Characterization of subgroup J avian Leukosis virus isolated from Chinese indigenous chickens.

BACKGROUND: In spite of the purification of the laying hens and broilers of avian leukosis virus (ALV) has made remarkable achievements, the infection of ALV was still serious in Chinese indigenous chickens. METHODS: In order to assess the epidemic state of avian leukosis virus in indigenous chickens in China, 10 novel strains of ALV subgroup J (ALV-J), named JS16JH01 to JS16JH10, were isolated and identified by virus isolation and immunofluorescence antibody assays from a Chinese local breed farm with a sporadic incidence of tumors. To understand their virological characteristics further, the proviral genome of ENV-LTR was sequenced and compared with the reference strains. RESULTS: The homology of the gp85 gene between the ten ALV-J strains and NX0101 was in the range from 89.7-94.8% at the nuclear acid level. In addition, their gp85 genes were quite varied, with identities of 92-98% with themselves at the nuclear acid level. There were several snp and indel sites in the amino acid sequence of gp85 genes after comparison with other reference strains of ALV. Interestingly, a novel insertion in the gp85 region was found in two strains, JS16JH01 and JS16JH07, compared with NX0101 and HPRS-103. DISCUSSION: At present, owing to the large-scale purification of ALV in China, laying hens and broiler chickens with ALV infection are rarely detected, but ALVs are still frequently detected in the local chickens, which suggests that more efforts should be applied to the purification of ALV from indigenous chickens.

A New Strategy for the Detection of Chicken Infectious Anemia Virus Contamination in Attenuated Live Vaccine by Droplet Digital PCR.

Chicken infectious anemia virus (CIAV) causes the atrophy of bone marrow hematopoietic and lymphoid tissues in chicks, leading to huge economic losses all over the world. The using of attenuated vaccine contaminated with CIAV increased the mortality and the pathogenicity of other diseases in many farms. However, it is difficult to detect the CIAV contamination by general detection technology due to the extremely low dose of CIAV in vaccines. In this study, we established a new method called droplet digital Polymerase Chain Reaction (ddPCR) to detect CIAV contamination of vaccines more sensitively and accurately. The lowest detection limitation of this method is 2.4 copies of CIAV plasmid or CIAV contamination at 0.1 EID50/1000 feathers in vaccines without any positive signals of other viruses. Besides, the sensitivity of ddPCR is 100 times greater than that of conventional PCR and 10 times greater than that of real-time PCR. The ddPCR technique is more sensitive and more intuitive. Therefore, it could be valuable for the detection of CIAV contamination in vaccines.

Molecular characteristics of avian leukosis viruses isolated from indigenous chicken breeds in China.

To assess the status of avian leukosis virus (ALV) infection in indigenous chicken breeds in China, 121 plasma samples collected from various indigenous chicken breeds were tested for the presence of ALV from 2015 to 2016. A total of 14 ALV strains were isolated and identified, including two ALV-A strains, one ALV-B strain, eight ALV-J strains, and three ALV-K strains. To study the genome structure, biological characteristics, and the evolutionary relationships of the ALV-K strains with other known subgroup strains from infected chickens, we determined the complete genome sequence of the three ALV-K strains and performed comparative analysis using the whole genome sequence or selected sequence elements. The replication rates of the three ALV-K strains were markedly lower than the rates of other ALVs, and they shared a common mutation in the pol gene, which had not been previously observed. In addition, nine putative recombinant events were detected in the genomes of the three newly isolated ALV-K strains, with high statistical support. This was the first report of an ALV-K reorganization event, which has contributed to its genetic evolution. In summary, we established a robust classification system for ALV, especially for ALV-K, and revealed additional genomic diversity for the ALV strains in indigenous chicken breeds. Therefore additional works are warranted to explore ALV genomics and epidemiology.

Co-infection of fowl adenovirus with different immunosuppressive viruses in a chicken flock.

In poultry, fowl adenovirus (FAdV) and immunosuppressive virus co-infection is likely to cause decreased egg production, inclusion body hepatitis, and pericardial effusion syndrome. In this study, fowl adenovirus infection was found in parental and descendent generations of chickens. We used quantitative polymerase chain reaction (PCR) and dot blot hybridization to detect the infection of reticuloendotheliosis (REV), avian leukosis virus (ALV), and chicken infectious anemia virus (CIAV) in 480 plasma samples. The test samples were 34.58% FADV-positive, 22.29% REV-positive, 7.5% CAV-positive, and 0.63% ALV-positive. Sequence analysis showed that FADV belonged to serotype 7, which can cause inclusion body hepatitis. The ALV strain was ALV-A, in which the homology of gp85 gene and SDAU09C1 was 97.3%. The positive rate was lower because of the purification of avian leukemia, whereas the phylogenetic tree analysis of REV showed that the highest homology was with IBD-C1605, which was derived from a vaccine isolate. Through pathogen detection in poultry we present, to our knowledge, the first discovery of fowl adenovirus type 7 infection in parental chickens and found that there was co-infection of FAdV and several immunosuppressive viruses, such as the purified ALV and CIAV. This indicates that multiple infection of different viruses is ever-present, and more attention should be given in the diagnosis process.