Éva Nagy

Sequence analysis of infectious pancreatic necrosis virus genome segment B and its encoded VP1 protein: A putative RNA-dependent RNA polymerase lacking the Gly-Asp-Asp motif

Abstract The genome segment B sequence of infectious pancreatic necrosis virus was determined for both the Jasper and Sp serotypes. The sequences are 2784 and 2630 by long, respectively, and contain a single large open reading frame encoding the VP1 protein, the putative RNA-dependent RNA polymerase (RdRp) of IPNV. The proteins exhibit an 88% homology with each other, but only 41% with infectious bursal disease virus (IBDV) VP1, another member of the Birnaviridae. Despite the low overall homology between the IPNV and IBDV VP1 proteins, homologous regions were detected within the central portion of the proteins. The carboxy-proximal regions of the VP1, which contain very low amino acid homology, displayed evidence of conservation in structural features such as a hydrophilic, highly basic domain. Consensus sequences associated with GTP-binding proteins and RdRps were also detected in VP1. However, unlike the RdRps associated with single-stranded plus RNA viruses, the birnavirus RdRp lacks the Gly-Asp-Asp motif characteristic of this enzyme family.

Phylogenetic analysis reveals extensive evolution of avian paramyxovirus type 1 strains of pigeons (Columba livia) and suggests multiple species transmission

Partial sequence and residue substitution analyses of the fusion protein gene were performed for 68 strains of avian paramyxovirus type 1 of pigeons (PPMV-1), an antigenic variant of Newcastle disease virus (NDV) of chickens, derived from 16 countries between 1978 and 2002. The majority of isolates clustered into a single genetic lineage, termed VIb/1, within genotype VI of NDV strains of chickens, whereas a small number of isolates that originated in Croatia after 1995, grouped in a highly diverged lineage, termed VIb/2, indicating a separate host-switching event from that of VIb/1 strains. Four distinct subgroups of lineage VIb/1, Iraqi (IQ), early European (EU/ea), North American (NA) and recent European (EU/re) have emerged and circulated in the past decades. Subgroup EU/ea and NA strains were responsible for the main streams of infection in the 1980s, while EU/re viruses for infections in the 1990s. The degree of genetic diversity of viruses in the early phase of the epizootic suggested a prolonged infection period of the pigeon-type viruses prior to the emergence of the disease in the early 1980s. Shared derived character analysis showed a close genetic relationship to Sudanese viruses from the mid-1970, suggesting that PPMV-1 viruses could be of African origin.

The complete nucleotide sequence of fowl adenovirus type 8.

The fowl adenovirus type 8 (FAdV-8) genome was sequenced and found to be 45063 nucleotides in length, the longest adenovirus (AdV) genome for which the complete nucleotide sequence has been determined so far. No regions homologous to early regions 1, 3 and 4 (E1, E3 and E4) of mastadenoviruses were recognized. Gene homologues for early region 2 (E2) proteins, intermediate protein IVa2 and late proteins were found by their similarities to protein sequences from other AdVs. However, sequences homologous to intermediate protein IX and late protein V could not be identified. Sequences for virus-associated RNA could also not be recognized. Two regions of repeated sequences were found on the FAdV-8 genome. The shorter repeat region contained five identical and contiguous direct repeats that were each 33 bp long, while the longer repeat region was made of 13 identical and contiguous, 135 bp long repeated subunits.

Peripheral neuritis in psittacine birds with proventricular dilatation disease

Necropsies were performed on 14 psittacine birds of various species suspected to have proventricular dilatation disease (PDD). Eight of the birds exhibited neurological signs (seizures, ataxia, tremors and uncoordinated movements) and digestive tract signs (crop stasis, regurgitation, inappetance and presence of undigested food in the faeces). At necropsy, the birds had pectoral muscle atrophy, proventricular and ventricular distention, thinning of the gizzard wall, and duodenal dilation. In addition, five birds had a transparent fluid (0.2 to 1.0 ml) in the subarachnoidal space of the brain, and one bird had dilatation of the right ventricle of the heart. The histological lesions differed from earlier reports of PDD in that peripheral (sciatic, brachial and vagal) neuritis was seen in addition to myenteric ganglioneuritis, myocarditis, adrenalitis, myelitis and encephalitis.

