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In October 2018, an outbreak of transmissible viral proventriculitis (TVP) occurred in 30-day-old commercial broiler chickens on a farm in Weifang, China. TVP, an infectious viral disease characterized by runting and stunting, is associated with many viruses, and has a significant economic impact on the global poultry industry. TVP is diagnosed according to clinical symptoms, gross and histological lesions, and negative PCR results for pathogenic bacteria, avian leukosis virus subgroup J, Marek's disease virus, reticuloendotheliosis virus, infectious bursa disease virus, avian reovirus, chicken anemia virus, infectious bronchitis virus, chicken proventricular necrosis virus, gyrovirus 3 and chicken circovirus. To further detect the possible causative pathogens of TVP, we used PacBio third-generation sequencing to examine proventricular samples. A dominant abundance of the novel cyclovirus (CyCV), chCyCV-SDAU-1, was identified in broilers with TVP. The complete chCyCV-SDAU-1 genome was verified via inverse PCR, was 1936 bp long, and consisted of Rep, Cp, and two intergenic regions. Phylogenetic tree analysis showed that chCyCV-SDAU-1 formed an independent branch with other cycloviruses. The homology of chCyCV-SDAU-1 with 20 others known cycloviruses was < 40%. Retrospective investigation showed that the CyCV infection rate in the broilers with TVP was 80% (16/20), while no CyCV was found in healthy chickens. In conclusion, a novel CyCV was identified in chickens with TVP, though its role in this disease is unclear.

Circoviruses are found in many species, including mammals, birds, lower vertebrates and invertebrates. To date, there are no reports of circovirus‐induced diseases in chickens. In this study, we identified a new strain of chicken circovirus (CCV) by PacBio third‐generation sequencing samples from chickens with acute gastroenteritis in a Shandong commercial broiler farm in China. The complete genome of CCV was verified by inverse PCR. Genomic analysis revealed that CCV codes two inverse open reading frames (ORFs), and a potential stem‐loop structure was present at the 5′ end with a structure typical of a circular virus. Phylogenetic tree analysis showed that CCV formed an independent branch between mammalian and avian circovirus, and homology analysis indicated that the homology of CCV with 21 other known circoviruses was less than 40%. Thus, this CCV strain represents a new species in the genus Circovirus. The infection rate of CCV in 12 chickens with diarrhoea was 100%, but no CCV was found in healthy chickens, thereby indicating that the novel CCV strain is highly associated with acute infectious gastroenteritis in chickens. The emergence of a novel CCV in commercial broiler chickens is highly concerning for the broiler industry.

Chicken anemia virus (CAV) and Gyrovirus homsa 1 (GyH1) are members of the Gyrovirus genus. The two viruses cause similar clinical manifestations in chickens, aplastic anemia and immunosuppression. Our previous investigation displays that CAV and GyH1 often co-infect chickens. However, whether they have synergistic pathogenicity in chickens remains elusive. Here, we established a co-infection model of CAV and GyH1 in specific pathogen-free (SPF) chickens to explore the synergy between CAV and GyH1. We discovered that CAV and GyH1 significantly inhibited weight gain, increased mortality, and hindered erythropoiesis in co-infected chickens. Co-infected chickens exhibited severe immune organ atrophy and lymphocyte exhaustion. The proventriculus and gizzard had severe hemorrhagic necrosis and inflammation. We also discovered that the viral loads and shedding levels were higher and lasted longer in CAV and GyH1 co-infected chickens than in mono-infected chickens. Our results demonstrate that CAV and GyH1 synergistically promote immunosuppression, pathogenicity, and viral replication in co-infected chicken, highlighting the interaction between CAV and GyH1 in the disease process and increasing potential health risk in the poultry breeding industry, and needs further attention.

