Infection with IBV DMV/1639 at a Young Age Leads to Increased Incidence of Cystic Oviduct Formation Associated with False Layer Syndrome.

Infectious bronchitis virus (IBV) is an avian coronavirus that causes respiratory disease but can affect the reproductive tract of laying-type chickens. If infection occurs in pullets, false layer syndrome, which is characterized by the development of large, fluid-filled cystic oviducts, can occur. Recently, IBV strain DMV/1639 has been detected in parts of Canada and the U.S., where false layer syndrome has occurred, though it is not clear if IBV is the sole cause or if age at infection is an influencing variable. Our study investigates the role and timing of IBV infection on the development of false layer syndrome, using the IBV types DMV/1639 and Massachusetts (Mass). Six groups of 120 SPF chickens were challenged at either three, seven, or fourteen days of age, using either DMV/1639 or Mass IBV. Cystic oviducts were seen in all the challenged groups, and the pullets challenged at 14 days of age had fewer cystic oviducts than pullets challenged at 3 or 7 days of age. The highest percentage of severe histology lesion scores were seen in the 3-day challenge groups. The data collected in this experiment confirm that IBV DMV/1639 causes cystic oviducts and indicate that age at infection plays a role in the pathogenesis of false layer syndrome.

Biological and molecular characterization of ArkGA: A novel Arkansas serotype vaccine that is highly attenuated, efficacious, and protective against homologous challenge.

Almost all commercial poultry are vaccinated against avian coronavirus infectious bronchitis virus (IBV) using live attenuated vaccines mass administered by spray at day of hatch. Although many different types of IBV vaccines are used successfully, the ArkDPI serotype vaccine, when applied by spray, does not infect and replicate sufficiently to provide protection against homologous challenge. In this study, we examined a different Ark vaccine strain (Ark99), which is no longer used commercially due to its reactivity in one day old chicks, to determine if it could be further attenuated by passage in embryonated eggs but still provide adequate protection. Further attenuation of the Ark99 vaccine was achieved by passage in embryonated eggs but ArkGA P1, P20, and P40 (designated ArkGA after P1) were still too reactive to be suitable vaccine candidates. However, ArkGA P60 when given by spray had little or no vaccine reaction in one day old broiler chicks, and it induced protection from clinical signs and ciliostasis following homologous challenge. In addition, vaccinated and challenged birds had significantly less challenge virus, an important measure of protection, compared to non-vaccinated and challenged controls. The full-length genomes of viruses from egg passages 1, 20, 40, and 60 were sequenced using the Illumina platform and the data showed single nucleotide polymorphisms (SNPs) had accumulated in regions of the genome associated with viral replication, pathogenicity, and cell tropism. ArkGA P60 accumulated the most SNPs in key genes associated with pathogenicity (polyprotein gene 1ab) and cell tropism (spike gene), compared to previous passages, which likely resulted in its more attenuated phenotype. These results indicate that the ArkGA P60 vaccine is safe for spray vaccination of broiler chicks and induces suitable protection against challenge with pathogenic Ark-type virus.

S1 gene-based phylogeny of infectious bronchitis virus: An attempt to harmonize virus classification.

Infectious bronchitis virus (IBV) is the causative agent of a highly contagious disease that results in severe economic losses to the global poultry industry. The virus exists in a wide variety of genetically distinct viral types, and both phylogenetic analysis and measures of pairwise similarity among nucleotide or amino acid sequences have been used to classify IBV strains. However, there is currently no consensus on the method by which IBV sequences should be compared, and heterogeneous genetic group designations that are inconsistent with phylogenetic history have been adopted, leading to the confusing coexistence of multiple genotyping schemes. Herein, we propose a simple and repeatable phylogeny-based classification system combined with an unambiguous and rationale lineage nomenclature for the assignment of IBV strains. By using complete nucleotide sequences of the S1 gene we determined the phylogenetic structure of IBV, which in turn allowed us to define 6 genotypes that together comprise 32 distinct viral lineages and a number of inter-lineage recombinants. Because of extensive rate variation among IBVs, we suggest that the inference of phylogenetic relationships alone represents a more appropriate criterion for sequence classification than pairwise sequence comparisons. The adoption of an internationally accepted viral nomenclature is crucial for future studies of IBV epidemiology and evolution, and the classification scheme presented here can be updated and revised novel S1 sequences should become available.

Enteric viruses detected by molecular methods in commercial chicken and turkey flocks in the United States between 2005 and 2006.

