A multiplex polymerase chain reaction (mPCR) assay was developed to detect and distinguish feline panleukopenia virus (FPV), feline bocavirus (FBoV) and feline astrovirus (FeAstV). Three pairs of primers were designed based on conserved regions in the genomic sequences of the three viruses and were used to specifically amplify targeted fragments of 237 bp from the VP2 gene of FPV, 465 bp from the NP1 gene of FBoV and 645 bp from the RdRp gene of FeAstV. The results showed that this mPCR assay was effective, because it could detect at least 2.25-4.04 × 104 copies of genomic DNA of the three viruses per μl, was highly specific, and had a good broad-spectrum ability to detect different genotypes of the targeted viruses. A total of 197 faecal samples that had been screened previously for FeAstV and FBoV were collected from domestic cats in northeast China and were tested for the three viruses using the newly developed mPCR assay. The total positive rate for these three viruses was 59.89% (118/197). From these samples, DNA from FPV, FBoV and FeAstV was detected in 73, 51 and 46 faecal samples, respectively. The mPCR testing results agreed with the routine PCR results with a coincidence rate of 100%. The results of this study show that this mPCR assay can simultaneously detect and differentiate FPV, FBoV and FeAstV and can be used as an easy, specific and efficient detection tool for clinical diagnosis and epidemiological investigation of these three viruses.

/pdf/development-and-application-of-a-multiplex-pcr-method-for-52jg3vmhh5.pdf

Development and application of a multiplex PCR method for the simultaneous detection and differentiation of feline panleukopenia virus, feline bocavirus, and feline astrovirus.

Multiple tissue necrosis is one of the morphological features of selenium deficiency-mediated injury. MicroRNA (miRNA) participates in the occurrence and development of necroptosis by regulating target genes. Necroptosis is a programmed form of necrosis, and it is closely related to lipopolysaccharide (LPS)-induced injury. Our aim was to investigate whether Se deficiency can promote tracheal injury caused by LPS through miRNA-induced necroptosis. By establishing models of tracheal injury in Se-deficient chickens, we verified the targeting relationship between chicken-derived miR-16-5p and PI3K through bioinformatics, qRT-PCR and WB analyses, and we measured the changes in the expression of genes related to the PI3K/AKT pathway, RIP3/MLKL pathway and MAPK pathway and of heat shock proteins. Under the condition of Se deficiency, the following results were observed: PI3K/AKT expression decreased with the upregulation of miR-16-5p, the expression of necroptosis-related factors (TNF-α, RIP1, FADD, RIP3 and MLKL) increased, and the expression of Caspase 8 significantly decreased (p < 0.05). Light microscopy observations indicated that cell necrosis was the main pathological change due to Se deficiency injury in the tracheal epithelium. The MAPK pathway was activated, and HSP expression was upregulated, indicating that the MAPK pathway and HSPs are both involved in Se deficiency-mediated necroptosis. In addition, Se deficiency promoted the expression of necroptosis-related genes in LPS-treated chickens (p < 0.05), and the pathological changes of cell necrosis were more obvious. In conclusion, we demonstrated that Se deficiency regulates the miR-16-5p-PI3K/AKT pathway and exacerbates LPS-induced necroptosis in chicken tracheal epithelial cells by activating necroptosis-related genes.

/pdf/selenium-deficiency-exacerbates-lps-induced-necroptosis-by-346zww5az8.pdf

Selenium deficiency exacerbates LPS-induced necroptosis by regulating miR-16-5p targeting PI3K in chicken tracheal tissue

Discovery of anti-flu substances and mechanism of Shuang-Huang-Lian water extract based on serum pharmaco-chemistry and network pharmacology

Feline kobuvirus (FeKoV) is a newly discovered organism, classified under the species Aichivirus A of the genus Kobuvirus. Since it was first reported in 2013, molecular evidence for FeKoV in the feline population has been restricted to two countries: Korea and Italy. In this study, we collected faecal samples from cats in southern China and detected the FeKoV RNA in these samples. A prevalence rate of 9.9% (8/81) was identified by RT-PCR, and all positive samples were obtained from diarrhoeic animals. In addition, FeKoV was shown positive associated with diarrhoea in cats, with a correlation coefficient of 0.25. Next, we designed three primer pairs with degenerate bases, which targeted the conservative overlapping region of the entire published FeKoV genome, and sequenced the near-complete genome of the first Chinese field FeKoV strain, WHJ-1, using long-fragment PCR. Finally, we analysed WHJ-1's homology and phylogeny using the polyprotein gene. The results indicated that FeKoV has rapidly mutated since it was first discovered. This study will help to better understand FeKoV's epidemiology, evolutionary pattern and genetic diversity.

