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ObjectiveThe aim of this study was to detect botulinum toxin and Clostridium botulinum （C.botulinum） in 30 batches of infant formula milk powder obtained from an enterprise and to analyze the whole genome of the strain of C. botulinum type B isolated from the samples.MethodsThe samples were subjected to C. botulinum isolation and botulinum toxin typing experiments according to the “National standard for food safety microbiological examination of C. botulinum and botulinum toxin” （GB 4789.12—2016）. The whole genomes of the isolated strains were sequenced and their genetic characteristics were analyzed.ResultsBotulinum toxin was not detected in any of the 30 batches of samples. After intraperitoneal injection of the enrichment solution into mice， four batches of milk powder samples induced symptoms of botulism， but C. botulinum was isolated from only one batch of milk powder samples. Genome sequencing revealed that the bacterium was C. botulinum group Ⅰ， type B， the toxin gene cluster was type Ha， and the toxin gene was subtype B2.ConclusionThe detection of C. botulinum in infant formula milk powder with a complex background of microorganisms should not be based on the isolation of bacteria as the gold standard but should be confirmed by a mouse toxicity test of the enrichment solution combined with a botulinum toxin antiserum protection test. Whole genome sequencing can accurately identify strains and analyze relevant genetic characteristics， providing reliable technical support for handling poisoning events.

The nucleoprotein (NP) of influenza A virus plays a crucial role in virus replication, infectivity, and host adaptation. As a major component of the viral ribonucleoprotein complexes (vRNP), NP initiates vRNP shuttling between the nucleus and cytoplasm in the host cell. However, the characteristics of the nucleocytoplasmic shuttling of NP from H1N1 influenza A virus still remain unclear. In the present study, the subcellular localization and the related key residues of the H1N1 influenza virus NP were identified and evaluated. The NP of influenza virus A/WSN/33 (H1N1; WSN) displayed a more obvious nuclear accumulation than A/Anhui/1/2013 (H7N9; AH) and A/chicken/Shandong/lx1023/2007 (H9N2; SD). NP residue K4, located in NLS1, and residue F253, located in NES3, from WSN NP are not conserved in H7N9 and H9N2, which instead encode Q4 and I253, respectively. Crucially, these residues are involved in the regulation of NP nucleocytoplasmic shuttling through interactions with CRM1 and importin-α. Moreover, residues at position 253 also play important roles in the replication of the virus, resulting in an increase in vRNP polymerase activity and an alteration of the cell tropism and pathogenicity in mice. The present data revealed a pivotal role of the Q4 and I253 residues of NP from H7N9 in enhancing the cytoplasmic accumulation of NP and vRNP activity compared to the K4 and F253 residues in WSN-NP. In addition, an F253I substitution in the NP of WSN altered the survival ratio of infected mice and the growth curve in infected avian-origin cells (DF-1). The current data indicate that the F253I mutation results in attenuated pathogenicity of the virus in mice and altered cell tropism. The present study demonstrated the dissimilarity in subcellular NP transport processes between H1N1 virus WSN and other influenza A virus strains, as well as uncovered the mechanism responsible for this difference. [ABSTRACT FROM AUTHOR]

IFIT family genes are a kind of interferon stimulated genes (ISGs), and play important roles in antiviral sector and immunity regulation. To study the regulatory effect of IFIT family genes during influenza A virus (IAV) infection, we used RNA-sequencing analysis (RNA-Seq) technique and found that when 293T cells were infected by A/WSN/33 (WSN), the concentration of IFIT family genes were increased. Further study reveals that overexpression of IFIT2 or IFIT3 could inhibit IAV replication and transcription, and cause the dose-dependent inhibition of polymerase activity of vRNP. In addition, IFIT2 and IFIT3 encoding protein could colocalize with NS1 in 293T cells infected by WSN, indicating that they might interact with each other. The results suggest that IFIT family genes can inhibit the replication and transcription of IAV, which contributes to our understanding of the regulatory effect of host factors during influenza virus infection.

Objective: To express and purify Porcine Reproductive and Respiratory Syndrome Virus Nsp2 protein and analyze the protease activity of Nsp2., Methods: N-terminus and C-terminus of nsp2 gene were amplified by PCR and inserted into expression vector pET21a(+), respectively. The recombinant protein (Nsp2-N and Nsp2-C) were over expressed in E. coli BL21 and purified by Ni-NTA agarose affinity chromatogram and gel filtration. There is a cysteine protease domain (CP) in Nsp2-N through genetic alignment. In this study, the protease activity of Nsp2-N in cis was analyzed by western blot. Additionally, the predicted peptide substrate were synthesized, the protease activity in trans was analyzed by peptide cleavage assay in vitro., Results: Two soluble recombinant protein were successfully expressed and purity reached up to 90% after purification. The putative protease domain of Nsp2-N couldn't cleave the predicted substrate through cis cleavage and trans cleavage., Conclusion: The putative protease domain in Nsp2-N may need other host factors to act as cofactor, which supply basis for further identification of biological activity and screening of anti-virus drug.