Differential innate immune responses to fowl adenovirus serotype 4 infection in Leghorn male hepatocellular and chicken embryo fibroblast cells.

Fowl adenovirus serotype 4 (FAdV-4) infections result in substantial economic losses in the poultry industry. Recent findings have revealed that FAdV-4 significantly suppresses the host immune response upon infection; however, the specific viral and host factors contributing to this immunomodulatory activity remain poorly characterized. Moreover, diverse cell types exhibit differential immune responses to FAdV-4 infection. To elucidate cell-specific host responses, we performed transcriptomic analysis of FAdV-4 infected leghorn male hepatocellular (LMH) and chicken embryo fibroblast (CEF) cells. Although FAdV-4 replicated more efficiently in LMH cells, it provoked limited interferon-stimulated gene induction. In contrast, FAdV-4 infection triggered robust antiviral responses in CEF cells, including upregulation of cytosolic DNA sensing and interferon-stimulated genes. Knockdown of key cytosolic DNA sensing molecules enhanced FAdV-4 replication in LMH cells while reducing interferon-stimulated gene expression. Our findings reveal cell-specific virus-host interactions that provide insight into FAdV-4 pathogenesis while identifying factors that mediate antiviral immunity against FAdV-4.

Fowl adenovirus serotype 4 enters leghorn male hepatocellular cells via the clathrin-mediated endocytosis pathway.

Hepatitis-hydropericardium syndrome (HHS) induced by fowl adenovirus serotype-4 (FAdV-4) has caused large economic losses to the world poultry industry in recent years. HHS is characterized by pericardial effusion and hepatitis, manifesting as a swollen liver with focal necroses and petechial haemorrhage. However, the process of FAdV-4 entry into hepatic cells remains largely unknown. In this paper, we present a comprehensive study on the entry mechanism of FAdV-4 into leghorn male hepatocellular (LMH) cells. We first observed that FAdV-4 internalization was inhibited by chlorpromazine and clathrin heavy chain (CHC) knockdown, suggesting that FAdV-4 entry into LMH cells depended on clathrin. By using the inhibitor dynasore, we showed that dynamin was required for FAdV-4 entry. In addition, we found that FAdV-4 entry was dependent on membrane cholesterol, while neither the knockdown of caveolin nor the inhibition of a tyrosine kinase-based signalling cascade affected FAdV-4 infection. These results suggested that FAdV-4 entry required cholesterol but not caveolae. We also found that macropinocytosis played a role, and phosphatidylinositol 3-kinase (PI3K) was required for FAdV-4 internalization. However, inhibitors of endosomal acidification did not prevent FAdV-4 entry. Taken together, our findings demonstrate that FAdV-4 enters LMH cells through dynamin- and cholesterol-dependent clathrin-mediated endocytosis, accompanied by the involvement of macropinocytosis requiring PI3K. Our work potentially provides insight into the entry mechanisms of other avian adenoviruses.

Characteristics of human papillomavirus infection among females and the genetic variations of HPV18 and HPV58 in Henan province, China.

The present study aims to investigate the genotype distribution of Human papillomavirus (HPV) and variations of HPV18 and HPV58 infection among 6538 females in Luoyang city during 2019-2021. The overall positive rate of females with HPV infection was 12.34%, with 9.74% were infected with single HPV and 2.60% with multiple HPVs. The prevalent rate of high-risk HPV (HR-HPV) was 9.85% and the top five HR-HPV genotypes were HPV52 (1.94%), HPV16 (1.93%), HPV58 (1.48%), HPV51 (1.02%) and HPVV39 (0.99%). Two peaks of HPV infections rates were observed in females aged ≤ 20 and 61-65 years old. To characterize mutations, 39 HPV18 and 56 HPV58 L1, E6 and E7 genes were sequenced and submitted to GenBank. In the HPV18 E6-E7-L1 sequences, 38 nucleotides changes were observed with 10/38 were non-synonymous mutations (5 in E6 gene, 1 in E7 gene and 4 in L1 gene). In the HPV58 E6-E7-L1 sequences, 53 nucleotides changes were observed with 23/53 were non-synonymous mutations (3 in E6 gene, 5 in E7 gene and 15 in L1 gene). Phylogenetic analysis based on L1 gene showed that 92.3% (36/39) of HPV18 isolates fell into sublineage A1 and 7.7% (3/39) belonged to A5. For HPV58, 75.0% (42/56) isolates belonged to sublineage A1 and 25.0% (14/56) were sublineage A2. There was no association between amino mutation and cervical lesions. The present study provides basic information about the distribution, genotypes and variations of HPV among females population in Luoyang city, which would assist in the formulation of HPV screening and vaccination programs and preventive strategies for HPV-attributable cancer in this region.

