Hepatitis-splenomegaly (HS) syndrome is an emerging disease in chickens in North America ; the cause of this disease is unknown. In this study, the genetic identification and characterization of a novel virus related to human hepatitis E virus (HEV) isolated from bile samples of chickens with HS syndrome is reported. Based upon the similar genomic organization and significant sequence identity of this virus with HEV, the virus has been tentatively named avian HEV in order to distinguish it from human and swine HEV. Electron microscopy revealed that avian HEV is a non-

We recently identified and characterized a novel virus, designated avian hepatitis E virus (avian HEV), from chickens with hepatitis-splenomegaly syndrome (HS syndrome) in the United States. Avian HEV is genetically related to but distinct from human and swine HEVs. To determine the extent of genetic variation and the seroprevalence of avian HEV infection in chicken flocks, we genetically identified and characterized 11 additional avian HEV isolates from chickens with HS syndrome and assessed the prevalence of avian HEV antibodies from a total of 1,276 chickens of different ages and breeds from 76 different flocks in five states (California, Colorado, Connecticut, Virginia, and Wisconsin). An enzyme-linked immunosorbent assay using a truncated recombinant avian HEV ORF2 antigen was developed and used to determine avian HEV seroprevalence. About 71% of chicken flocks and 30% of chickens tested in the study were positive for antibodies to avian HEV. About 17% of chickens younger than 18 weeks were seropositive, whereas about 36% of adult chickens were seropositive. By using a reverse transcription-PCR (RT-PCR) assay, we tested 21 bile samples from chickens with HS syndrome in California, Connecticut, New York, and Wisconsin for the presence of avian HEV RNA. Of the 21 bile samples, 12 were positive for 30-to 35-nm HEV-like virus particles by electron microscopy (EM). A total of 11 of the 12 EM-positive bile samples and 6 of the 9 EM-negative bile samples were positive for avian HEV RNA by RT-PCR. The sequences of a 372-bp region within the helicase gene of 11 avian HEV isolates were determined. Sequence analyses revealed that the 11 field isolates of avian HEV had 78 to 100% nucleotide sequence identities to each other, 79 to 88% identities to the prototype avian HEV, 76 to 80% identities to chicken big liver and spleen disease virus, and 56 to 61% identities to other known strains of human and swine HEV. The data from this study indicated that, like swine and human HEVs, avian HEV isolates are genetically heterogenic and that avian HEV infection is enzoonotic in chicken flocks in the United States.

Avian astroviruses were detected by reverse transcriptase and polymerase chain reaction in intestinal contents collected from commercial chickens and turkeys from throughout the United States from 2003 through 2005. Astroviruses were detected in birds from both healthy and poorly performing flocks with or without enteric disease. Phylogenetic analysis was performed with sequence data from the polymerase (ORF-1b) genes of 41 turkey-origin astroviruses and 23 chicken-origin astroviruses. All currently available avian astrovirus sequence data and selected mammalian astrovirus sequence data were included in the analysis. Four groups of avian astroviruses were observed by phylogenetic analysis: turkey astrovirus type 1 (TAstV-1)-like viruses, turkey astrovirus type 2 (TAstV-2)-like viruses, both detected in turkeys; avian nephritis virus (ANV)-like viruses, detected in both chickens and turkeys; and a novel group of chicken-origin astroviruses (CAstV). Among these four groups, amino acid identity was between 50.1% and 73.8%, and was a maximum of 49.4% for all avian isolates when compared with the mammalian astroviruses. There were multiple phylogenetic subgroups within the TAstV-2, ANV, and CAstV groups based on 9% nucleotide sequence divergence. Phylogenetic analysis revealed no clear assortment by geographic region or isolation date. Furthermore, no correlation was observed between the detection of a particular astrovirus and the presence of enteric disease or poor performance. Based on these data, a revision of the present taxonomic classification for avian astroviruses within the genus Avastrovirus is warranted.

Isolates from the 2002-2003 virulent Newcastle disease virus (v-NDV) outbreak in southern California, Nevada, Arizona, and Texas in the United States were compared to each other along with recent v-NDV isolates from Mexico and Central America and reference avian paramyxovirus type 1 strains. Nucleotide sequencing and phylogenetic analyses were conducted on a 1,195-base genomic segment composing the 3′ region of the matrix (M) protein gene and a 5′ portion of the fusion (F) protein gene including the M-F intergenic region. This encompasses coding sequences for the nuclear localization signal of the M protein and the F protein cleavage activation site. A dibasic amino acid motif was present at the predicted F protein cleavage activation site in all v-NDVs, including the California 2002-2003, Arizona, Nevada, Texas, Mexico, and Central America isolates. Phylogenetic analyses demonstrated that the California 2002-2003, Arizona, Nevada, and Texas viruses were most closely related to isolates from Mexico and Central America. An isolate from Texas obtained during 2003 appeared to represent a separate introduction of v-NDV into the United States, as this virus was even more closely related to the Mexico 2000 isolates than the California, Arizona, and Nevada viruses. The close phylogenetic relationship between the recent 2002-2003 U.S. v-NDV isolates and those viruses from countries geographically close to the United States warrants continued surveillance of commercial and noncommercial poultry for early detection of highly virulent NDV

