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Although the incidence of severe fever with thrombocytopenia syndrome virus (SFTSV) infection has increased from its discovery with a mortality rate of 10–20%, no effective vaccines are currently available. Here we describe the development of a SFTSV DNA vaccine, its immunogenicity, and its protective efficacy. Vaccine candidates induce both a neutralizing antibody response and multifunctional SFTSV-specific T cell response in mice and ferrets. When the vaccine efficacy is investigated in aged-ferrets that recapitulate fatal clinical symptoms, vaccinated ferrets are completely protected from lethal SFTSV challenge without developing any clinical signs. A serum transfer study reveals that anti-envelope antibodies play an important role in protective immunity. Our results suggest that Gn/Gc may be the most effective antigens for inducing protective immunity and non-envelope-specific T cell responses also can contribute to protection against SFTSV infection. This study provides important insights into the development of an effective vaccine, as well as corresponding immune parameters, to control SFTSV infection., Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging tick-borne virus with no specific treatment or vaccine available. Here, the authors develop a DNA vaccine for SFTSV that is protective against lethal challenge in ferrets and show that anti-envelope antibodies are important for protection.

Because H5N1 influenza viruses continuously threaten the public health, the WHO has prepared various clades of H5N1 mock-up vaccines as one of the measures for pandemic preparedness. The recent worldwide outbreak of H5Nx virus which belongs to clade 2.3.4.4 and of which H5N6 subtype belongs and already caused human infection also increases the need of pandemic vaccine for such novel emerging viruses. In this study, we evaluated the protective efficacy and immunogenicity of an egg-based and inactivated whole-virus H5N8 (IDCDC-RG43A) developed by CDC containing HA and NA gene of the parent virus A/gyrfalcon/Washington/41088-6/2014. Mice vaccinated two times elicited low to moderate antibody titer in varying amount of antigen doses against the homologous H5N8 vaccine virus and heterologous intra–clade 2.3.4.4 H5N6 (A/Sichuan/26221/2014) virus. Mice immunized with at least 3.0 µg/dose of IDCDC-RG43A with aluminum hydroxide adjuvant were completely protected from lethal challenge with the mouse-adapted H5N8 (A/Environment/Korea/ma468/2015, maH5N8) as well as cleared the viral replication in tissues including lung, brain, spleen, and kidney. Vaccinated ferrets induced high antibody titers against clade 2.3.4.4 H5N8/H5N6 viruses and the antibody showed high cross-reactivity to clade 2.2 H5N1 but not to clade 1 and 2.3.4 viruses as measured by hemagglutinin inhibition and serum neutralization assays. Furthermore, administration of the vaccine in ferrets resulted in attenuation of clinical disease signs and virus spread to peripheral organs including lung, spleen, and kidney from high dose challenge with maH5N8 virus. The protective and immunogenic characteristic of the candidate vaccine are essential attributes to be considered for further clinical trials as a pre-pandemic vaccine for a potential pandemic virus.

Recent canine influenza outbreaks have raised concerns about the generation of pathogenic variants that may pose a threat to public health. Here, we examine avian-like H3N2 canine influenza viruses (CIVs) isolated from 2009 to 2013 in South Korea from dogs. Phylogenetic analysis revealed that these viruses are closely related to strains previously isolated from dogs in Korea and China. However, molecular characterization demonstrated non-synonymous mutations between the canine viruses, particularly in the putative H3 antigenic sites, NA stalk regions, and in the internal genes of the 2012-2013 isolates compared with the 2009 isolate. Animal experiments showed that three representative isolates (A/canine/Korea/AS-01/2009(AS-01/09), A/canine/Korea/AS-05/2012(AS-05/12) and A/canine/Korea/AS-11/2013(AS-11/13), were readily droplet transmitted between dogs, whereas AS-05/12 induced more severe clinical disease and was lethal in dogs compared with AS-01/09. Although all viruses were able to infect ferrets, AS-05/12 consistently yielded higher nasal wash titers and was transmissible to ferrets via airborne droplets. Using reverse genetics, we show that the NA, NP, and M genes of CIV are critical for the adaptation of avian H3N2 viruses, and the resulting reassortant genotypes promote viral growth in dogs in a manner similar to that of the wild-type AS-01/09 virus. Taken together, these results demonstrate that CIVs continuously evolve in dogs thereby allowing them to gain a foothold in mammalian hosts. Importantly, we elucidated the genetic contributions of the NA, NP, and M genes to the adaptability of CIVs derived from the avian H3N2 virus.

During the outbreaks of highly pathogenic avian influenza (HPAI) H5N6 viruses in 2016 in South Korea, novel H5N8 viruses were also isolated from migratory birds. Phylogenetic analysis revealed that the HA gene of these H5N8 viruses belonged to clade 2.3.4.4, similarly to recent H5Nx viruses, and originated from A/Brk/Korea/Gochang1/14(H5N8), a minor lineage of H5N8 that appeared in 2014 and then disappeared. At least four reassortment events occurred with different subtypes (H5N8, H7N7, H3N8 and H10N7) and a chicken challenge study revealed that they were classified as HPAI viruses according to OIE criteria.

