Role and Importance of NS1 Protein of Avian Influenza Virus to Grow in the Presence of Interferon and Evaluation of the NS1 Mutant Viruses as Potential DIVA Vaccines

A proper vaccination program can play a critical role in prevention and control of avian influenza (AI) in commercial poultry. Low pathogenic avian influenza viruses (LPAIV) of H5 and H7 AI subtypes cause serious economic losses to the poultry industry and have the potential to mutate to highly pathogenic AI (HPAI) strains. Due to trade implications, differentiation of infected from vaccinated animals (DIVA) is an important issue in the control of AI. Therefore, the development and characterization of vaccine candidates with DIVA properties is critical in improving vaccination programs. Keeping these aspects in mind, we investigated the role of an NS1 mutant virus as a potential live attenuated DIVA vaccine. The NS1 protein of influenza virus plays a major role in blocking the host's antiviral response. Using an eight-plasmid reverse genetics system, we recovered the low pathogenic parental (H5N3) and NS1 mutant (H5N3/NS1/144) viruses. H5N3/NS1/144 expresses only the first 144 amino acids of the NS1 protein compared to the 230 of the parental H5N3. The growth properties of H5N3 and H5N3/NS1/144 were compared in cell culture and in different age embryonated chicken eggs. Our results confirmed that NS1 is involved in down regulation of interferon as shown by IFN-beta mRNA expression analysis and by the inability of H5N3/NS1-144 to efficiently grow in older age, interferon competent, chicken embryos. However with regards to safety the virus reverted to virulence within five back passages in chickens and was therefore not a safe vaccine candidate. However the killed form of H5N3/NS1-144 was a safer alternative and it also induced antibody titers and protection not significantly different from the parental H5N3 as vaccine. To further understand the reversion of H5N3/NS1/144 to virulence, we carried out 3 independent serial passages of H5N3/NS1/144 in increasing age of embryonated chicken eggs and examined the NS1 gene for presence of mutations. RT-PCR and sequence analysis of the NS gene in all three lineages showed the presence of a 54 amino acid deletion resulting in the generation of a 87 amino acids long NS1 ORF with a point mutation (L80V) at the site of deletion. In addition, the NS1 ORF in lineages L2 and L3 presented two additional point mutations in the RNA binding domain (Q40R and T73M). To determine if these mutations played a role in increased virulence, recombinant viruses expressing these mutant NS1 proteins in the background of parental virus were generated by reverse genetics and their replication properties and pathogenicity was examined in vitro, in ovo and in vivo systems. Our results showed that the 87 amino acid long NS1 protein clearly increased virus replication and virulence specifically in interferon competent systems. In addition, the two point mutations in the RNA binding domain of NS1 ORF expressing 87 a protein slightly increased the virus virulence. Overall this study reinforces the role of NS1 in influenza virus pathogenicity and supports the use of killed inactivated NS1 mutant virus vaccines as potential DIVA vaccines

IL-2 and IL-6 cooperate to enhance the generation of influenza-specific CD8 T cells responding to live influenza virus in aged mice and humans

An age-related decline in cytolytic activity has been described in CD8+ T cells and we have previously shown that the poor CD8+ effector T cell responses to influenza A/H3N2 challenge result from a decline in the proportion and function of these cytolytic T lymphocytes (CTL). Here, we describe that addition of exogenous cytokines to influenza-stimulated PBMC from both aged mice and humans, enhances the generation of influenza specific CD8 CTL by increasing their proliferation and survival. Our data show that the addition of IL-2 and IL-6 to splenocytes from mice previously infected with influenza virus restores the aged CD8+ T cell response to that observed in young mice. In humans, IL-2 plus IL-6 also reduces the proportion of apoptotic effector CD8+ T cells to levels resembling those of younger adults. In HLA-A2+ donors, MHC Class I tetramer staining showed that adding both exogenous IL-2 and IL-6 resulted in greater differentiation into influenza-specific effector CD8+ T cells. Since this effect of IL-2/IL-6 supplementation can be reproduced with the addition of Toll-like receptor agonists, it may be possible to exploit this mechanism and design new vaccines to improve the CD8 T cell response to influenza vaccination in older adults

