Yeun-Kyung Shin

SH3 Binding Motif 1 in Influenza A Virus NS1 Protein Is Essential for PI3K/Akt Signaling Pathway Activation

ABSTRACT Recent studies have demonstrated that influenza A virus infection activates the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway by binding of influenza NS1 protein to the p85 regulatory subunit of PI3K. Our previous study proposed that two polyproline motifs in NS1 (amino acids 164 to 167 [PXXP], SH3 binding motif 1, and amino acids 213 to 216 [PPXXP], SH3 binding motif 2) may mediate binding to the p85 subunit of PI3K. Here we performed individual mutational analyses on these two motifs and demonstrated that SH3 binding motif 1 contributes to the interactions of NS1 with p85β, whereas SH3 binding motif 2 is not required for this process. Mutant viruses carrying NS1 with mutations in SH3 binding motif 1 failed to interact with p85β and induce the subsequent activation of PI3K/Akt pathway. Mutant virus bearing mutations in SH3 binding motif 2 exhibited similar phenotype as the wild-type (WT) virus. Furthermore, viruses with mutations in SH3 binding motif 1 induced more severe apoptosis than did the WT virus. Our data suggest that SH3 binding motif 1 in NS1 protein is required for NS1-p85β interaction and PI3K/Akt activation. Activation of PI3K/Akt pathway is beneficial for virus replication by inhibiting virus induced apoptosis through phosphorylation of caspase-9.

The PI3K/Akt pathway inhibits influenza A virus-induced Bax-mediated apoptosis by negatively regulating the JNK pathway via ASK1.

It has previously been reported that influenza A virus infection activates the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. In addition, it has been shown that the mutant influenza A virus PR8-SH3-mf-1, which is unable to activate the PI3K/Akt pathway, is more pro-apoptotic than the wild-type (WT) virus. However, the molecular pathways involved in regulating this process remain unknown. Here, it is reported that, although both WT and PR8-SH3-mf-1 viruses induced apoptosis, the PR8-SH3-mf-1 virus consistently showed greater potential to induce mitochondrial membrane disruption, cytochrome c release, and translocation and conformational change of Bax than the WT virus. Furthermore, the PR8-SH3-mf-1 virus was unable to phosphorylate apoptosis signal-regulating kinase 1 (ASK1) but induced higher levels of c-jun N-terminal kinase (JNK) phosphorylation than the WT virus. Blocking JNK activity could inhibit virus-induced Bax activation and apoptosis. These results reveal that, during influenza A virus infection, the PI3K/Akt pathway negatively regulates the JNK pathway via ASK1, thereby inhibiting JNK-dependent, Bax-mediated apoptosis.

Pandemic H1N1 influenza virus-like particles are immunogenic and provide protective immunity to pigs.

The outbreak of the 2009 influenza pandemic underscored the important role of swine in influenza virus evolution and the emergence of novel viruses with pandemic potential. Vaccination is the most common practice to control swine influenza in swine industry. Influenza virus-like particle (VLP) vaccines are an alternative approach and have been demonstrated to be immunogenic and confer protection against influenza virus challenge in chickens, mice and ferrets. In this study, we generated VLPs consisting of HA, NA and M1 proteins derived from pandemic virus A/California/04/2009 in insect cells. The immunogenicity and efficacy following vaccination of VLPs were evaluated in swine. Our data showed that vaccination using VLPs elicited robust levels of serum IgG, mucosal IgA, and viral neutralizing antibodies against A/Sw/Manitoba/MAFRI32/2009 H1N1. Following challenge with pandemic H1N1 2009, vaccinated pigs were protected, displaying reduced lung lesions, virus shedding and inhibition of virus replication in the lungs compared to non-vaccinated control pigs. Thus, VLPs can serve as a promising vaccination strategy to control influenza in swine.