To investigate nationwide severe fever with thrombocytopenia syndrome virus (SFTSV) infection status, we isolated SFTSVs from patients with suspected severe fever with thrombocytopenia syndrome (SFTS) in 207 hospitals throughout South Korea between 2013 and April 2017. A total of 116 SFTSVs were isolated from 3137 SFTS-suspected patients, with an overall 21.6% case fatality rate. Genetic characterization revealed that at least 6 genotypes of SFTSVs were co-circulating in South Korea, with multiple reassortments among them. Of these, the genotype B-2 strains were the most prevalent, followed by the A and F genotypes. Clinical and epidemiologic investigations revealed that genotype B strains were associated with the highest case fatality rate, while genotype A caused only one fatality among 10 patients. Further, ferret infection studies demonstrated varying clinical manifestations and case mortality rates with different strains of SFTSV, which suggests this virus could exhibit genotype-dependent pathogenicity.

To investigate cross-protective vaccine efficacy of highly-pathogenic avian influenza H5N1 viruses against a recent HPAI H5N8 virus, we immunized C57BL/6 mice and ferrets with three alum-adjuvanted inactivated whole H5N1 vaccines developed through reverse-genetics (Rg): [Vietnam/1194/04xPR8 (clade 1), Korea/W149/06xPR8 (clade 2.2), and Korea/ES223N/03xPR8 (clade 2.5)]. Although relatively low cross-reactivities (10-40 HI titer) were observed against heterologous H5N8 virus, immunized animals were 100% protected from challenge with the 20 mLD50 of H5N8 virus, with the exception of mice vaccinated with 3.5μg of Rg Vietnam/1194/04xPR8. Of note, the Rg Korea/ES223N/03xPR8 vaccine provided not only effective protection, but also markedly inhibited viral replication in the lungs and nasal swabs of vaccine recipients within five days of HPAI H5N8 virus challenge. Further, we demonstrated that antibody-dependent cell-mediated cytotoxicity (ADCC) of an antibody-coated target cell by cytotoxic effector cells also plays a role in the heterologous protection of H5N1 vaccines against H5N8 challenge.

Severe fever with thrombocytopenia syndrome phlebovirus (SFTSV) has a wide host range. Not only has it been found in humans, but also in many wild and domesticated animals. The infection of breeding deer on farms is a particularly worrisome public health concern due to the large amount of human contact and the diverse use of deer products, including raw blood. To investigate the prevalence of breeding domesticated deer, we examined the SFTSV infection rate on deer farms in South Korea from 2015 to 2017. Of the 215 collected blood samples, 0.9% (2/215) were found to be positive for viral RNA by PCR, and sequence analysis showed the highest homology with the KADGH human isolate. Both SFTSV-specific recombinant N and Gn protein-based ELISAs revealed that 14.0% (30/215) and 7.9% (17/215) of collected blood specimens were positive for SFTSV antibody. These results demonstrate that the breeding farm deer are exposed to SFTSV and could be a potential infection source for humans through direct contact or consumption of byproducts.

As shown during the 2009 pandemic H1N1 (A(H1N1)pdm09) outbreak, egg-based influenza vaccine production technology is insufficient to meet global demands during an influenza pandemic. Therefore, there is a need to adapt cell culture-derived vaccine technology using suspended cell lines for more rapid and larger-scale vaccine production. In this study, we attempted to generate a high-growth influenza vaccine strain in MDCK cells using an A/Puerto/8/1934 (H1N1) vaccine seed strain. Following 48 serial passages with four rounds of virus plaque purification in MDCK cells, we were able to select several MDCK-adapted plaques that could grow over 10⁸ PFU/ml. Genetic characterization revealed that these viruses mainly had amino acid substitutions in internal genes and exhibited higher polymerase activities. By using a series of Rg viruses, we demonstrated the essential residues of each gene and identified a set of high-growth strains in MDCK cells (PB1D153N, M1A137T, and NS1N176S). In addition, we confirmed that in the context of the high-growth A/PR/8/34 backbone, A/California/7/2009 (H1N1), A/Perth/16/2009 (H3N2), and A/environment/Korea/deltaW150/2006 (H5N1) also showed significantly enhanced growth properties (more than 10⁷ PFU/ml) in both attached- and suspended-MDCK cells compared with each representative virus and the original PR8 vaccine strain. Taken together, this study demonstrates the feasibility of a cell culture-derived approach to produce seed viruses for influenza vaccines that are cheap and can be grown promptly and vigorously as a substitute for egg-based vaccines. Thus, our results suggest that MDCK cell-based vaccine production is a feasible option for producing large-scale vaccines in case of pandemic outbreaks.