Pathogenicity and complete genome sequence of a fowl adenovirus serotype 8 isolate.

In this study we determined and analyzed the complete nucleotide sequence of the genome of a fowl adenovirus serotype 8 (FAdV-8) isolate and examined its pathogenicity in chickens. The full genome of FAdV-8 was 44,055 nucleotides in length with a similar organization to that of FAdV-1 and FAdV-9 genomes. No regions homologous to early regions E1, E3 and E4 of mastadenoviruses were recognized. Along with FAdV-9, FAdV-8 has only one fiber gene and with regard to sequence composition and genome organization, FAdV-8 is closer to FAdV-9 than to FAdV-1. Moreover, our findings suggest that FAdV-1 of species Fowl adenovirus A as the current type species despite its historical priority is not representative of the genus Aviadenovirus, and that FAdV-8 or FAdV-9 in species Fowl adenovirus E and Fowl adenovirus D, respectively, would be more suitable for that designation. Additionally, pathogenicity of FAdV-8 was studied in specific pathogen free chickens following oral and intramuscular inoculations. Despite lack of clinical signs and pathological changes virus was found in tissues and cloacal swabs of all birds with the highest viral copy numbers present in the cecal tonsils. The highest virus titers in the feces for orally and intramuscularly inoculated chickens were recorded at days 10 and 3 post-infection, respectively.

Coding potential and transcript analysis of fowl adenovirus 4: insight into upstream ORFs as common sequence features in adenoviral transcripts.

Recombinant fowl adenoviruses (FAdVs) have been successfully used as veterinary vaccine vectors. However, insufficient definitions of the protein-coding and non-coding regions and an incomplete understanding of virus-host interactions limit the progress of next-generation vectors. FAdVs are known to cause several diseases of poultry. Certain isolates of species FAdV-C are the aetiological agent of inclusion body hepatitis/hydropericardium syndrome (IBH/HPS). In this study, we report the complete 45667 bp genome sequence of FAdV-4 of species FAdV-C. Assessment of the protein-coding potential of FAdV-4 was carried out with the Bio-Dictionary-based Gene Finder together with an evaluation of sequence conservation among species FAdV-A and FAdV-D. On this basis, 46 potentially protein-coding ORFs were identified. Of these, 33 and 13 ORFs were assigned high and low protein-coding potential, respectively. Homologues of the ancestral adenoviral genes were, with few exceptions, assigned high protein-coding potential. ORFs that were unique to the FAdVs were differentiated into high and low protein-coding potential groups. Notable putative genes with high protein-coding capacity included the previously unreported fiber 1, hypothetical 10.3K and hypothetical 10.5K genes. Transcript analysis revealed that several of the small ORFs less than 300 nt in length that were assigned low coding potential contributed to upstream ORFs (uORFs) in important mRNAs, including the ORF22 mRNA. Subsequent analysis of the previously reported transcripts of FAdV-1, FAdV-9, human adenovirus 2 and bovine adenovirus 3 identified widespread uORFs in AdV mRNAs that have the potential to act as important translational regulatory elements.

Growth characteristics of fowl adenovirus type 8 in a chicken hepatoma cell line

Fowl adenoviruses, many of which appear to be non-pathogenic, are ubiquitous in birds. In addition, the genome of these viruses is large, making them ideal candidates for construction as vectors for foreign genes. Current methods to cultivate fowl adenoviruses use primary cell cultures derived from embryonated chicken eggs. In order to provide a more suitable culture method, the growth of fowl adenovirus type 8 (FAdV-8) was investigated in CH-SAH, a continuous hepatoma cell line. A one step growth curve demonstrated release of extracellular virus beginning by 18 h p.i. and with a final yield about 100 fold higher than that in chicken embryo liver cells. Viral DNA synthesis was first detected 8 h prior to this. The CH-SAH cell line supported the production of progeny viruses similar to the wild-type virus after being transfected with purified FAdV-8 DNA. This study demonstrated that the continuous hepatoma cell line is an appropriate in vitro host for FAdV-8.

Prophylactic treatment with Toll-like receptor ligands enhances host immunity to avian influenza virus in chickens.