First report and genetic characterization of feline kobuvirus in diarrhoeic cats in China.

Host miRNAs are known to modulate the cell response to virus infections. We characterized the miRNA-targeted transcriptome of porcine alveolar macrophages (PAMs) at early times after infection with a subtype 1.1 strain of PRRSV (Porcine Reproductive and Respiratory Syndrome Virus). We performed the immunoprecipitation of RISC (RNA-induced Silencing Complex) followed by microarray analysis of the RISC-bound miRNA targets (RIP-Chip) to evaluate the relative enrichment or depletion of expressed genes in RISC. The miRNA-mediated regulation occurred early after PRRSV infection and decreased fast (1,241 and 141 RISC-bound genes at 7 h and 10 h post-infection, respectively); it affected several cell functions with evidence of miRNA buffering of upregulated interferon-related genes. Eight miRNAs were highly enriched in RISC of both control and infected cells with no evidence of differential expression. Although miR-335-5p was the miRNA with most predicted targets among enriched RISC-bound genes, no effects on surface markers, cytokine expression and PRRSV replication were detected upon miR-335-5p mimics of primary PAMs. Our results do not point to specific miRNA-driven mechanisms regulating the early response to infection with this PRRSV 1.1 strain and indicate that the miRNome expressed by steady-state PAMs reacts promptly to counterbalance PRRSV infection by a pervasive modulation of host functions.

/pdf/the-mirna-targeted-transcriptome-of-porcine-alveolar-3qdtl57m11.pdf

The miRNA-targeted transcriptome of porcine alveolar macrophages upon infection with Porcine Reproductive and Respiratory Syndrome Virus.

Comparative pathogenesis of H3N2 canine influenza virus in beagle dogs challenged by intranasal and intratracheal inoculation.

The canine influenza virus (CIV) outbreaks have raised concerns as they pose a threat to the health of dogs. The successful construction of a canine influenza (CI) infection model is essential to study the CIV. Here we investigated the pathogenicity of different infectious doses of H3N2 CIV in Beagle dogs. Thirty-seven healthy Beagle dogs were used in the experiment and were infected with 103, 104, 105, and 106 50% egg-infectious doses (EID50). Compared to the dogs in the other three groups, those in the 106 EID50 group presented with obvious clinical symptoms, high virus titer, and typical pathological changes. Considering the ensemble of clinical scores, body temperature, virus shedding, lung lesions, pathological section scores, and visceral virus titers, we determined that 106 EID50 is the minimum infectious dose for the Beagle infection model. The other three infectious doses had almost no clinical symptoms. These results indicate that 106 EID50 is the minimum infectious dose of H3N2 CIV that can cause obvious clinical manifestations and pathological changes associated with CI in Beagle dogs. The theoretical framework developed in this research will guide the establishment of an infection model of CIV for future research.

/pdf/comparison-of-pathogenicity-of-different-infectious-doses-of-189niik7hx.pdf

Comparison of Pathogenicity of Different Infectious Doses of H3N2 Canine Influenza Virus in Dogs.

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.

/pdf/integrated-lung-and-tracheal-mrna-seq-and-mirna-seq-analysis-6ha4dh660a.pdf

Integrated Lung and Tracheal mRNA-Seq and miRNA-Seq Analysis of Dogs with an Avian-Like H5N1 Canine Influenza Virus Infection

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Papers

First report and genetic characterization of feline kobuvirus in diarrhoeic cats in China.

Comparative pathogenesis of H3N2 canine influenza virus in beagle dogs challenged by intranasal and intratracheal inoculation.

Comparison of Pathogenicity of Different Infectious Doses of H3N2 Canine Influenza Virus in Dogs.

Integrated Lung and Tracheal mRNA-Seq and miRNA-Seq Analysis of Dogs with an Avian-Like H5N1 Canine Influenza Virus Infection