Highly pathogenic avian influenza (HPAI) A(H5N6) and A(H5N8) virus infections resulted in the culling of more than 37 million poultry in the Republic of Korea during the 2016/17 winter season. Here we characterize two representative viruses, A/Environment/Korea/W541/2016 [Em/W541(H5N6)] and A/Common Teal/Korea/W555/2017 [CT/W555(H5N8)], and evaluate their zoonotic potential in various animal models. Both Em/W541(H5N6) and CT /W555(H5N8) are novel reassortants derived from various gene pools of wild bird viruses present in migratory waterfowl arising from eastern China. Despite strong preferential binding to avian virus–type receptors, the viruses were able to grow in human respiratory tract tissues. Em/W541(H5N6) was found to be highly pathogenic in both chickens and ducks, while CT/W555(H5N8) caused lethal infections in chickens but did not induce remarkable clinical illness in ducks. In mice, both viruses appeared to be moderately pathogenic and displayed limited tissue tropism relative to HPAI H5N1 viruses. Em/W541(H5N6) replicated to moderate levels in the upper respiratory tract of ferrets and was detected in the lungs, brain, spleen, liver, and colon. Unexpectedly, two of three ferrets in direct contact with Em/W541(H5N6)-infected animals shed virus and seroconverted at 14 dpi. CT/W555(H5N8) was less pathogenic than the H5N6 virus in ferrets and no transmission was detected. Given the co-circulation of different, phenotypically distinct, subtypes of HPAI H5Nx viruses for the first time in South Korea, detailed virologic investigations are imperative given the capacity of these viruses to evolve and cause human infections.

Coinfection of ferrets with H5N1 and pH1N1 viruses resulted in two predominate genotypes in the lungs containing surface genes of highly pathogenic avian influenza H5N1 virus in the backbone of pandemic H1N1 2009 (pH1N1). Compared to parental strains, these reassortants exhibited increased growth and virulence in vitro and in mice but failed to be transmitted indirectly to naive contact ferrets. Thus, this demonstrates a possible natural reassortment following coinfection as well as the pathogenicity of the potential reassortants.

Avian influenza A virus (AIV) is commonly isolated from domestic poultry and wild migratory birds, and the H9N2 subtype is the most prevalent and the major cause of severe disease in poultry in Korea. In addition to the veterinary concerns regarding the H9N2 subtype, it is also considered to be the next potential human pandemic strain due to its rapid evolution and interspecies transmission. In this study, we utilize serial lung-to-lung passage of a low pathogenic avian influenza virus (LPAI) H9N2 (A/Ck/Korea/163/04, WT163) (Y439-lineage) in mice to increase pathogenicity and investigate the potential virulence marker. Mouse-adapted H9N2 virus obtained high virulence (100% mortality) in mice after 98 serial passages. Sequence results show that the mouse adaptation (ma163) possesses several mutations within seven gene segments (PB2, PA, HA, NP, NA, M, and NS) relative to the wild-type strain. The HA gene showed the most mutations (at least 11) with one resulting in the loss of an N-glycosylation site (at amino acid 166). Moreover, reverse genetic studies established that an E627K substitution in PB2 and the loss of the N-glycosylation site in the HA protein (aa166) are critical virulence markers in the mouse-adapted H9N2 virus. Thus, these results add to the increasing body of mutational analysis data defining the function of the viral polymerase and HA genes and their roles in mammalian host adaptation. To our knowledge, this is first report of the generation of a mammalian-adapted Korea H9N2 virus (Y493-lineages). Therefore, this study offers valuable insights into the molecular evolution of the LPAI Korean H9N2 in a new host and adds to the current knowledge of the molecular markers associated with increased virulence.

We investigated influenza A(H5N6) viruses from migratory birds in Chungnam and Gyeonggi Provinces, South Korea following a reported die-off of poultry in nearby provinces in November 2017. Genetic analysis and virulence studies in chickens and ducks identified our isolate from December 2017 as a novel highly pathogenic avian influenza virus. It resulted from reassortment between the highly virulent H5N8 strain from Korea with the N6 gene from a low-pathogenic H3N6 virus from the Netherlands.

Several subtypes of avian influenza viruses (AIVs) are emerging as novel human pathogens, and the frequency of related infections has increased in recent years. Although neuraminidase (NA) inhibitors (NAIs) are the only class of antiviral drugs available for therapeutic intervention for AIV-infected patients, studies on NAI resistance among AIVs have been limited, and markers of resistance are poorly understood. Previously, we identified unique NAI resistance substitutions in AIVs of the N3, N7, and N9 NA subtypes. Here, we report profiles of NA substitutions that confer NAI resistance in AIVs of the N4, N5, N6, and N8 NA subtypes using gene-fragmented random mutagenesis. We generated libraries of mutant influenza viruses using reverse genetics (RG) and selected resistant variants in the presence of the NAIs oseltamivir carboxylate and zanamivir in MDCK cells. In addition, two substitutions, H274Y and R292K (N2 numbering), were introduced into each NA gene for comparison. We identified 37 amino acid substitutions within the NA gene, 16 of which (4 in N4, 4 in N5, 4 in N6, and 4 in N8) conferred resistance to NAIs (oseltamivir carboxylate, zanamivir, or peramivir) as determined using a fluorescence-based NA inhibition assay. Substitutions conferring NAI resistance were mainly categorized as either novel NA subtype specific (G/N147V/I, A246V, and I427L) or previously reported in other subtypes (E119A/D/V, Q136K, E276D, R292K, and R371K). Our results demonstrate that each NA subtype possesses unique NAI resistance markers, and knowledge of these substitutions in AIVs is important in facilitating antiviral susceptibility monitoring of NAI resistance in AIVs.IMPORTANCEThe frequency of human infections with avian influenza viruses (AIVs) has increased in recent years. Despite the availability of vaccines, neuraminidase inhibitors (NAIs), as the only available class of drugs for AIVs in humans, have been constantly used for treatment, leading to the inevitable emergence of drug-resistant variants. To screen for substitutions conferring NAI resistance in AIVs of N4, N5, N6, and N8 NA subtypes, random mutations within the target gene were generated, and resistant viruses were selected from mutant libraries in the presence of individual drugs. We identified 16 NA substitutions conferring NAI resistance in the tested AIV subtypes; some are novel and subtype specific, and others have been previously reported in other subtypes. Our findings will contribute to an increased and more comprehensive understanding of the mechanisms of NAI-induced inhibition of influenza virus and help lead to the development of drugs that bind to alternative interaction motifs.