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Background Gyrovirus homsa1 (GyH1) (also known as Gyrovirus 3, GyV3) is a non-enveloped, small, single-stranded DNA virus, which was first identified in children with acute diarrhea, and was subsequently detected in marketed chickens, broilers with transmissible viral proventriculitis (TVP), and mammals. GyH1 is a pathogenic virus in chickens, causing aplastic anemia, immunosuppression, and multisystem damage. However, the seroepidemiology of GyH1 infection in chickens remains unclear. Here, we investigated the seroprevalence of GyH1 in chickens by ELISA to reveal the endemic status of GyH1 in China. Results An indirect ELISA with high sensitivity and specificity was developed for investigation of seroepidemiology of GyH1 in chickens in China. The seropositive rate of GyH1 ranged from 0.6% to 7.7% in thirteen provinces, and ranged from 4.1% to 8.1% in eight species chickens. The seropositive rate of GyH1 in broiler breeders was significantly higher than that of in layers. There was a negative correlation between seropositive rate and age of chickens. The highest and lowest seropositive rate were present in chickens at 30–60 days and over 180 days, respectively. Conclusions The seroepidemiological investigation results demonstrated that natural GyH1 infection is widespread in chickens in China. Different species showed different susceptibility for GyH1. Aged chickens showed obvious age-resistance to GyH1. GyH1 has shown a high risk to the poultry industry and should be highly concerned.

Background Transmissible viral proventriculitis (TVP) causes significant economic loss to the poultry industry. However, the exact causative agents are obscure. Here we examine the virome of proventriculus from specified pathogen free (SPF) chickens that reproduced by infection of proventricular homogenate from broiler chicken with TVP using long read sequencing of the Pacific Biosciences RSII platform. The normal SPF chickens were used as control. Results Our investigation reveals a virome of proventriculitis, including three Gyrovirus genera of the Aneloviridae: Gyrovirus homsa1 (GyH1) (also known as Gyrovirus 3, GyV3) (n = 2662), chicken anemia virus (CAV) (n = 482) and Gyrovirus galga1 (GyG1) (also known as avian Gyrovirus 2, AGV2) (n = 11); a plethora of novel CRESS viral genomes (n = 26) and a novel genomovirus. The 27 novel viruses were divided into three clusters. Phylogenetic analysis showed that the GyH1 strain was more closely related to the strains from chicken (MG366592) than mammalian (human and cat), the GyG1 strain was closely related to the strains from cat in China (MK089245) and from chicken in Brazil (HM590588), and the CAV strain was more closely related to the strains from Germany (AJ297684) and United Kingdom (U66304) than that previously found in China. Conclusion In this study, we revealed that Gyrovirus virome showed high abundance in chickens with TVP, suggesting their potential role in TVP, especially GyH1. This study is expected to contribute to the knowledge of the etiology of TVP.

Gyrovirus 3 (GyV3), the third novel emerging species of the genus Gyrovirus of the Anelloviridae family, has been described in multiple hosts. Epidemiologically, there are suggestions that GyV3 is associated with diarrhea/proventriculitis, however, no direct causal evidence exists between GyV3 infection and specific clinical diseases. Herein, we infected special pathogen-free (SPF) chickens with GyV3, and then assessed the pathogenicity and tissue tropism. The results revealed that GyV3 induced persistent infection characterized by diarrhea, aplastic anemia, immunosuppression, and persistent systemic lymphocytic inflammation. Clinically, the infected chickens presented ruffled feathers, diarrhea, anemia, and weight loss. Aplastic anemia was characterized by progressive depletion of hematopoietic cells in the bone marrow, immunosuppression was associated with atrophy of the thymus, spleen, and bursa of Fabricious, progressive lymphocytic inflammations were characterized by proventriculitis, adrenalitis, pancreatitis, hepatitis, nephritis, and bronchitis. Viral loads of GyV3 in tissues exhibited “M”, “N”, “W” or “V” type dynamic changes. The highest level of viral loads was reported in bone marrow at 7dpi, followed by the adrenal gland at 2 dpi, the sciatic nerve at 7 dpi, and bile at 35 dpi. The bone marrow and kidney demonstrate the strongest immunostaining of GyV3-VP1 antigen and were suggested as the target tissues of GyV3. Collectively, GyV3 is an immunosuppressive pathogenic virus that targets the bone marrow and kidney in chickens. Exploring the pathogenicity and tissue tropism of GyV3 will guide the basic understanding of the biology of GyV3 and its pathogenesis in chickens.