Background Five isolates (JS09GY2, JS09GY3, JS09GY4, JS09GY5, and JS09GY6) of avian leukosis virus subgroup J (ALV-J) were isolated from six infected commercial layer flocks displaying both hemangioma and myeloid leukosis (ML), which shared the same parental line, in China in 2009. Results All six of the commercial layer chickens examined showed hemangiomas on their body surface or feet. Some developed hemangiomas in their internal organs, causing hepatorrhexis and blood loss. Histopathologically different stages of hemangiomas with ML in the liver, heart, and spleen, were observed. Five viral isolates were obtained from infected DF1 cells incubated with the spleen tissue or serum of the birds from the six flocks. By full genome sequences analysis, a 19-nucleotide repeat sequence was identified in the primer binding site (PBS)-leader region of isolates JS09GY3 and JS09GY6, located between sites 249 and 250 according to the sequence of reference strain HPRS103, and also present in Rous sarcoma virus strain Schmidt–Ruppin B (RSV-SRB), Rous associated virus type 1 (RAV-1), and Rous associated virus type 2 (RAV-2). The predicted Gp85 proteins of isolates JS09GY2, JS09GY3, JS09GY5, and JS09GY6 were highly variable. Interestingly, the E elements of these four examined isolates showed a key deletion at site 30, which produced a new c-Ets-1 binding site. An 11-bp insertion was also found in the E element of isolate JS09GY3 located between bp 66 and 67 according to the sequence of reference strain HPRS103, while almost all previously reported Chinese strains showed an almost identical deletion of 127 bp in the same region. Conclusions Five ALV-J isolates were obtained from six field infected commercial layer chickens. Coexistence of hemangioma and ML were observed in these infected cases both macro- and microscopically. Complete proviral genome sequences of two isolates (JS09GY3 and JS09GY6) and the partial sequences of the other two isolates (JS09GY2 and JS09GY5) were determined. The isolates were found to be recombinants of ALV-J with a PBS-leader sequence originating from other retroviruses. The Gp85 protein with an amino acid deletion, a contiguous 11-bp insertion mutation in the E element, and a novel binding site, were noted in the proviral genomes.

Background: Gametocyte proteins ofEimeria(E.) spp. are important components of the oocyst wall and some have been used to develop transmission-blocking vaccines against avian coccidiosis.Methods: Total RNA isolated fromE. necatrixgametocytes was utilized as templates for RT-PCR amplification and sequencing of cDNA encoding a gametocyte protein using gene-specific primers. The cDNA was cloned into the bacterial expression vector pET28a(+) and expressed inE. coliBL21 cells. The antigenicity of the recombinant gametocyte protein and its localization in differentE. necatrixlife-cycle stages were determined by western blot and indirect immunofluorescence analyses, respectively.Results: A 731-nucleotide sequence of cDNA [GenBank: KF649255] ofE. necatrixhad 97.7% identity to that of Etgam22ofE. tenella. The cDNA ORF encoded a 186-amino acid protein containing a histidine-proline-rich region. The recombinant gametocyte protein (rEnGAM22) was predominately expressed in the insoluble inclusion body and recognized by antiserum from chickens immunized with oocysts ofE. necatrix,E. maximaandE. tenella. A specific antibody to the rEnGAM22 protein recognized the wall-forming bodies in macrogametocytes and the walls of oocysts and sporocysts.Conclusions: The gene cloned fromE. necatrixgametocytes is an ortholog to Etgam22ofE. tenellaand presents a potential target for future recombinant subunit vaccines against coccidiosis.

Background: Chicken anemia virus (CAV) is the causative agent of chicken infectious anemia. CAV putative intergenotypic recombinants have been reported previously. This fact is based on the previous classification of CAV sequences into three genotypes. However, it is unknown whether intersubtype recombination occurs between the recently reported four CAV genotypes and five subtypes of genome sequences. Results: Phylogenetic analysis, together with a variety of computational recombination detection algorithms, was used to investigate CAV approximately full genomes. Statistically significant evidence of intersubtype recombination was detected in the parent-like and two putative CAV recombinant sequences. This event was shown to occur between CAV subgroup A1 and A2 sequences in the phylogenetic trees. Conclusions: We revealed that intersubtype recombination in CAV genome sequences played a role in generating genetic diversity within the natural population of CAV.

Rothia nasimurium was known previously as an opportunistic pathogen of animals. However, there are few reports regarding the pathogenicity of Rothia nasimurium. In September 2020, geese contracted a disease of unknown cause which brought economic losses to a farm in Jiangsu Province, China, prompting a series of investigations. The bacterium was isolated, cultured, and purified, and then identified using Gram staining, biochemical tests, matrix-assisted laser desorption/ionization time of flight mass spectrometry, and 16S rRNA sequence analysis. After determining the obtained bacteria species, antibiotic susceptibility tests and animal regression experiments were carried out. A strain of bacterium was successfully isolated from the livers of the diseased geese, which was identified as a strain of the Gram-positive bacterium Rothia nasimurium according to the 16S rRNA sequencing results. By indexing references, no goose was reported to have been infected with Rothia nasimurium. The antibiotic susceptibility testing showed that only four antibiotics (amikacin, cefazolin, fosfomycin, and ampicillin/sulbactam) could effectively inhibit the growth of the Rothia nasimurium strain. The animal regression experiments showed that the novel isolated strain could infect goslings, and it also causes serious depilation of goslings. The results of the manuscript expanded the range of pathogenic microorganisms in geese, which is helpful to develop methods for avian endemic control.

Background: Porcine epidemic diarrhea virus (PEDV) is an α-coronavirus that causes highly contagious intestinal infectious disease, involving clinically characterized by diarrhea, dehydration, vomiting, and high mortality to suckling piglets. As a strategy for antiviral therapy, artificial microRNA (amiRNA) mediated suppression of viral replication has recently become increasingly important. In this study, we evaluated the advantages of using an amiRNA vector against PEDV. Methods: In this study, we evaluated the advantages of using an amiRNA vector against PEDV. We designed two single amiRNA sequences for different conserved sequences of the PEDV S and N genes, and tested their inhibitory effects on PEDV in Vero cells. Results: It was obvious from the CCK-8 results that the transient transfection of amiRNA was non-toxic to the cells. In addition, our results showed that the transient expression of two amiRNAs (amiRNA-349 and amiRNA-1447) significantly reduced the expression of viral RNA and protein in the cells. The TCID₅₀ results showed that the release of virus particles into the culture supernatant was significantly reduced, with an effect as high as 90%. To avoid virus mutation escape, the above two single amiRNA sequences were tandem in this study (amiRNA-349 + 1447), enabling a single microRNA to be expressed simultaneously. The real-time PCR and Western blot results showed that the inhibitory effect was significantly enhanced in each of the different time periods. The TCID₅₀ results showed that the release of virus particles in the culture supernatant was significantly reduced at the different time periods. Conclusions: In summary, these results suggest that an RNAi based on amiRNA targeting the conserved region of the virus is an effective method to improve PEDV nucleic acid inhibitors and provide a novel treatment strategy for PEDV infection.