Neuraminidase inhibitors (NAIs) have been widely used to control influenza virus infection, but their increased use could promote the global emergence of resistant variants. Although various mutations associated with NAI resistance have been identified, the amino acid substitutions that confer multidrug resistance with undiminished viral fitness remain poorly understood. We therefore screened a known mutation(s) that could confer multidrug resistance to the currently approved NAIs oseltamivir, zanamivir, and peramivir by assessing recombinant viruses with mutant NA-encoding genes (catalytic residues R152K and R292K, framework residues E119A/D/G, D198N, H274Y, and N294S) in the backbones of the 2009 pandemic H1N1 (pH1N1) and highly pathogenic avian influenza (HPAI) H5N1 viruses. Of the 14 single and double mutant viruses recovered in the backbone of pH1N1, four variants (E119D, E119A/D/G-H274Y) exhibited reduced inhibition by all of the NAIs and two variants (E119D and E119D-H274Y) retained the overall properties of gene stability, replicative efficiency, pathogenicity, and transmissibility in vitro and in vivo . Of the nine recombinant H5N1 viruses, four variants (E119D, E119A/D/G-H274Y) also showed reduced inhibition by all of the NAIs, though their overall viral fitness was impaired in vitro and/or in vivo . Thus, single mutations or certain combination of the established mutations could confer potential multidrug resistance on pH1N1 or HPAI H5N1 viruses. Our findings emphasize the urgency of developing alternative drugs against influenza virus infection. IMPORTANCE There has been a widespread emergence of influenza virus strains with reduced susceptibility to neuraminidase inhibitors (NAIs). We screened multidrug-resistant viruses by studying the viral fitness of neuraminidase mutants in vitro and in vivo . We found that recombinant E119D and E119A/D/G/-H274Y mutant viruses demonstrated reduced inhibition by all of the NAIs tested in both the backbone of the 2009 H1N1 pandemic (pH1N1) and highly pathogenic avian influenza H5N1 viruses. Furthermore, E119D and E119D-H274Y mutants in the pH1N1 background maintained overall fitness properties in vitro and in vivo . Our study highlights the importance of vigilance and continued surveillance of potential NAI multidrug-resistant influenza virus variants, as well as the development of alternative therapeutics.

Severe fever with thrombocytopenia syndrome (SFTS) is a newly emerging tick-borne infectious disease caused by the SFTS virus (SFTSV). To investigate the prevalence of SFTSV in domestic goats in South Korea, we collected blood samples in commercial slaughterhouses in Chungbuk Province in 2017. Of the 207 samples tested, 4 (2%) were found to be positive for viral RNA by RT-PCR and 30 (14.4%) were positive for SFTSV antibody as detected by a nucleocapsid (NP) protein-based ELISA. Phylogenetic analysis of the non-structural protein (NS) sequences showed that all viruses belonged to the genotype B, although they were clustered into two different sublineages that showed the highest homology with the KR612076-JP01 and KY789441-CB3 human isolate from South Korea. Further, we confirmed the specificity of seropositive goat sera by FRNT50 and western blotting analysis and found differential cross-reactivity of the sera with genotype A and B SFTSV strains. Collectively, this study suggests that relatively high numbers of goats are infected by antigenically different SFTSV strains, which might have a potential for zoonotic infection.