Molecular epidemiological and serological studies of bovine leukemia virus (BLV) infection in Thailand cattle

BLV is the etiological agent of enzootic bovine leucosis. BLV has negative effects on animal health and causes economic losses worldwide. However, epidemiological studies on BLV are relatively unknown in many parts of Asian countries. Thus, this study sought to explore BLV infections in cattle in Thailand to determine the extent of the geographic distribution of BLV and to measure its prevalence rates. For this study, 744 cattle from 11 farms in 9 provinces of Thailand were screened in 2013 and 2014 by ELISA and nested PCR. Of those cattle, 41 BLVs were genetically characterized using 188 BLV gp51 env gene sequences available in GenBank. The BLV prevalence in Thailand was high, ranging from 5.3% to 87.8%, as determined by PCR and 11.0% to 100% as determined by ELISA, according to geographical region. Phylogenetic analysis showed that Thailand BLVs belonged to genotypes 1 and 6 and a new genotype 10, which are sporadically observed across Thailand with a prevalence of 31.7%, 19.5%, and 48.8%, respectively. A significant number of amino acid substitutions were also found in the gp51 sequences, of which unique changes in genotype 10 have not been reported previously. Briefly, the majority of substitutions were confined to CD4+/CD8+ T-cell epitopes, neutralizing domains, and E-D-A epitopes. Those observations indicate that BLV infections in Thailand cattle are prevalent and that the geographic distribution of BLV is dynamic, with a high level of genetic diversity. This distribution implies a long-term BLV infection in cattle populations and the movement of infected cattle. In sum, this study suggests that intensive surveillance and effective prevention strategies are required to determine the prevalence of BLV in Thailand and control continuous infections with BLVs.

Sequencing and phylogenetic analysis of the gp51 gene from Korean bovine leukemia virus isolates

BackgroundBovine Leukemia virus (BLV) infection of cattle has been reported in Korea for more than three decades. However, to date, there have been few studies regarding Korean BLV since 1980s. Thus, the purpose of this study is to perform a diagnosis and molecular characterization of BLV strains circulating in Korea and to estimate genetic diversity of different genotypes of BLV.MethodTo investigate the distribution of BLV variants in the world and assess the evolutionary history of Korean BLV isolates, a comprehensive molecular analysis of the BLV env gp51 gene was conducted using recent worldwide BLV isolates. The isolates included 50 samples obtained from two cattle farms in southeastern Korea in 2014.ResultsSequence and phylogenetic analyses of partial 444-nt fragment sequences and complete gp51 sequences of BLV revealed eight distinct genotypes of BLV showing geographic distribution of the world. Most Korean BLV isolates were found to belong to genotype 1 which is a major genotype prevailed throughout the world, and only four isolates from one farm were classified as genotype 3 related to the US and Japan isolates. Analysis of amino acids of Korean BLV isolates showed several sequence substitutions in the leader peptide, conformational epitope, and neutralizing domain regions. The observations suggest the possibility of affecting on viral infectivity and formation.ConclusionKorean BLV isolates showed the close relationship to genotype 1 and 3. Further study to identify the diversity of BLV circulating in Korea is necessary with samples collected nationwide because this study is the first report of BLV genotype 3 being in circulation in Korea.

Genetic diversity of porcine reproductive and respiratory syndrome virus in Korea.

The high genetic diversity of porcine reproductive and respiratory syndrome virus (PRRSV) has been an obstacle to developing an effective vaccine for porcine reproductive and respiratory syndrome (PRRS). This study was performed to assess the degree of genetic diversity among PRRSVs from Korean pig farms where wasting and respiratory syndrome was observed from 2005 to 2009. Samples from 786 farms were tested for the presence of PRRSV using reverse transcription PCR protocol. A total of 117 farms were positive for type 1 PRRSV while 198 farms were positive for type 2. Nucleotide sequences encoding the open reading frame (ORF) 5 were analyzed and compared to those of various published PRRSV isolates obtained worldwide. Sequence identity of the ORF 5 in the isolates was 81.6~100% for type 1 viruses and 81.4~100% for type 2 viruses. Phylogenetic analysis of the ORF 5 sequences showed that types 1 and 2 PRRSVs from Korea were mainly classified into three and four clusters, respectively. The analyzed isolates were distributed throughout the clusters independent of the isolation year or geographical origin. In conclusion, our results indicated that the genetic diversity of PRRSVs from Korean pig farms is high and has been increasing over time.

