Eighteen isolates of infectious bronchitis virus (IBV) from

Article summary line: Phylogenetic and epidemiologic evidence shows incursion of HPAIV into the food chain.

Broiler chickens with clinical signs of uneven growth, depression, and dull feathers were submitted to our laboratory and, at necropsy, lesions in proventriculus, gizzard, and intestines were detected. Fowl adenovirus serotype 1 (FAdV-1) was isolated from digestive tissues. The virus, assigned as FAdV-PL/G068/08, showed 99.5% nucleotide homology and 99.2% amino acid homology in hexon gene with chicken embryo lethal orphan (CELO) strain classified as the European reference of FAdV-1. One-day-old and 21-d-old SPF chickens were inoculated with FAdV-PL/068/08 by both nasal and ocular routes and then observed daily and examined by necropsy at 6, 10, and 14 d postinoculation. Experimental infection with isolated virus was fatal for younger chickens and major lesions occurred in the gizzards. No clinical or pathological changes were observed in chickens infected at 21 d of age, but the presence of intranuclear inclusion bodies in gizzard epithelial cells was detected. Molecular characterization was based on the long and short fibers genes sequencing and comparison of obtained sequences with other FAdV-1 strains. The homology between FAdV-PL/G068/08 and other sequences available in GenBank was between 98.9 and 99.8% (short fiber region) and 99.0 and 99.7% (long fiber region) at nucleotide level and between 98.4 and 100% (short fiber region) and 99.3 and 99.9% (long fiber region) at amino acid level. No correlation between identified amino acid changes in short and long fiber proteins and pathogenicity of studied FAdV-1 strains was observed. Although short and long proteins were indicated as factors influencing virus pathogenicity, the role of identified sequence differences in infectivity determination remain unclear.

Chicken astrovirus (CAstV) was recently indicated as the factor of the "white chicks" condition associated not only with increased embryo/chick mortality but also with weakness and white plumage of hatched chicks. In February 2014, organ samples (livers and kidneys) from dead-in-shell embryos, as well as 1-day-old whitish and normal chicks, were delivered from one hatchery in Poland for disease diagnosis. The samples originated from the same 30-week-old breeder flock in which the only observed abnormal signs were 4-5% decrease in the number of hatched chickens and the presence (about 1%) of weaker chicks with characteristic whitish plumage among normal ones. CAstV was detected in submitted samples and was then isolated in 10-day-old embryonated specific pathogen free (SPF) chicken eggs. We also reproduced an infection model for the "white chicks" condition in SPF layer chickens using the isolated PL/G059/2014 strain as the infectious agent. Results of experimental reproduction of the "white chicks" condition were somewhat more serious than field observation. The administration of the CAstV material into the yolk sac of 8-day-old SPF chicken eggs caused delay and prolongation of hatching, as well as death of embryos/chicks, and also a change of plumage pigmentation. Only two chicks of a total of 10 inoculated SPF eggs survived and were observed for 2 months. A gradual elimination of the CAstV genome was noted in this period. Moreover, a few contact-naive SPF chicks, which had been placed in the same cage, were infected with CAstV. Molecular characterization of detected CAstV was performed by nucleotide sequencing of the full ORF2 region encoding the capsid precursor protein gene. Phylogenetic studies showed that the PL/G059/2014 isolate clustered in the subgroup Aiii of CAstV. In the light of the new classification rules, the Polish PL/G059/2014 CAstV isolate could be assigned to a new species of the Avastrovirus genus.

Persistence of H5N1 high pathogenicity avian influenza virus (HPAIV), isolated during the epidemic in wild birds in Poland in 2006, was evaluated in three water samples derived from the sources known to host wild water birds (city pond, Vistula river mouth, and Baltic Sea). The virus was tested at two concentrations (10(4) and 10(6) median tissue culture infective dose per milliliter) and at three temperatures (4 C, 10 C, and 20 C), representing average seasonal temperatures in Poland. All tested water samples were filtered before virus inoculation, and one unfiltered sample (Baltic seawater) was also tested. Infectivity was determined twice a week over a 60-day trial period by microtiter endpoint titration. The persistence of the virus varied considerably depending on its concentration and also on physico-chemical parameters of the water, such as temperature and salinity. Avian influenza virus survival was the highest at 4 C and the lowest at 20 C. Prolonged infectivity of the virus in Baltic seawater (brackish, 7.8 ppt) was also seen. In distilled water, the virus retained its infectivity beyond the 60-day study period. Interestingly, a devastating effect of the unfiltered fraction of seawater was seen as the virus disappeared in this fraction the quickest in all studied combinations; thus, biologic factors may also affect infectivity of HPAIV.