Due to increasing concerns about human infection by various H7 influenza viruses, including recent H7N9 viruses, we evaluated the genetic relationships and cross-protective efficacies of three different Eurasian H7 avian influenza viruses. Phylogenic and molecular analyses revealed that recent Eurasian H7 viruses can be separated into two different lineages, with relatively high amino acid identities within groups (94.8 to 98.8%) and low amino acid identities between groups (90.3 to 92.6%). In vivo immunization with representatives of each group revealed that while group-specific cross-reactivity was induced, cross-reactive hemagglutination inhibition (HI) titers were approximately 4-fold lower against heterologous group viruses than against homologous group viruses. Moreover, the group I (RgW109/06) vaccine protected 100% of immunized mice from various group I viruses, while only 20 to 40% of immunized mice survived lethal challenge with heterologous group II viruses and exhibited high viral titers in the lung. Moreover, while the group II (RgW478/14) vaccine also protected mice from lethal challenge with group II viruses, it failed to elicit cross-protection against group I viruses. However, it is noteworthy that vaccination with RgAnhui1/13, a virus of a sublineage of group I, cross-protected immunized mice against lethal challenge with both group I and II viruses and significantly attenuated lung viral titers. Interestingly, immune sera from RgAnhui1/13-vaccinated mice showed a broad neutralizing spectrum rather than the group-specific pattern observed with the other viruses. These results suggest that the recent human-infective H7N9 strain may be a candidate broad cross-protective vaccine for Eurasian H7 viruses. IMPORTANCE Genetic and phylogenic analyses have demonstrated that the Eurasian H7 viruses can be separated into at least two different lineages, both of which contain human-infective fatal H7 viruses, including the recent novel H7N9 viruses isolated in China since 2013. Due to the increasing concerns regarding the global public health risk posed by H7 viruses, we evaluated the genetic relationships between Eurasian H7 avian influenza viruses and the cross-protective efficacies of three different H7 viruses: W109/06 (group I), W478/14 (group II), and Anhui1/13 (a sublineage of group I). While each vaccine induced group-specific antibody responses and cross-protective efficacy, only Anhui1/13 was able to cross-protect immunized hosts against lethal challenge across groups. In fact, the Anhui1/13 virus induced not only cross-protection but also broad serum neutralizing antibody responses against both groups of viruses. This suggests that Anhui1/13-like H7N9 viruses may be viable vaccine candidates for broad protection against Eurasian H7 viruses.

In order to produce a dually effective vaccine against H9 and H5 avian influenza viruses that aligns with the DIVA (differentiating infected from vaccinated animals) strategy, we generated a chimeric H9/H5N2 recombinant vaccine that expressed the whole HA1 region of A/CK/Korea/04163/04 (H9N2) and the HA2 region of recent highly pathogenic avian influenza (HPAI) A/MD/Korea/W452/14 (H5N8) viruses. The chimeric H9/H5N2 virus showed in vitro and in vivo growth properties and virulence that were similar to those of the low-pathogenic avian influenza (LPAI) H9 virus. An inactivated vaccine based on this chimeric virus induced serum neutralizing (SN) antibodies against both H9 and H5 viruses but induced cross-reactive hemagglutination inhibition (HI) antibody only against H9 viruses. Thus, this suggests its compatibility for use in the DIVA strategy against H5 strains. Furthermore, the chimeric H9/H5N2 recombinant vaccine protected immunized chickens against lethal challenge by HPAI H5N8 viruses and significantly attenuated virus shedding after infection by both H9N2 and HPAI H5N8 viruses. In mice, serological analyses confirmed that HA1- and HA2 stalk-specific antibody responses were induced by vaccination and that the DIVA principle could be employed through the use of an HI assay against H5 viruses. Furthermore, each HA1- and HA2 stalk-specific antibody response was sufficient to inhibit viral replication and protect the chimeric virus-immunized mice from lethal challenge with both mouse-adapted H9N2 and wild-type HPAI H5N1 viruses, although differences in vaccine efficacy against a homologous H9 virus (HA1 head domain immune-mediated protection) and a heterosubtypic H5 virus (HA2 stalk domain immune-mediated protection) were observed. Taken together, these results demonstrate that the novel chimeric H9/H5N2 recombinant virus is a low-pathogenic virus, and this chimeric vaccine is suitable for a DIVA vaccine with broad-spectrum neutralizing antibody against H5 avian influenza viruses. IMPORTANCE Current influenza virus killed vaccines predominantly induce antihemagglutinin (anti-HA) antibodies that are commonly strain specific in that the antibodies have potent neutralizing activity against homologous strains but do not cross-react with HAs of other influenza virus subtypes. In contrast, the HA2 stalk domain is relatively well conserved among subtypes, and recently, broadly neutralizing antibodies against this domain have been isolated. Therefore, in light of the need for a vaccine strain that applies the DIVA strategy utilizing an HI assay and induces broad cross-protection against H5N1 and H9N2 viruses, we generated a novel chimeric H9/H5N1 virus that expresses the entire HA1 portion from the H9N2 virus and the HA2 region of the heterosubtypic H5N8 virus. The chimeric H9/H5N2 recombinant vaccine protected immunized hosts against lethal challenge with H9N2 and HPAI H5N1 viruses with significantly attenuated virus shedding in immunized hosts. Therefore, this chimeric vaccine is suitable as a DIVA vaccine against H5 avian influenza viruses.