Mycoplasma synoviae LP78 is a fibronectin/plasminogen binding protein, putative adhesion, and potential diagnostic antigen.

Mycoplasma synoviae (M. synoviae) is one of the major poultry pathogens causing infectious synovitis, airsacculitis, a high incidence of shell breakage, and egg production loss. However, the pathogenesis of M. synoviae remains unclear. Adhesion of mycoplasmas to host cells is a crucial step in infection and colonization. The purpose of this study was to determine the adhesive function of a putative P80 family lipoprotein (LP78) and evaluate its application in the detection of antibodies against M. synoviae. Recombinant LP78 (rLP78) was expressed in the supernatant component of Escherichia coli and mouse anti-rLP78 serum was prepared. Bioinformatic analysis and western blotting results revealed that LP78 was conservative among M. synoviae strains. It was distributed not only in the cytoplasm but also on the membrane of M. synoviae through western blotting and indirect immunofluorescence (IFA). The adherence of M. synoviae to DF-1 cells was significantly inhibited by mouse anti-rLP78 serum (p < 0.01). IFA revealed that rLP78 adhered to DF-1 cells, and this adherence was prevented by mouse anti-rLP78 serum. Furthermore, rLP78 was found to bind to the DF-1 cells membrane proteins in a dose-dependent manner by enzyme-linked immunosorbent assay (ELISA). Screening of DF-1 cells membrane proteins by western blotting showed that proteins with molecular weight of 35-40 kDa and 55-70 kDa bound to rLP78. Moreover, rLP78 was identified to be a fibronectin/plasminogen binding protein. The sensitivity and specificity of rLP78-based iELISA were 85.7 and 94.1%, respectively. The maximum dilution of positive serum (HI titer, 1:128) detected via rLP78-based iELISA was 1:6,400, whereas that detected using a commercial ELISA kit was 1:12,800-1:25,600. Both rLP78-based iELISA and the commercial ELISA kit detected seroconversion after 7 days of challenge and immunization. No cross-reactivity with positive sera against other avian pathogens was observed in rLP78-based iELISA. Collectively, these results indicate that LP78 is a fibronectin/plasminogen-binding adhesion protein of M. synoviae and a potential diagnostic antigen. The present study will facilitate a better understanding of the pathogenesis of M. synoviae and the development of new diagnostic.

Evaluation of the protective efficacy of six major immunogenic proteins of Mycoplasma Synoviae.

Mycoplasma synoviae (MS) is a primary avian pathogen prevalent worldwide that causes airsacculitis and synovitis in birds. Vaccination is recommended as the most cost-effective strategy in the control of MS infection. Novel alternative vaccines are needed for eradicating and controlling MS infection in flocks. DnaK, enolase, elongation factor Tu (EF-Tu), MSPB, NADH oxidase and LP78 are the major immunogenic antigens of MS and are promising targets for subunit vaccine candidates. In the present study, genes encoding DnaK, enolase, EF-Tu, MSPB, LP78, and NADH oxidase were cloned and expressed in Escherichia coli. Enzyme-linked immunosorbent assay showed that the six recombinant proteins were recognized by convalescent sera, indicating that they were expressed during infection. Two injections of the six subunit vaccines induced a robust antibody response and increased the concentrations of IFN-γ and IL-4, especially rEnolase and rEF-Tu. The proliferation of peripheral blood lymphocytes was enhanced in all of the immunized groups. Chickens immunized with rEnolase, rEF-Tu, rLP78, and rMSPB conferred significant protection against MS infection, as indicated by significantly lower DNA copies in the trachea, lower scores of air sac lesions, and lesser tracheal mucosal thickness than that in the challenge control. Especially, rEnolase provided the best protective efficacy, followed by rEF-Tu, rMSPB, and rLP78. Our finds demonstrate that the subunit vaccines and bacterin can only reduce the lesions caused by MS infection, but not prevent colonization of the organism. Our findings may contribute to the development of novel vaccine agents against MS infection.

Long noncoding RNA IRF1-AS is associated with peste des petits ruminants infection.

