis (14%), over a 10-year period from 1986 to 1995 in Belgium. Among them, 65% of the investigated flocks had not been vaccinated against infectious bronchitis. Type-specific reverse transcriptase polymerase chain reactions (RT-PCRs) were used after propagation of the isolates in embryonated eggs in order to detect and differentiate Massachusetts, D274, B1648 and 793/B types. The incidence of these types was approximately 50, 38, 11 and 1%, respectively. In 16% of cases, two or three types of IBV were detected, representing mostly combinations of Massachusetts and D274. The majority of the Massachusetts and D274 isolates (68 and 69%, respectively) were recovered from non-vaccinated flocks, confirming that such flocks are at greatest risk of infection by these types of IBV. Interestingly, the B1648 type was isolated from more vaccinated flocks (14%) than non-vaccinated flocks (7.6%). Most surprising was the very low incidence (1%) of the 793/B type, which was the dominant type in some neighbouring countries, during the period of investigation. The DNA derived by RT-PCR from 24 of the Massachusetts-type isolates from 12 vaccinated and 12 non-vaccinated flocks was sequenced and compared with the sequence of Massachusetts vaccines used in Belgium. This revealed that the sequence of four of the isolates (two from vaccinated and two from non-vaccinated flocks) was identical to that of a Massachusetts vaccine strain. Similar results were obtained for D274 isolates when compared with the sequence of D274 vaccines. These sequencing results demonstrate a co-circulation of vaccine and wild-type infectious bronchitis viruses in broilers, and are further justification for permanent monitoring of circulating strains in order to rationally modify vaccination strategies to make them appropriate to the field situation.

Differential effect of ultraviolet light on the induction of simian virus 40 and a cellular mutator phenotype in transformed mammalian cells.

UV irradiation of simian virus 40 (SV40)-transformed human and hamster cells induced them both to express a mutator phenotype and to produce SV40. The mutator could also be activated indirectly by transfecting unirradiated cells with UV-damaged calf thymus DNA. In contrast, UV-damaged exogenous DNA failed to rescue SV40 from unirradiated transformed cells. These results suggest that the expression of transforming viruses and of cellular mutator functions is regulated by at least partially independent mechanisms. Unlike the activation of a cellular mutator phenotype, the rescue of SV40 from virus-transformed mammalian cells by UV light might require that the integrated viral DNA and/or specific cellular sequences are directly damaged.

Acute pancreatitis in chickens due to non-virulent Newcastle disease virus.

A non-virulent Newcastle disease virus (strain APMV-1 96/89 VB) was isolated from a broiler chicken from a backyard flock. Using monoclonal antibodies, the virus was shown to be different from the vaccinal virus strains Hitchner, La Sota and Ulster. The virus was shown to replicate in the pancreas of one-day-old specific pathogen-free chickens infected orally, and the histological lesions observed in the pancreas of chickens inoculated with the fourth chicken passage of the virus five to nine days after infection were consistent with an acute pancreatitis.

Antigenic relationships between fowl enteroviruses.

An antigenical cross-relationship was observed by immunofluorescence and seroneutralisation tests between the G-4260 strain of avian nephritis virus and three other entero-like particles, one isolated by McNulty et al. (Avian Pathology, 13: 429) and the entero PV2 and entero 3 both isolated in our laboratory. No cross reactions were observed between those viruses and the avian encephalomyelitis virus.

An ELISA for the detection of antibodies to avian nephritis virus and related entero-like viruses.

An ELISA for the detection of antibodies against avian nephritis virus (ANV) and related entero-like viruses was developed. Different antigenic preparations made from chicken kidney cells infected with the G-4260 strain of ANV were compared. Crude antigen obtained by fluorocarbon treatment of infected cells was found to be appropriate and to give reproducible results with antisera directed against ANV and three entero-like particles (ELPs): the Belgian entero PV2 and entero 3, and the Irish ELP-1. A cut-off value was determined using 30 specific pathogen-free (SPF) sera and the absence of antigenic relationships with nine different reference sera directed against avian viruses was demonstrated. The cross antigenic relationships between avian encephalomyelitis virus (AEV), ANV and the three ELPs was investigated by ELISA and confirms the classification of those fowl enteroviruses into two serotypes: the first including ANV and ELPs and the second including AEV. A strong correlation (94%) was demonstrated between ELISA and a seroneutralisation test. Using ELISA, antibodies against ANV and related ELPs were demonstrated in 13/14 breeding and 8/10 broiler flocks from Belgium.