While the effect of the influenza A virus non-structural protein (NS) on cytokine production during viral infection is well known, inconsistent results have been observed with some other influenza A virus backbone studied. In this study, in order to focus on the impact of the avian NS gene segments on viral virulence, the NS genes encoded by different strains of avian influenza A viruses were incorporated into an identical [A/Puerto Rico/8/1934(H1N1), PR8] virus background to generate various NS recombinant viruses. Thus, PR8NS, PR8 × [A/Hong Kong/483/97(H5N1) 483NS, PR8 × [A/Ck/Korea/150/03(H9N2) 150NS, and PR8 × [A/EM/Korea/W149/06(H5N1) W149NS were constructed utilizing reverse genetics. Here, we show the effects of each of these recombinant viruses upon viral pathogenesis and cytokine production during viral replication in vivo . In this regard, we found that infection of mice with the PR8 × 150NS recombinant virus resulted in the lowest pathogenicity (6.0 × 10 4 MLD 50 ), yet elicited the highest levels of TNF-α production in bronchoalveolar lavage (BAL) fluid compared to infection with the other recombinant influenza viruses. In contrast, infection with the PR8 virus showed the highest pathogenicity (1.0 × 10 2 MLD 50 ) as well as relatively high cytokine levels (IL-1α, IL-1β, IL-17, and eotaxin) in mouse BAL fluid. In addition, the PR8 and PR8 × 483NS viruses induced severe and extensive inflammation in infected lungs compared with that of PR8 × 150 NS recombinant virus-infected mice. These results clearly demonstrate that the NS genes of diverse influenza A strains can variable impact pathogenicity, histopathology, and cytokine production in mice even when expressed in an identical genetic background.

Background Human-to-swine transmission of the pandemic H1N1 2009 [A(H1N1)pdm09] virus in pig populations resulted in reassortment events with endemic swine influenza viruses worldwide. Objective We investigated whether A(H1N1)pdm09-derived reassortant viruses are present in South Korea and sought to determine the pathogenic potential of the novel swine viruses. Methods Pig lung tissues were collected from commercially slaughtered pigs. Isolated swine influenza viruses were genetically analyzed and characterized in vitro and in vivo. Results We identified reassortant H3N2 (H3N2pM-like) and H3N1 swine viruses containing A(H1N1)pdm09-like segments in Korean pigs that are genetically closely related to strains recently detected in pigs and humans in North America. Although the H3N2pM-like and novel H3N1 reassortants demonstrated efficient replication in mice and ferrets, all the H3N1 strains exhibited growth advantage over the representative H3N2pM-like virus in human airway cells. Interestingly, A/swine/Korea/CY02-07/2012(H3N1) and A/swine/Korea/CY03-13/2012(H3N1) reassortants were more readily transmitted to respiratory-droplet-contact ferrets compared with the H3N2pM-like (A/swine/Korea/CY02-10/2012) isolate. Furthermore, serologic evaluation showed poor antigenicity to contemporary reference human seasonal H3N2 vaccine strains. Conclusions We report here for the first time the isolation of H3N2pM-like viruses outside North America and of novel reassortant swine H3N1 viruses with A(H1N1)pdm09-derived genes. Apart from further complicating the genetic diversity of influenza A viruses circulating in domestic pigs, our data also indicate that these strains could potentially pose threat to public health asserting the need for continuous virus monitoring in these ecologically important hosts.

A novel genotype of H5N6 influenza viruses was isolated from migratory birds in South Korea during November 2016. Domestic outbreaks of this virus were associated with die-offs of wild birds near reported poultry cases in Chungbuk province, central South Korea. Genetic analysis and animal studies demonstrated that the Korean H5N6 viruses are highly pathogenic avian influenza (HPAI) viruses and that these viruses are novel reassortants of at least three different subtypes (H5N6, H4N2 and H1N1).

The continuous worldwide spread of highly pathogenic avian influenza (HPAI) H5N8 viruses among wild birds and poultry is a potential threat to public health. In the present study, we investigated the genetic characteristics of recent H5N8 viruses continuously isolated from migratory birds over two winters (2013-2014 and 2014-2015) in South Korea. Genetic and phylogenetic analysis demonstrated that the 2014-2015 HPAI H5N8 viruses are closely related to the 2013-2014 viruses, including virulence markers; however, all eight gene segments of 2014-2015 H5N8 viruses clustered in different phylogenetic branches from 2013-2014 H5N8 viruses, except the A/Em/Korea/W492/2015 virus. The H5N8 viruses of Europe and North America belong to sublineages of the 2013-2014 Korean H5N8 viruses but differ from the 2014-2015 Korean H5N8 viruses. Further hemagglutination inhibition (HI) assay results showed that there were 2-to-4 fold differences in HI titer between 2013-2014 and 2014-2015 H5N8 viruses. Taken together, our results suggested that the 2014-2015 Korean H5N8 viruses were genetically and serologically different from those of 2013-2014 winter season H5N8 viruses, including those from Europe and North America.