Intestinal samples collected from 43 commercial broiler and 33 commercial turkey flocks from all regions of the United States during 2005 and 2006 were examined for the presence of astrovirus, rotavirus, reovirus, and coronavirus by reverse transcription-polymerase chain reaction (PCR), and for the presence of groups 1 and 2 adenovirus by PCR. Phylogenetic analysis was performed to further characterize the viruses and to evaluate species association and geographic patterns. Astroviruses were identified in samples from 86% of the chicken flocks and from 100% of the turkey flocks. Both chicken astrovirus and avian nephritis virus (ANV) were identified in chicken samples, and often both viruses were detected in the same flock. Turkey astrovirus type-2 and turkey astrovirus type-1 were found in 100% and 15.4% of the turkey flocks, respectively. In addition, 12.5% of turkey flocks were positive for ANV. Rotaviruses were present in 46.5% of the chicken flocks tested and in 69.7% of the turkey flocks tested. Based upon the rotavirus NSP4 gene sequence, the chicken and turkey origin rotaviruses assorted in a species-specific manner. The turkey origin rotaviruses also assorted based upon geographical location. Reoviruses were identified in 62.8% and 45.5% of chicken and turkey flocks, respectively. Based on the reovirus S4 gene segment, the chicken and turkey origin viruses assorted separately, and they were distinct from all previously reported avian reoviruses. Coronaviruses were detected in the intestinal contents of chickens, but not turkeys. Adenoviruses were not detected in any chicken or turkeys flocks. Of the 76 total chicken and turkey flocks tested, only three chicken flocks were negative for all viruses targeted by this study. Most flocks were positive for two or more of the viruses, and overall no clear pattern of virus geographic distribution was evident. This study provides updated enteric virus prevalence data for the United States using molecular methods, and it reinforces that enteric viruses are widespread in poultry throughout the United States, although the clinical importance of most of these viruses remains unclear.

Using DNA shuffling to create novel infectious bronchitis virus S1 genes: implications for S1 gene recombination.

We employed the staggered extension process (StEP) to shuffle the S1 genes from four infectious bronchitis virus (IBV) strains representing four unique serotypes. Upon creating a shuffled S1 gene library, we randomly selected 25 clones and analyzed them by DNA sequencing. In total, eleven clones contained novel S1 gene recombinants. Based on sequence data, each recombinant was unique and contained a full-length open reading frame. The average number of crossovers per recombinant was 5 and the average number of point mutations was 1.3, leading mostly to non-synonymous amino acid changes. No recombinant contained sequences from all four parental genes and no recombinant contained any sequence from the distantly related Delaware 072 strain. Our data suggests that recombination between distantly related IBV strains within the S1 gene probably does not readily occur. This finding is extremely important in light of the common industry vaccination practice of mixing different live-attenuated IBV strains.

Polymerase chain reaction for detection of avian leukosis virus subgroup J in feather pulp.

Feather pulp from experimentally infected chickens was used as a source of DNA for polymerase chain reaction (PCR) amplification of avian leukosis virus subgroup J (ALV-J) proviral DNA. A primer set that produces a large amplicon (approximately 2,125) was used to detect ALV-J proviral DNA. This primer set was used in lieu of previously published primers because it allows for sequencing of the entire envelope gene and because it was able to detect diagnostically a number of North American ALV-J isolates that could not be detected with previously published primers and PCR conditions. ALV-J proviral DNA was detected in feather pulp at 7 days of age in more than 90% of birds infected as embryos and 7 days postinoculation in over 50% of chickens infected at 3 days of age. The results obtained with PCR on feather pulp were compared with those of virus isolation. In the embryo-inoculated birds, the percentages of agreement between PCR and virus isolation were 92.5% at 7 days of age and 100% at 28, 42, 49, and 56 days of age. However, the overall sensitivity of virus isolation in embryo-infected birds was higher, particularly at 7 and 56 days of age. In chickens inoculated at 3 days of age, the percentages of agreement of detection between PCR and virus isolation ranged from 75% at 10 days of age to 100% at 42 days of age. Agreement of negative results of ALV-J detection by PCR and virus isolation in chickens infected posthatch ranged between 66.6% and 100% between the ages of 10 and 42 days. Virus isolation requires chicken embryo fibroblasts of specific genetic lines, and the process takes onaverage 7-9 days. Aseptic collection of blood and tissues for virus isolation and molecular detection of ALV-J requires sterile necropsy instruments as well as syringes and needles for each individual chicken, whereas sterile microcentrifuge tubes and gloves are the only equipment necessary for aseptic feather pulp collection for ALV-J detection by PCR. PCR-based detection of ALV-J in feather pulp is especially suitable when ALV-J infection must be diagnosed rapidly and unequivocally without killing the chicken(s) and in situations where crucial reagents or suitable virus propagation substrates are not readily available for isolation and propagation of ALV-J in cell culture.