We examined 884 wild birds mainly from the Anseriformes, Charadriiformes and Galliformes orders for infectious bronchitis (IBV)-like coronavirus in Poland between 2008 and 2011. Coronavirus was detected in 31 (3.5%) of the tested birds, with detection rates of 3.5% in Anseriformes and 2.3% in Charadriiformes and as high as 17.6% in Galliformes. From the 31 positive samples, only 10 gave positive results in molecular tests aimed at various IBV genome fragments: five samples were positive for the RdRp gene, four for gene 3, eight for gene N and eight for the 3'-untranslated region fragment. All analysed genome fragments of the coronavirus strains shared different evolutionary branches, resulting in a different phylogenetic tree topology. Most detected fragment genes seem to be IBV-like genes of the most frequently detected lineages of IBV in this geographical region (i.e. Massachusetts, 793B and QX). Two waves of coronavirus infections were identified: one in spring (April and May) and another in late autumn (October to December). To our knowledge this is the first report of the detection of different fragment IBV-like genes in wild bird populations.

In April/May 2013, four outbreaks of avian influenza virus (AIV) infections caused by H9N2 subtype were diagnosed in Poland in fattening turkey flocks exhibiting a drop in feed and water intake, depression, respiratory signs and mortality. The subsequent serological survey carried out on samples collected between June 2012 and September 2013 from 92 poultry flocks detected positive sera in two additional meat turkey flocks located in the same province. The analysis of amino acids in the haemagglutinin and neuraminidase glycoproteins revealed that the detected H9N2 viruses possessed molecular profiles suggestive of low pathogenicity, avian-like SAα2,3 receptor specificity and adaptation to domestic poultry. Phylogenetic studies showed that these H9N2 AIVs grouped within the Eurasian clade of wild bird-origin AIVs and had no relationship with H9N2 AIV circulating in poultry in the Middle East and Far East Asia over the past decade. Experimentally infected SPF chickens with the index-case H9N2 virus remained healthy throughout the experiment. On the other hand, ten 3-week-old commercial turkeys infected via the oculonasal route showed respiratory signs and mortality (2/10 birds). Additional diagnostic tests demonstrated the consistent presence of DNA/RNA of Ornithobacterium rhinotracheale, Bordetella avium and, less frequently, of astro-, rota-, reo-, parvo- and adenoviruses in turkeys both from field outbreaks and laboratory experiment. Although no microbiological culture was performed, we speculate that these secondary pathogens could play a role in the pathogenicity of the current H9N2 infections.

The highly pathogenic (HP) H5N1 avian influenza viruses (AIVs) cause a mortality rate of up to 100% in infected chickens and pose a permanent pandemic threat. Attempts to obtain effective vaccines against H5N1 HPAIVs have focused on hemagglutinin (HA), an immunodominant viral antigen capable of eliciting neutralizing antibodies. The vast majority of vaccine projects have been performed using eukaryotic expression systems. In contrast, we used a bacterial expression system to produce vaccine HA protein (bacterial HA) according to our own design. The HA protein with the sequence of the H5N1 HPAIV strain was efficiently expressed in Escherichia coli, recovered in the form of inclusion bodies and refolded by dilution between two chromatographic purification steps. Antigenicity studies showed that the resulting antigen, referred to as rH5-E. coli, preserves conformational epitopes targeted by antibodies specific for H5-subtype HAs, inhibiting hemagglutination and/or neutralizing influenza viruses in vitro. The proper conformation of this protein and its ability to form functional oligomers were confirmed by a hemagglutination test. Consistent with the biochemical characteristics, prime-boost immunizations with adjuvanted rH5-E. coli protected 100% and 70% of specific pathogen-free, layer-type chickens against challenge with homologous and heterologous H5N1 HPAIVs, respectively. The observed protection was related to the positivity in the FluAC H5 test (IDVet) but not to hemagglutination-inhibiting antibody titers. Due to full protection, the effective contact transmission of the homologous challenge virus did not occur. Survivors from both challenges did not or only transiently shed the viruses, as established by viral RNA detection in oropharyngeal and cloacal swabs. Our results demonstrate that vaccination with rH5-E. coli could confer control of H5N1 HPAIV infection and transmission rates in chicken flocks, accompanied by reduced virus shedding. Moreover, the role of H5 subtype-specific neutralizing antibodies in anti-influenza immunity and a novel correlate of protection are indicated.