Infectious bursal disease virus (IBDV) causes an economically important, immunosuppressive disease in chickens. There are two serotypes of the virus that contain a bi-segmented double-stranded RNA genome. In December 2008, the first very virulent (vv)IBDV was identified in California, USA and in 2009 we isolated reassortant viruses in two different locations. Genome segment A of these reassortants was typical of vvIBDV serotype 1 but genome segment B was most similar to IBDV serotype 2. The CA-K785 reassortant caused 20% mortality in chickens but no morbidity or mortality in commercial turkey poults despite being infectious. There have been previous reports of natural reassortants between vvIBDV and other serotype 1 strains, but a natural reassortant between IBDV serotypes 1 and 2 has not been described. The apparent reassorting of California vvIBDV with an endemic serotype 2 virus indicates a common host and suggests vvIBDV may have entered California earlier than originally thought.

The avian influenza (AI) virus is usually isolated and propagated by inoculating either swab or tissue samples from infected birds into the chorioallantoic sac of embryonating chicken eggs. This is the accepted method, but occasionally an isolation may only be successful when inoculated either into the yolk sac or onto the chorioallantoic membrane of embryonating chicken eggs. Chorioallantoic fluid is harvested from eggs with dead or dying embryos and is tested for the presence of hemagglutinating antigen. If hemagglutination-positive, this indicates that the isolate may be the AI virus. The presence of the AI virus may be confirmed by either an agar gel immunodiffusion (AGID) assay, RT-PCR specific for AI virus, or a commercially available immunoassay kit specific for type A influenza. Instructions for AI virus primary isolation and propagation, preparing antigen for an AGID test, setting up an AGID test, and interpreting results are given.

During 2000, 2001, and January 2002, avian influenza virus was isolated from chickens from 12 different locations in California. All the isolates were typed as H6N2 and determined to be of low pathogenicity for chickens. Nine of the isolates came from commercial layer flocks; one from a backyard flock; one from a mixed age flock, where ducks and squabs were also present; and one from a primary broiler breeder. Although a drop in egg production and increased mortality were among the disease signs reported in the layer flocks, the pathological changes observed in the early cases were primarily associated with mild respiratory infections. It was not until August 2001 that yolk peritonitis was observed; this has been a feature of all the remaining cases through 2001 and 2002. All the isolates clustered as a unique group separate from other influenza viruses based upon sequence data of the H6, neuraminidase (N2), and matrix (MA) genes, indicating a common ancestor for these three gene segments. However, sequencing of the nonstructural (NS) gene indicates introductions from two separate origins. With the first isolate CK/CA/431/00 as the index case, the N2, MA, and NS genes are more closely related to North American isolates, as is the NP gene of CK/CA/650/00. In contrast, the H6 gene is more closely related to a Eurasian influenza isolate. Comparison of amino acid sequences of the N2 and MA genes of these isolates with available type A influenza viruses identified two unique changes in the MA gene and nine in the N2 gene, as well as four progressive changes. These results are discussed in relation to available clinical and epidemiological data.

West Nile virus (WNV) infection was diagnosed in 38 psittacine birds based on histology, immunohistochemistry, and reverse transcriptase polymerase chain reaction (RT-PCR). Rosellas (Platycercus spp, n = 13), conures (Enicognathus, Aratinga, and Nandayus spp, n = 6), and lorikeets (Trichoglossus spp, n = 6) represented the most commonly affected species. Clinical signs ranged from lethargy, ruffled feathers, anorexia, and weight loss in most birds to sudden death in others. Except for mild to moderate enlargement of liver and spleen, there were no significant gross lesions at necropsy. Histopathologic findings included lymphoplasmacytic and histiocytic hepatitis, interstitial nephritis, myocarditis, splenitis, enteritis, pancreatitis, and occasionally, encephalitis. Viral antigen was detected by immunohistochemistry in 34 of 35 hearts (97.1%), 29 of 32 pancreata (90.6%), 33 of 37 kidneys (89.2%), 31 of 35 intestines (88.6%), 27 of 33 gizzards (81.8%), 8 of 10 ovaries (80%), 27 of 34 spleens (79.4%), 30 of 38 livers (78.9%), 23 of 32 lungs (71.9%), 21 of 31 proventriculi (67.7%), 14 of 21 adrenals (66.7%), 10 of 16 testes (62.5%), 17 of 30 brains (56.7%), 15 of 27 skins (55.5%), 3 of 6 oviducts (50%), 15 of 34 skeletal muscles (44.1%), 11 of 27 crop or esophagus (40.7%), and 1 of 6 thymuses (16.7%). Kidney was positive for WNV by RT-PCR in all the cases tested. In conclusion, Psittaciformes are susceptible to West Nile virus infection, and WNV infections are often associated with nonspecific clinical signs and widespread viral distribution in this order of birds.