Identification of differentially expressed miRNAs in chicken lung and trachea with avian influenza virus infection by a deep sequencing approach

<p>Abstract</p> <p>Background</p> <p>MicroRNAs (miRNAs) play critical roles in a wide spectrum of biological processes and have been shown to be important effectors in the intricate host-pathogen interaction networks. Avian influenza virus (AIV) not only causes significant economic losses in poultry production, but also is of great concern to human health. The objective of this study was to identify miRNAs associated with AIV infections in chickens.</p> <p>Results</p> <p>Total RNAs were isolated from lung and trachea of low pathogenic H5N3 infected and non-infected SPF chickens at 4 days post-infection. A total of 278,398 and 340,726 reads were obtained from lung and trachea, respectively. And 377 miRNAs were detected in lungs and 149 in tracheae from a total of 474 distinct chicken miRNAs available at the miRBase, respectively. Seventy-three and thirty-six miRNAs were differentially expressed between infected and non-infected chickens in lungs and tracheae, respectively. There were more miRNAs highly expressed in non-infected tissues than in infected tissues. Interestingly, some of these differentially expressed miRNAs, including miR-146, have been previously reported to be associated with immune-related signal pathways in mammals.</p> <p>Conclusion</p> <p>To our knowledge, this is the first study on miRNA gene expression in AIV infected chickens using a deep sequencing approach. During AIV infection, many host miRNAs were differentially regulated, supporting the hypothesis that certain miRNAs might be essential in the host-pathogen interactions. Elucidation of the mechanism of these miRNAs on the regulation of host-AIV interaction will lead to the development of new control strategies to prevent or treat AIV infections in poultry.</p

Integrated analysis of microRNA expression and mRNA transcriptome in lungs of avian influenza virus infected broilers

Abstract Background Avian influenza virus (AIV) outbreaks are worldwide threats to both poultry and humans. Our previous study suggested microRNAs (miRNAs) play significant roles in the regulation of host response to AIV infection in layer chickens. The objective of this study was to test the hypothesis if genetic background play essential role in the miRNA regulation of AIV infection in chickens and if miRNAs that were differentially expressed in layer with AIV infection would be modulated the same way in broiler chickens. Furthermore, by integrating with parallel mRNA expression profiling, potential molecular mechanisms of host response to AIV infection can be further exploited. Results Total RNA isolated from the lungs of non-infected and low pathogenic H5N3 infected broilers at four days post-infection were used for both miRNA deep sequencing and mRNA microarray analyses. A total of 2.6 M and 3.3 M filtered high quality reads were obtained from infected and non-infected chickens by Solexa GA-I Sequencer, respectively. A total of 271 miRNAs in miRBase 16.0 were identified and one potential novel miRNA was discovered. There were 121 miRNAs differentially expressed at the 5% false discovery rate by Fisher’s exact test. More miRNAs were highly expressed in infected lungs (108) than in non-infected lungs (13), which was opposite to the findings in layer chickens. This result suggested that a different regulatory mechanism of host response to AIV infection mediated by miRNAs might exist in broiler chickens. Analysis using the chicken 44 K Agilent microarray indicated that 508 mRNAs (347 down-regulated) were differentially expressed following AIV infection. Conclusions A comprehensive analysis combining both miRNA and targeted mRNA gene expression suggests that gga-miR-34a, 122–1, 122–2, 146a, 155, 206, 1719, 1594, 1599 and 451, and MX1, IL-8, IRF-7, TNFRS19 are strong candidate miRNAs or genes involved in regulating the host response to AIV infection in the lungs of broiler chickens. Further miRNA or gene specific knock-down assay is warranted to elucidate underlying mechanism of AIV infection regulation in the chicken