A recombinant adenovirus bicistronically expressing porcine interferon-α and interferon-γ enhances antiviral effects against foot-and-mouth disease virus

Foot-and-mouth disease (FMD) is a virulent and economically costly disease in domestic livestock. Since the current vaccine available against FMD provides no protection until 7days postvaccination, the only alternative method to halt the spread of the FMD virus (FMDV) during outbreaks is by the application of anti-viral agents. The combination of recombinant adenovirus expressing type I interferon (IFN-α) and adenovirus expressing type II IFN (IFN-γ) has been reported to be an effective anti-viral treatment strategy against FMDV. Nevertheless, the recombinant adenovirus mixture may be inefficient because of the low anti-viral efficiency of IFN-γ compared to that of IFN-α. In this study, we generated a recombinant adenovirus co-expressing porcine IFN-α and IFN-γ in tandem using an FMDV 2A sequence to mediate effective cleavage of the two proteins (referred to as Ad-porcine IFN-αγ). We demonstrated that both recombinant porcine IFN-α and IFN-γ were expressed and interferon stimulated gene (ISG)s related with IFN-α and IFN-γ were induced in porcine kidney (IBRS-2) cells infected with Ad-porcine IFN-αγ. Additionally, the anti-viral effects of Ad-porcine IFN-αγ against FMDV were enhanced both in IBRS-2 cells and in CD-1 (ICR) suckling mice compared to that of adenovirus expressing only a single protein. We propose that Ad-porcine IFN-αγ could be a rapid, highly efficient, convenient anti-viral agent against FMDV.

Reemergence of Foot-and-Mouth Disease, South Korea, 2000–2011

Five outbreaks of foot-and-mouth disease have occurred in South Korea during 2000–2011. Macro-analysis of these outbreaks showed a correlation with outbreaks in countries in eastern Asia. Genetic analyses of food-and-mouth disease viruses in South Korea showed a correlation with viruses that are prevalent in neighboring countries.

Genetic and pathogenic characteristics of H1 avian and swine influenza A viruses.

This study examined the potential for cross-species transmission of influenza viruses by comparing the genetic and pathogenic characteristics of H1 avian influenza viruses (AIVs) with different host origins in Korea. Antigenic and phylogenetic analyses of H1 AIVs circulating in Korea provided evidence of genetic similarity between viruses that infect domestic ducks and those that infect wild birds, although there was no relationship between avian and swine viruses. However, there were some relationships between swine and human viral genes. The replication and pathogenicity of the H1 viruses was assessed in chickens, domestic ducks and mice. Viral shedding in chickens was relatively high. Virus was recovered from both oropharyngeal and cloacal swabs up to 5-10 days post-inoculation. The titres of domestic duck viruses in chickens were much higher than those of wild-bird viruses. Both domestic duck and wild-bird viruses replicated poorly in domestic ducks. None of the swine viruses replicated in chickens or domestic ducks; however, six viruses showed relatively high titres in mice, regardless of host origin, and induced clinical signs such as ruffled fur, squatting and weight loss. Thus, although the phylogenetic and antigenic analyses showed no evidence of interspecies transmission between birds and swine, the results suggest that Korean H1 viruses have the potential to cause disease in mammals. Therefore, we should intensify continuous monitoring of avian H1 viruses in mammals and seek to prevent interspecies transmission.

Design and testing of multiplex RT-PCR primers for the rapid detection of influenza A virus genomic segments: Application to equine influenza virus

The avian influenza A virus causes respiratory infections in animal species. It can undergo genomic recombination with newly obtained genetic material through an interspecies transmission. However, the process is an unpredictable event, making it difficult to predict the emergence of a new pandemic virus and distinguish its origin, especially when the virus is the result of multiple infections. Therefore, identifying a novel influenza is entirely dependent on sequencing its whole genome. Occasionally, however, it can be time-consuming, costly, and labor-intensive when sequencing many influenza viruses. To compensate for the difficulty, we developed a rapid, cost-effective, and simple multiplex RT-PCR to identify the viral genomic segments. As an example to evaluate its performance, H3N8 equine influenza virus (EIV) was studied for the purpose. In developing this protocol to amplify the EIV eight-segments, a series of processes, including phylogenetic analysis based on different influenza hosts, in silico analyses to estimate primer specificity, coverage, and variation scores, and investigation of host-specific amino acids, were progressively conducted to reduce or eliminate the negative factors that might affect PCR amplification. Selectively, EIV specific primers were synthesized with dual priming oligonucleotides (DPO) system to increase primer specificity. As a result, 16 primer pairs were selected to screen the dominantly circulating H3N8 EIV 8 genome segments: PA (3), PB2 (1), PA (3), NP (3), NA8 (2), HA3 (1), NS (1), and M (2). The diagnostic performance of the primers was evaluated with eight sets composing of four segment combinations using viral samples from various influenza hosts. The PCR results suggest that the multiplex RT-PCR has a wide range of applications in detection and diagnosis of newly emerging EIVs. Further, the proposed procedures of designing multiplex primers are expected to be used for detecting other animal influenza A viruses.