The threat of a highly pathogenic avian influenza (HPAI) H5N1 virus causing the next pandemic remains a major concern. In this study, we evaluated the immunogenicity and efficacy of an inactivated whole-virus H5N1 pre-pandemic vaccine (MG1109) formulated by Green Cross Co., Ltd containing the hemagglutinin (HA) and neuraminidase (NA) genes of the clade 1 A/Vietnam/1194/04 virus in the backbone of A/Puerto Rico/8/34 (RgVietNam/04xPR8/34). Administration of the MG1109 vaccine (2-doses) in mice and ferrets elicited high HI and SN titers in a dose-dependent manner against the homologous (RgVietNam/04xPR8/34) and various heterologous H5N1 strains, (RgKor/W149/06xPR8/34, RgCambodia/04xPR8/34, RgGuangxi/05xPR8/34), including a heterosubtypic H5N2 (A/Aquatic bird/orea/W81/05) virus. However, efficient cross-reactivity was not observed against heterosubtypic H9N2 (A/Ck/Korea/H0802/08) and H1N1 (PR/8/34) viruses. Mice immunized with 1.9 μg HA/dose of MG1109 were completely protected from lethal challenge with heterologous wild-type HPAI H5N1 A/EM/Korea/W149/06 (clade 2.2) and mouse-adapted H5N2 viruses. Furthermore, ferrets administered at least 3.8 μg HA/dose efficiently suppressed virus growth in the upper respiratory tract and lungs. Vaccinated mice and ferrets also demonstrated attenuation of clinical disease signs and limited virus spread to other organs. Thus, this vaccine provided immunogenic responses in mouse and ferret models even against challenge with heterologous HPAI H5N1 and H5N2 viruses. Since the specific strain of HPAI H5N1 virus that would potentially cause the next outbreak is unknown, pre-pandemic vaccine preparation that could provide cross-protection against various H5 strains could be a useful approach in the selection of promising candidate vaccines in the future.

The continued spread of a highly pathogenic avian influenza (HPAI) H5N1 virus among wild birds and poultry has posed a potential threat to human public health. In the present study, we report the isolation of HPAI H5N1 viruses (A/Md/Korea/W401/11 and A/Md/Korea/W404/11) from fecal samples of migratory birds. Genetic and phlyogenetic analyses demonstrated that these viruses are genetically identical possessing gene segments from avian virus origin and showing highest sequence similarities (as high as 99.8%) to A/Ws/Hokkaido/4/11 and 2009-2010 Mongolian-like clade 2.3.2 isolates rather than previous Korean H5N1 viruses. Both viruses possess the polybasic motif (QRERRRK/R) in HA but other genes did not bear additional virulence markers. Pathogenicity of A/Md/Korea/W401/11 was assessed and compared with a 2006 clade 2.2 HPAI H5N1 migratory bird isolate (A/EM/Korea/W149/06) in chickens, ducks, mice and ferrets. Experimental infection in these hosts showed that both viruses have high pathogenic potential in chickens (2.3-3.0 LD(50)s) and mice (3.3-3.9 LD(50)s), but A/Md/Korea/W401/11 was less pathogenic in duck and ferret models. Despite recovery of both infection viruses in the upper respiratory tract, efficient ferret-to-ferret transmission was not observed. These data suggest that the 2011 Korean HPAI wild bird H5N1 virus could replicate in mammalian hosts without pre-adaptation but could not sustain subsequent infection. This study highlights the role of migratory birds in the perpetuation and spread of HPAI H5N1 viruses in Far-East Asia. With the changing pathobiology caused by H5N1 viruses among wild and poultry birds, continued surveillance of influenza viruses among migratory bird species remains crucial for effective monitoring of high-pathogenicity or pandemic influenza viruses.

To investigate whether currently circulating H9N2 avian influenza viruses (AIVs) in domestic poultry have evolved in Korean poultry since 2007, genetic and serological comparisons were conducted of H9N2 isolates from poultry slaughterhouses from January 2008 to December 2009. The isolation rate was relatively low in 2008 but increased gradually from January 2009 onwards. Genetic and phylogenetic analyses revealed that reassortant viruses had emerged, generating at least five novel genotypes, mostly containing segments of a previously prevalent domestic H9N2 virus lineage (Ck/Korea/04116/04-like). It was noteworthy that the N2 genes of some H9N2 isolates (genotypes D, E and F) were derived from those of H3N2-like viruses commonly isolated among domestic ducks in live-poultry markets. Animal challenge studies demonstrated that the pathogenicity of Ck/Korea/SH0906/09 (genotype B) and Ck/Korea/SH0912/09 (genotype F) in domestic avian species was altered due to reassortment. Furthermore, serological analysis revealed that the isolates were antigenically distinct from previous Korean H9N2 viruses including Ck/Korea/01310/01. Such antigenic diversity was illustrated further in experiments using H9N2-immunized chickens, which could not inhibit the replication and transmission of challenge viruses from each genotype. These results suggest that H9N2 viruses from domestic poultry have undergone substantial evolution since 2007 by immune selection as a result of vaccinal and natural immunity, coupled with reassortment. Taken together, this study demonstrates that periodical updating of vaccine strains, based on continuous surveillance data, is an important issue in order to provide sufficient protectivity against AIV infections.

