Data_Sheet_1_Equine Parvovirus-Hepatitis in China: Characterization of Its Genetic Diversity and Evidence for Natural Recombination Events Between the Chinese and American Strains.docx

Equine parvovirus-hepatitis (EqPV-H) was first reported in a horse that died of equine serum hepatitis in the USA in 2018, and was determined having a strong association with equine serum hepatitis in the following studies. As a newly discovered virus, the genomic sequences of only seven EqPV-H strains have been reported. Considering this, an epidemiological study was performed to investigate the prevalence of EqPV-H in equines in Guangdong Province in China, and obtain genomic sequences of the field prevalent EqPV-H strains. The detection rate of EqPV-H was finally determined to be 8.33% (95% CI: 2.8–18.4%), and EqPV-H's coinfection with equine hepacivirus and equine pegivirus was also determined. Then, the genomes of the Chinese field EqPV-H strains were obtained by PCR, sequencing, and assembly. Through bootscanning analysis, Simplot analysis, and phylogenetic analysis, strong evidence for natural recombination events were found in two Chinese field EqPV-H strains. The natural recombination events occurred between the Chinese and American strains, and were determined within VP protein. Finally, the genetic distance of EqPV-H strains was investigated. Nucleotide identities of 97.1–99.9% and 95.2–100% were found for NS and VP between EqPV-H strains, respectively. Together with other molecular evidence obtained in the present study, the genetic diversity of EqPV-H was determined. Taken together, the results of the present study expand our knowledge on the epidemiological characteristics, genetic variability, and evolution of EqPV-H.</p

Data_Sheet_1_Effects of the PA-X and PB1-F2 Proteins on the Virulence of the 2009 Pandemic H1N1 Influenza A Virus in Mice.docx

There have been several previous reports showing that PA-X and PB1-F2 proteins can regulate innate immune responses and may play roles in the adaptation of influenza viruses to new hosts. In this research, we investigated, for the first time, the combined effects of PA-X and PB1-F2 proteins on viral virulence in mice. Based on the 2009 pH1N1 A/Guangdong/1057/2010 virus backbone, four viruses encoding different combinations of full-length or truncated PA-X and PB1-F2 proteins were rescued by a reverse genetic engineering system. We analyzed viral replication, host-shutoff activity, in vitro viral pathogenicity and in vivo host immune response. We found that simultaneously expressing the full-length PA-X and PB1-F2 proteins enhanced viral replication in vitro through increasing the accumulation of the RNP complex protein and enhanced viral pathogenicity in mice during the early stage of infection. Furthermore, PA-X and PB1-F2 simultaneously regulated the host innate response, and different forms of PB1-F2 proteins may have impacts on the host shutoff activity induced by the PA-X protein. Our results provide a better understanding of the mechanisms of PA-X and PB1-F2 proteins during viral replication, pathogenicity and host immune response.</p

Additional file 1 of Congenital heart diseases with airway stenosis: a predictive nomogram to risk-stratify patients without airway intervention

Supplementary Material

Additional file 2 of Congenital heart diseases with airway stenosis: a predictive nomogram to risk-stratify patients without airway intervention

Supplementary Material

DataSheet_1_Investigating Influenza Virus Polymerase Activity in Feline Cells Based on the Influenza Virus Minigenome Replication System Driven by the Feline RNA Polymerase I Promoter.doc

Emerging influenza virus poses a health threat to humans and animals. Domestic cats have recently been identified as a potential source of zoonotic influenza virus. The influenza virus minigenome replication system based on the ribonucleic acid (RNA) polymerase I (PolI) promoter is the most widely used tool for investigating polymerase activity. It could help determine host factors or viral proteins influencing influenza virus polymerase activity in vitro. However, influenza virus polymerase activity has never been studied in feline cells thus far. In the present study, the feline RNA PolI promoter was identified in the intergenic spacer regions between adjacent upstream 28S and downstream 18S rRNA genes in the cat (Felis catus) genome using bioinformatics strategies. The transcription initiation site of the feline RNA PolI promoter was predicted. The feline RNA PolI promoter was cloned from CRFK cells, and a promoter size of 250 bp contained a sequence with sufficient PolI promoter activity by a dual-luciferase reporter assay. The influenza virus minigenome replication system based on the feline RNA PolI promoter was then established. Using this system, the feline RNA PolI promoter was determined to have significantly higher transcriptional activity than the human and chicken RNA PolI promoters in feline cells, and equine (H3N8) influenza virus presented higher polymerase activity than human (H1N1) and canine (H3N2) influenza viruses. In addition, feline myxovirus resistance protein 1 (Mx1) and baloxavir were observed to inhibit influenza virus polymerase activity in vitro in a dose-dependent manner. Our study will help further investigations on the molecular mechanism of host adaptation and cross-species transmission of influenza virus in cats.</p

Additional file 3 of Congenital heart diseases with airway stenosis: a predictive nomogram to risk-stratify patients without airway intervention