Avian influenza viruses (AIV) pose a threat towards the health of both poultry and humans. To interrupt the transmission of the virus, novel prophylactic strategies must be considered which may reduce the shedding of AIV. One potential is the prophylactic use of Toll-like receptor (TLR) ligands. Many cells of the immune system express TLRs, and cellular responses to TLR stimulation include activation and the production of cytokines. TLR ligands have been employed as prophylactic treatments to enhance host resistance to pathogens both in mammals and chickens. Therefore, the present study was conducted to determine whether TLR ligands may be used prophylactically in chickens to enhance host immunity to AIV. Chickens received intramuscular injections of either low or high doses of the TLR ligands poly I:C, lipopolysaccharide (LPS) and CpG ODN. Twenty-four hours post-treatment, chickens were infected with the low pathogenic avian influenza virus H4N6, and both oropharyngeal and cloacal virus shedding were assessed on days 4 and 7 post-infection. To identify potential correlates of immunity, spleen and lungs were collected on days 2, 4 and 7 post-infection for RNA extraction. The results suggested that all of the TLR ligand treatments induced a significant reduction in virus shedding, with the TLR3 ligand poly I:C conferring the greatest AIV immunity compared to control birds, followed by CpG ODN and LPS. Furthermore, transcriptional analysis of gene expression in the spleen and lungs suggest IFN-α and IL-8 as correlates of immunity conferred by poly I:C, and IFN-γ for CpG ODN and LPS. In conclusion, TLR ligands, have the ability to enhance host immunity against AIV, and future studies should consider exploring the combinatory effects of poly I:C and CpG ODN prophylaxis in conjunction with AIV vaccination.

Antibody response and virus tissue distribution in chickens inoculated with wild-type and recombinant fowl adenoviruses

We demonstrated that the long tandemly repeated region (TR-2) is dispensable for in vitro replication of fowl adenovirus 9 (FAdV-9). The TR-2-deleted recombinant FAdV-9 expressing the enhanced green fluorescence protein was further characterized for in vivo effects. Groups of chickens were exposed to recombinant or wild-type FAdV-9 by intramuscular injection, through the feed or drinking water and one group served as a negative control. The antibody (Ab) response, evaluated by ELISA and a plaque reduction test depended on the virus, dosage and the route of inoculation. Although the highest levels of anti-viral Ab were detected in chickens inoculated intramuscularly (i.m.) with wild-type FAdV-9, the deletion of TR-2 did not have a significant effect on the immune response. The tissue distribution of the virus was examined by the polymerase chain reaction (PCR) and was similar for both wild-type and recombinant viruses. Based on these results the TR-2 was dispensable for viral replication in vivo and did not influence virus distribution, and the recombinant FAdV-9 induced the same immune response as the wild-type virus.

Avian Influenza Virus H13 Circulating in Ring-Billed Gulls (Larus delawarensis) in Southern Ontario, Canada

Abstract Avian influenza virus (AIV) was studied in ring-billed gulls (Larus delawarensis) in one breeding colony on Lake Erie in 2000, and two on Lake Ontario in both 2000 and 2004. Antibodies to H13 AIV were detected in 92% of adults in 2000 and 82% in 2004. Antibody prevalence in 3-wk-old chicks was 5%–30% (overall 15%) in 2000 and 21% and 76% (overall 48%) in 2004. In 5-wk-old chicks, antibody prevalence was 23%–75% (overall 53%) in 2000 and 53% and 79% (overall 66%) in 2004. Geometric mean antibody titers at 3 and 5 wk did not differ in 2000, but increased significantly at one colony in 2004. In 2000, overall prevalence of AIV isolation from cloaca in embryonated chicken eggs was 32% (3 wk old), 13% (5 wk old), and 0 (adults), but AIV was also isolated from kidney and lung in a high proportion of tissues cultured from 3-wk-old birds in one colony. Isolates from cloaca were characterized as subtype H13 by serology; all 15 tested for neuraminidase were H13N6. However, three AIV detections considered on the basis of nucleotide sequence to be subtype H16 were among the 28 detected retrospectively by PCR in archived cloacal swabs; the remainder were subtype H13. Outcome of virus isolation was not related to presence of antibody titers in chicks. The presence of antibody to AIV in chicks was associated significantly with inflammation in heart, kidney, pancreas, and liver. AIV was not isolated in 2004. AIV infected chicks annually within the first 3 wk of life, ultimately infecting the majority of birds in most colonies, but did not appear to cause clinical disease.