Combined insertion of basic and non-basic amino acids at hemagglutinin cleavage site of highly pathogenic H7N9 virus promotes replication and pathogenicity in chickens and mice.

Since mid-2016, the low pathogenic H7N9 influenza virus has evolved into a highly pathogenic (HP) phenotype in China, raising many concerns about public health and poultry industry. The insertion of a "KRTA" motif at hemagglutinin cleavage site (HACS) occurred in the early stage of HP H7N9 variants. During the co-circulation, the HACS of HP-H7N9 variants were more polymorphic in birds and humans. Although HP-H7N9 variants, unlike the H5 subtype virus, exhibited the insertions of basic and non-basic amino acids, the underlying function of those insertions and substitutions remains unclear. The results of bioinformatics analysis indicated that the PEVPKRKRTAR/G motif of HACS had become the dominant motif in China. Then, we generated six H7N9 viruses bearing the PEIPKGR/G, PEVPKGR/G, PEVPKRKRTAR/G, PEVPKGKRTAR/G, PEVPKGKRIAR/G, and PEVPKRKRR/G motifs. Interestingly, after the deletion of threonine and alanine (TA) at HACS, the H7N9 viruses manifested decreased thermostability and virulence in mice, and the PEVPKRKRTAR/G-motif virus is prevalent in birds and humans probably due to its increased transmissibility and moderate virulence. By contrast, the insertion of non-basic amino acid isoleucine and alanine (IA) decreased the transmissibility in chickens and virulence in mice. Remarkably, the I335V substitution of H7N9 virus enhanced infectivity and transmission in chickens, suggesting that the combination of mutations and insertions of amino acids at the HACS promoted replication and pathogenicity in chickens and mice. The ongoing evolution of H7N9 increasingly threatens public health and poultry industry, so, its comprehensive surveillance and prevention of H7N9 viruses should be pursued.

Development and evaluation of a meat mitochondrial metagenomic (3MG) method for composition determination of meat from fifteen mammalian and avian species.

BACKGROUND: Bioassessment and biomonitoring of meat products are aimed at identifying and quantifying adulterants and contaminants, such as meat from unexpected sources and microbes. Several methods for determining the biological composition of mixed samples have been used, including metabarcoding, metagenomics and mitochondrial metagenomics. In this study, we aimed to develop a method based on next-generation DNA sequencing to estimate samples that might contain meat from 15 mammalian and avian species that are commonly related to meat bioassessment and biomonitoring. RESULTS: In this project, we found the meat composition from 15 species could not be identified with the metabarcoding approach because of the lack of universal primers or insufficient discrimination power. Consequently, we developed and evaluated a meat mitochondrial metagenomics (3MG) method. The 3MG method has four steps: (1) extraction of sequencing reads from mitochondrial genomes (mitogenomes); (2) assembly of mitogenomes; (3) mapping of mitochondrial reads to the assembled mitogenomes; and (4) biomass estimation based on the number of uniquely mapped reads. The method was implemented in a python script called 3MG. The analysis of simulated datasets showed that the method can determine contaminant composition at a proportion of 2% and the relative error was < 5%. To evaluate the performance of 3MG, we constructed and analysed mixed samples derived from 15 animal species in equal mass. Then, we constructed and analysed mixed samples derived from two animal species (pork and chicken) in different ratios. DNAs were extracted and used in constructing 21 libraries for next-generation sequencing. The analysis of the 15 species mix with the method showed the successful identification of 12 of the 15 (80%) animal species tested. The analysis of the mixed samples of the two species revealed correlation coefficients of 0.98 for pork and 0.98 for chicken between the number of uniquely mapped reads and the mass proportion. CONCLUSION: To the best of our knowledge, this study is the first to demonstrate the potential of the non-targeted 3MG method as a tool for accurately estimating biomass in meat mix samples. The method has potential broad applications in meat product safety.

[Rapid detection of novel avian influenza virus subtype H7N9 by multiplex real-time RT-PCR].

In order to develop a rapid detection kit for novel avian influenza virus (AIV) subtype H7N9, two sets of specific primers and probes were designed based on the nucleotide sequences of hemagglutinin antigen (HA) and neuraminidase antigen (NA) of novel H7N9 virus (2013) available in GenBank to establish the method of TaqMan probe-based multiplex real-time RT-PCR for rapid detection of AIV subtype H7N9. The primer and probe of HA were for all H7 subtype AIVs, while the primer and probe of NA were only for novel N9 subtype AIVs. The results showed that this method had high sensitivity and specificity. This method was applicable to the testing of positive standard sample with a minimum concentration of 10 copies/microL; it not only distinguished H7 subtype from H1, H3, H5, H6, and H9 subtypes, but also distinguished novel N9 subtype from traditional N9 subtype. A total of 2700 samples from Zhuhai, China were tested by this method, and the results were as expected. For the advantages of sensitivity and specificity, the method holds promise for wide application.

[Inhibition of proliferation of H5N1 subtype AIV in CEF by chemosynthetic siRNA].

In order to study the proliferation inhibition effect of H5N1 subtype avian influenza virus (AIV) with small interfere RNA (siRNA), a total of 4 siRNAs were designed in accordance with the NP and PA genes of H5N1 subtype AIV, the siRNAs were then transfected to chicken embryo fibroblast(CEF), CEF was infected with H5N1 subtype AIV after 6 hrs. Virus titer of cell supernatant was tested at 16-56hrs post infection, and pathological changes of the cells was observed; mRNA levels of NP, PA, HA and p13-actin gene were tested at 36hrs post infection. The results showed that these 4 siRNAs could inhibit the prolif-eration of H5N1 subtype AIV in CEF in varying degrees, and one siRNA targeting PA was best per-formed. The experimental results also showed that the inhibition effect was decreased with the time prolonged. This research provides a basis for further studying RNAi on AIV prevention and control.