Supplementary Material

Data_Sheet_2.docx

<p>Avian-like H5N1 canine influenza virus (CIV) causes severe respiratory infections in dogs. However, the mechanism underlying H5N1 CIV infection in dogs is unknown. The present study aimed to identify differentially expressed miRNAs and mRNAs in the lungs and trachea in H5N1 CIV-infected dogs through a next-generation sequencing-based method. Eighteen 40-day-old beagles were inoculated intranasally with CIV, A/canine/01/Guangdong/2013 (H5N1) at a tissue culture infectious dose 50 (TCID₅₀) of 10⁶, and lung and tracheal tissues were harvested at 3 and 7 d post-inoculation. The tissues were processed for miRNA and mRNA analysis. By means of miRNA-gene expression integrative negative analysis, we found miRNA–mRNA pairs. Lung and trachea tissues showed 138 and 135 negative miRNA–mRNA pairs, respectively. One hundred and twenty negative miRNA–mRNA pairs were found between the different tissues. In particular, pathways including the influenza A pathway, chemokine signaling pathways, and the PI3K-Akt signaling pathway were significantly enriched in all groups in responses to virus infection. Furthermore, dysregulation of miRNA and mRNA expression was observed in the respiratory tract of H5N1 CIV-infected dogs and notably, TLR4 (miR-146), NF-κB (miR-34c) and CCL5 (miR-335), CCL10 (miR-8908-5p), and GNGT2 (miR-122) were found to play important roles in regulating pathways that resist virus infection. To our knowledge, the present study is the first to analyze miRNA and mRNA expression in H5N1 CIV-infected dogs; furthermore, the present findings provide insights into the molecular mechanisms underlying influenza virus infection.</p

Data_Sheet_1.XLSX

<p>Avian-like H5N1 canine influenza virus (CIV) causes severe respiratory infections in dogs. However, the mechanism underlying H5N1 CIV infection in dogs is unknown. The present study aimed to identify differentially expressed miRNAs and mRNAs in the lungs and trachea in H5N1 CIV-infected dogs through a next-generation sequencing-based method. Eighteen 40-day-old beagles were inoculated intranasally with CIV, A/canine/01/Guangdong/2013 (H5N1) at a tissue culture infectious dose 50 (TCID₅₀) of 10⁶, and lung and tracheal tissues were harvested at 3 and 7 d post-inoculation. The tissues were processed for miRNA and mRNA analysis. By means of miRNA-gene expression integrative negative analysis, we found miRNA–mRNA pairs. Lung and trachea tissues showed 138 and 135 negative miRNA–mRNA pairs, respectively. One hundred and twenty negative miRNA–mRNA pairs were found between the different tissues. In particular, pathways including the influenza A pathway, chemokine signaling pathways, and the PI3K-Akt signaling pathway were significantly enriched in all groups in responses to virus infection. Furthermore, dysregulation of miRNA and mRNA expression was observed in the respiratory tract of H5N1 CIV-infected dogs and notably, TLR4 (miR-146), NF-κB (miR-34c) and CCL5 (miR-335), CCL10 (miR-8908-5p), and GNGT2 (miR-122) were found to play important roles in regulating pathways that resist virus infection. To our knowledge, the present study is the first to analyze miRNA and mRNA expression in H5N1 CIV-infected dogs; furthermore, the present findings provide insights into the molecular mechanisms underlying influenza virus infection.</p

Identification and genetic characterization of a novel parvovirus associated with serum hepatitis in horses in China

A novel equine parvovirus, equine parvovirus-hepatitis (EqPV-H), was first discovered in a horse that died of equine serum hepatitis in the USA in 2018. EqPV-H was shown to be a novel etiological agent associated with equine serum hepatitis. Following this initial report, no additional studies on EqPV-H have been published. In this study, a total of 143 serum samples were collected from racehorses at 5 separate farms in China and were analyzed to detect EqPV-H DNA via nested PCR. The results indicated a high prevalence of EqPV-H (11.9%, 17/143) in the studied animals. In addition, a remarkably high coinfection rate (58.8%, 10/17) with 2 equine flaviviruses (equine hepacivirus and equine pegivirus) was observed in the EqPV-H positive equines. However, all equines tested negative for Theiler’s disease-associated virus, an etiological agent associated with equine serum hepatitis. The genomes of six field EqPV-H strains were sequenced and analyzed, with the results indicating that the Chinese EqPV-H strains have low genetic diversity and high genetic similarity with the USA EqPV-H strain BCT-01. A phylogenetic analysis demonstrated that the Chinese EqPV-H strains clustered with BCT-01 in the genus Copiparvovirus but were distantly related to another equine parvovirus identified in horse cerebrospinal fluid. In addition, liver enzyme levels were detected in the EqPV-H positive serum samples, and all the values were in the normal range, indicating that infection can occur without concurrent liver disease. This study will promote an understanding of the geographical distribution, genetic diversity, and pathogenicity of EqPV-H.</p

Data_Sheet_1_Comparative Analysis of Whole-Transcriptome RNA Expression in MDCK Cells Infected With the H3N2 and H5N1 Canine Influenza Viruses.ZIP

This study aimed to detect changes in the complete transcriptome of MDCK cells after infection with the H5N1 and H3N2 canine influenza viruses using high-throughput sequencing, search for differentially expressed RNAs in the transcriptome of MDCK cells infected with H5N1 and H3N2 using comparative analysis, and explain the differences in the pathogenicity of H5N1 and H3N2 at the transcriptome level. Based on the results of our comparative analysis, significantly different levels of expression were found for 2,464 mRNAs, 16 miRNAs, 181 lncRNAs, and 262 circRNAs in the H3N2 infection group and 448 mRNAs, 12 miRNAs, 77 lncRNAs, and 189 circRNAs in the H5N1 infection group. Potential functions were predicted by performing Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses of the target genes of miRNAs, lncRNAs and circRNAs, and the ncRNA-mRNA regulatory network was constructed based on differentially expressed RNAs. A greater number of pathways regulating immune metabolism were altered in the H3N2 infection group than in the H5N1 infection group, which may be one reason why the H3N2 virus is less pathogenic than is the H5N1 virus. This study provides detailed data on the production of ncRNAs during infection of MDCK cells by the canine influenza viruses H3N2 and H5N1, analyzed differences in the total transcriptomes between H3N2- and H5N1-infected MDCK cells, and explained these differences with regard to the pathogenicity of H3N2 and H5N1 at the transcriptional level.</p