In Vivo Therapy with M2e-Specific IgG Selects for an Influenza A Virus Mutant with Delayed Matrix Protein 2 Expression

Broadly protective influenza vaccine candidates may have a higher barrier to immune evasion compared to conventional influenza vaccines. We used Illumina MiSeq deep sequence analysis to study the mutational patterns in A/Puerto Rico/8/34 viruses that evolve in chronically infected SCID mice that were treated with different M2e-specific MAbs.

Influenza A viruses serially passaged in different MDCK cell lines exhibit limited sequence variation across their genomes, with the exception of the hemagglutinin gene

ABSTRACTNew methods for deep sequence analysis provide an opportunity to follow the emergence and dynamics of virus mutations in real time. Although viruses are commonly grown in cell culture for research and for vaccine development, the cells used to grow the virus are often not derived from the same tissue or even the same host that the virus naturally replicates in. The selective pressures of culturing virus in vitro are still only partially understood. MDCK cells are the standard cell for growing influenza viruses yet are derived from the epithelium of the canine kidney and are also heterogenous. We passaged human H3N2, H1N1 pandemic, and canine H3N2 influenza A viruses (IAV) in different lineages of MDCK cells, as well as lines engineered to express variant Sia receptors, including α2,3- and α2,6-linkages or N-glycolylneuraminic acid (Neu5Gc) or N-acetylneuraminic acid (Neu5Ac) forms. MDCK-Type II cells had lower infection efficiency and virus production, and infection appeared more dependent on protease activation of the virus. When viruses were passaged in the different cells, they exhibited only small numbers of consensus-level mutations, and most were within the HA gene. Both human IAVs showed selection for single nucleotide minority variants in the HA stem across cell types, as well as low frequency variants in the HA receptor binding site of virus passaged in cells expressing Neu5Gc. Canine H3N2 also showed minority variants near the receptor-binding site in cells expressing Neu5Gc and also in those expressing α2,6-linkages.IMPORTANCEThe genetic variation and adaptability of viruses are fundamental properties that allow their evolutionary success in the face of differing host environments and immune responses. The growth of viruses in cell culture is widely used for their study and for preparing vaccines. However, the selection pressures that cell passaging imposes on viruses are often poorly understood. We used deep sequence analysis to define, in detail, how three different influenza A viruses respond to passaging in different lineages of canine MDCK cells that are commonly used for their growth, as well as in variant cells engineered to express different forms of their cell surface receptor, sialic acid. This analysis revealed that most mutations occur in the HA gene and few sequence changes in the virus population reached high proportions. This is relevant for understanding the selective pressures of virus growth in cell culture and how it shapes evolutionary patterns.

The reduction of miR146b-5p in monocytes and T cells could contribute to the immunopathogenesis of hepatitis C virus infection

It has been reported that various kinds of miRNAs could affect the pathogenesis of hepatitis C virus infection. Recently, our group reported that deep-sequencing analysis was useful to detect disease-specific miRNAs. The aim of this study is to identify the HCV-specific miRNAs that could contribute to the immunopathogenesis of HCV by using clinical samples and in vitro analysis. Five miRNAs (hsa-miR181a-2-3p, hsa-miR-374a-3p, hsa-miR374a-5p, hsa-miR-204-5p and hsa-miR146b-5p) were shown to be significantly downregulated in CH-C by deep sequence analysis. The average ratio (PBMCs miRNAs/serum miRNAs) of hsa-miR146b-5p was highest among all the miRNAs. Moreover, serum hsa-miR146b-5p was significantly down-regulated in CH-C patients in comparison to CH-B patients and healthy subjects. The expression of hsa-miR146b-5p in CD3+ T cells and CD14+ monocytes of CH-C patients was significantly lower than that of the other groups. The hsa-miR146b-5p expression in CD14+ monocytes of SVR patients treated with Peg-IFN/RBV was significantly higher than in those of non-SVR patients treated with Peg IFN/RBV. However, the hsa-miR146b-5p expression in CD14+ monocytes of SVR patients treated with DCV and ASV was comparable to that in monocytes of non-SVR patients treated with DCV and ASV. Moreover, the expression levels of hsa-miR146b-5p in CD14+ monocytes were significantly increased after achieving SVR and 1(OH)Vitamin D3 treatment. Further, the expression of HCV-Core could suppress miR146b-5p expression in immune cells and affect the expression of various kinds of cytokines by affecting the NF-κB signaling. In conclusion, the reduction of miR146b-5p in monocytes and T cells could contribute to the immunopathogenesis of hepatitis C virus infection.