Peste des petits ruminants (PPR) is an acute and highly contagious disease and has long been a significant threat to small ruminant productivity worldwide. However, the molecular mechanism underlying host-PPRV interactions remains unclear and the long noncoding RNAs (lncRNAs) regulation of PPR virus (PPRV) infection has rarely been reported so far. Here, we first demonstrated that PPRV infection can induce an obvious innate immune response in caprine endometrial epithelial cells (EECs) at 48 h post-infection (hpi) with an MOI of 3. Subsequently, we determined that PPRV infection is associated with 191 significantly differentially expressed (SDE) lncRNAs, namely, 137 upregulated and 54 downregulated lncRNAs, in caprine EECs compared with mock control cells at 48 hpi by using deep sequencing technology. Importantly, bioinformatics preliminarily analyses revealed that these DE lncRNAs were closely related to the immune response. Furthermore, we identified a system of lncRNAs related to the immune response and focused on the role of lncRNA 10636385 (IRF1-AS) in regulating the innate immune response. Interestingly, we found that IRF1-AS was a potent positive regulator of IFN-ß and ISG production, which can significantly inhibit PPRV replication in host cells. In addition, our data revealed that IRF1-AS was positively correlated with its potential target gene, IRF1, which enhanced the activation of IRF3 and the expression of ISGs and interacted with IRF3. This study suggests that IRF1-AS could be a new host factor target for developing antiviral therapies against PPRV infection.

Prevalence and distribution of human papillomavirus (HPV) in Luoyang city of Henan province during 2015-2021 and the genetic variability of HPV16 and 52.

BACKGROUND: Persistent high-risk Human papillomavirus (HPV) subtypes infection has been implicated as a causative of cervical cancer. Distribution and genotypes of HPV infection among females and their variations would assist in the formulation of preventive strategy for cervical cancer. The purpose of the present study is to investigate the prevalence of HPV among females in central China. METHODS: The distribution and genotypes of HPV among 9943 females attending the gynecological examinations in central of China during 2015-2021 were investigated. HPV genotypes were detected using a commercial kit. Nucleotides sequences of L1, E6 and E7 genes in HPV16 or HPV52 positive samples collected in 2021 were amplified by polymerase chain reaction (PCR). Variations of L1, E6 and E7 in HPV16 and HPV52 were gained by sequencing and compared with the reference sequence. Sublineages of HPV16 and HPV52 were determined by the construction of phylogenetic tree based on L1 gene. RESULTS: The overall prevalence of HPV infection was 22.81%, with the infection rate of high-risk human papillomavirus (HR-HPV) was 19.02% and low-risk human papillomavirus (LR-HPV) was 6.40%. The most top five genotypes of HPV infection were HPV16 (7.49%), HPV52 (3.04%), HPV58 (2.36%), HPV18 (1.65%) and HPV51 (1.61%). Plots of the age-infection rate showed that the single HPV, multiple HPV, HR-HPV, LR-HPV infection revealed the same tendency with two peaks of HPV infection were observed among females aged ≤ 20 year-old and 60-65 year-old. The predominant sublineage of HPV16 was A1 and B2 for HPV52. For HPV16, The most prevalent mutations were T266A (27/27) and N181T (7/27) for L1, D32E for E6 and S63F for E7 in HPV16. For HPV52, all of the nucleotide changes were synonymous mutation in L1 (except L5S) and E7 genes. The K93R mutation was observed in most HPV52 E6 protein. CONCLUSIONS: The present study provides basic information about the distribution, genotypes and variations of HPV among females population in Henan province, which would assist in the formulation of preventive strategies and improvements of diagnostic probe and vaccine for HPV in this region.

Establishment and application of a competitive enzyme-linked immunosorbent assay differentiating PCV2 antibodies from mixture of PCV1/PCV2 antibodies in pig sera.

BACKGROUND: Porcine cirovirus type 1 (PCV1) and type 2 (PCV2) are circulating in Chinese pig herds and the infected pigs develop antibodies to both viruses. Current commercial available ELISA kits cannot differentiate PCV2-specific antibodies from the mixtures of PCV1 and PCV2 antibodies in PCV1/2-infected or PCV2-vaccinated pigs. Therefore, the need for developing PCV2-specific ELISA methods is urgent to evaluate PCV2 antibody level in exclusion of PCV1 antibody interference after PCV2 vaccination. RESULTS: Virus-like particles (VLPs) of PCV2 based on the recombinant Cap protein were expressed in Escherichia coli. A competing ELISA was established by using the VLPs as coating antigen and a PCV2-specific monoclonal antibody as the competing antibody. The competing ELISA was compared with the results obtained by using an immunoperoxidase monolayer assay on 160 serum samples. The sensitivity and specificity of this competing ELISA were determined as 96.5 and 96.0 %, at 2 standard deviation from the mean or 91.8 and 100 % at 3 standard deviations from the mean. Next, a serological survey of 1297 vaccinated serum samples collected from commercial pig herds in Beijing, Hunan and Henan provinces in China was conducted. The results showed that 85.9 % of sera having positive PCV2 antibodies. CONCLUSIONS: The competing ELISA we developed in this study was both sensitive and specific to PCV2 and was suitable for large-scale PCV2 antibody monitoring in exclusion of PCV1 antibody interference after PCV2 vaccination.