Epidemiological study of enteric viruses in broiler chickens: comparison of tissue culture and direct electron microscopy.

The intestinal and caecal contents of chickens from 102 broiler flocks affected by enteric and associated problems were analysed. The second week of life was found to be the most important in the onset of clinical signs of malabsorption shown by the presence of uneven flocks, growth retardation and enteritis problems, but one-week-old flocks frequently presented similar problems. Some cases of feather aberration were observed, mainly in flocks of two weeks of age. From the third week, enteric problems were less acute. Viral particles were found in 67% of the samples by examination by electron microscope and in 52% by cell culture isolation. By complementing the two methods the viral recovery was increased to 85% of the samples. Four virus types were identified: reovirus, entero-like virus, rotavirus and adenovirus. Entero-like virus was mainly found in the first two weeks of life, whilst reovirus and rotavirus were principally found from the second week on. Adenovirus was found infrequently but this may have been a reflection of the mean age of the affected flocks which was only 12 days.

Significance of parvoviruses, entero-like viruses and reoviruses in the aetiology of the chicken malabsorption syndrome.

Specific-pathogen-free White Leghorn chickens and commercial broilers were inoculated orally at 1 day of age with different intestinal preparations containing a chicken parvovirus, an entero-like virus associated with a reovirus from field materials, or the entero-like viruses and reovirus alone. Despite viral multiplication in inoculated birds, no clinical signs or growth retardation were observed in SPF and broiler chickens infected with the reo or parvoviruses. Abnormal faeces and reduction in weight gains were observed after infection with the field materials and the entero-like viruses. Some easily sedimentable particles could be involved with the entero-like virus in the aetiology of runting syndrome. Proventriculitis was present in chickens inoculated with one of the field materials and with the entero-like virus isolated from that material. Specific-pathogen-free White Leghorn chickens were as susceptible as commercial broiler chickens to weight gain depression after oral inoculation with crude homogenates at 1 day of age.

Pathogenicity of fowl enteroviruses.

The pathogenicity of avian nephritis virus, entero-like particles described by McNulty et al. (Avian Pathology, 13: 429), the entero PV2 and entero 3 isolated in our laboratory, was studied by oral inoculation of one-day-old specific pathogen-free chickens. All viruses were shown by immunofluorescence and transmission electron microscopy to multiply in the cytoplasm of enterocytes but histological lesions of the intestine were only observed in chickens infected with McNulty's entero-like particles, entero PV2 and entero 3. Those lesions were present from 3 days post inoculation but were most prominent on the 7th day post inoculation. Variable histological lesions of the pancreas, proventriculus or kidneys were observed 14 days post inoculation with McNulty's entero-like particles, entero PV2 or entero 3. Avian nephritis virus principally induced kidney lesions. This demonstrates that members of a same species of fowl enteroviruses may have different tropisms and could induce different clinical signs and pathology as nephritis or malabsorption syndrome.

Evolution of pigeon Newcastle disease virus strains.

Twenty-seven Newcastle disease virus isolates obtained during the years 1998 and 1999 from racing pigeons were shown to be antigenically indistinguishable from the pigeon paramyxovirus type 1 (PPMV-1) viruses isolated in the years 1983 and 1984. Partial sequencing of 240 base pairs of the F gene demonstrated at least 94.7% identity at the nucleotide level between isolates from 1983 and 1984, and more recent viruses isolated in 1998 and 1999. Most of the nucleotide changes observed were silent mutations as only six amino acid changes were observed. Three amino acid substitutions were observed in the F2/F1 cleavage site. The sequence of the F2/F1 cleavage site of all isolates was typical for pathogenic paramyxovirus 1 viruses. Amino acids at the F2/F1 cleavage site changed from (112)GRQKRF(117) to (112)RRQKRF(117), (112)RRKKRF(117) or (112)RRRKRF(117). The motif (112)RRQKRF(117) was present in the majority of the isolates but the intracerebral pathogenicity indexes of PPMV-1 isolates having this motif was highly variable but largely lower (mean, 0.69) than that reported for PPMV-1 viruses isolated in the years 1983 and 1984 (mean, 1.44).