Migratory waterfowls are the natural reservoir of influenza A viruses. However, interspecies transmission had occasionally caused outbreaks in various hosts including humans. To characterize the genetic origins of H5 avian influenza viruses isolated from migratory birds in South Korea, phylogenetic analysis were conducted. A total of 53 H5 viruses were isolated between October 2005 and November 2008. Full genetic characterization indicated that most of these viruses belong to the Eurasian-like avian lineage. However, some segments of the AB/Korea/W235/07 and the AB/Korea/W236/07 isolates were clustered with North American lineage viruses rather than those of the Eurasian lineage, suggesting the occurrence of reassortment between these two avian virus lineages. Phylogenetic analysis further demonstrated that the H5N2 and H5N3 virus isolates were of the low pathogenicity H5 phenotype. The H5 viruses appear to be antigenically similar to each other, but could be distinguished from a recent HPAI H5N1 (EM/Korea/W149/06) virus by hemagglutinin inhibition (HI) assays. Experimental inoculation of representative viruses indicated that certain isolates, particularly AB/Korea/W163/07 (H5N2), could be detected in trachea and lungs of chickens but none could be transmitted by direct contact. Furthermore, all of the viruses could be detected in mice lung without prior adaptation which is indicative of their pathogenic potential in a mammalian host. Overall, our results emphasize the important role that migratory birds play in the perpetuation, transport, and reassortment of avian influenza viruses stressing the need for continued surveillance of influenza virus activity in these avian populations.

Emerging outbreaks of newly found, highly pathogenic avian influenza (HPAI) A(H5N8) viruses have been reported globally. Previous studies have indicated that H5N8 pathogenicity in mice is relatively moderate compared with H5N1 pathogenicity. However, detailed mechanisms underlying avian influenza pathogenicity are still undetermined. We used a high-throughput RNA-seq method to analyse host and pathogen transcriptomes in the lungs of mice infected with A/MD/Korea/W452/2014 (H5N8) and A/EM/Korea/W149/2006 (H5N1) viruses. Sequenced numbers of viral transcripts and expression levels of host immune-related genes at 1 day post infection (dpi) were higher in H5N8-infected than H5N1-infected mice. Dual sequencing of viral transcripts revealed that in contrast to the observations at 1 dpi, higher number of H5N1 genes than H5N8 genes was sequenced at 3 and 7 dpi, which is consistent with higher viral titres and virulence observed in infected lungs in vivo. Ingenuity pathway analysis revealed a more significant upregulation of death receptor signalling, driven by H5N1 than with H5N8 infection at 3 and 7 dpi. Early induction of immune response-related genes may elicit protection in H5N8-infected mice, which correlates with moderate pathogenicity in vivo. Collectively, our data provide new insight into the underlying mechanisms of the differential pathogenicity of avian influenza viruses.

In 2011–2012, contemporary North American-like H3N2 swine influenza viruses (SIVs) possessing the 2009 pandemic H1N1 matrix gene (H3N2pM-like virus) were detected in domestic pigs of South Korea where H1N2 SIV strains are endemic. More recently, we isolated novel reassortant H1N2 SIVs bearing the Eurasian avian-like swine H1-like hemagglutinin and Korean swine H1N2-like neuraminidase in the internal gene backbone of the H3N2pM-like virus. In the present study, we clearly provide evidence on the genetic origins of the novel H1N2 SIVs virus through genetic and phylogenetic analyses. In vitro studies demonstrated that, in comparison with a pre-existing 2012 Korean H1N2 SIV [A/swine/Korea/CY03-11/2012 (CY03-11/2012)], the 2013 novel reassortant H1N2 isolate [A/swine/Korea/CY0423/2013 (CY0423-12/2013)] replicated more efficiently in differentiated primary human bronchial epithelial cells. The CY0423-12/2013 virus induced higher viral titers than the CY03-11/2012 virus in the lungs and nasal turbinates of infected mice and nasal wash samples of ferrets. Moreover, the 2013 H1N2 reassortant, but not the intact 2012 H1N2 virus, was transmissible to naive contact ferrets via respiratory-droplets. Noting that the viral precursors have the ability to infect humans, our findings highlight the potential threat of a novel reassortant H1N2 SIV to public health and underscore the need to further strengthen influenza surveillance strategies worldwide, including swine populations.

To investigate the molecular changes that allow influenza B viruses to adapt to new mammalian hosts, influenza B/Florida/04/2006 was serially passaged in BALB/c mice until highly virulent. The viral factors underlying this transition were then investigated in mice and ferrets. Five viruses, including the wild-type virus (P0), three intermediate viruses (P5, P9 and P12) and a lethal mouse-adapted virus (P17 (MA)), harbored one to five amino acid substitutions in the hemagglutinin, M, NP and PA segments suggesting that these mutations enhance virulence. The P17 (MA) virus replicated significantly more efficiently than the P0 virus both in vitro and in vivo (P 50: 105.25TCID50) while the P0, P5 and P9 viruses did not kill any infected mice (MLD50 > 106.0TCID50). Furthermore, the P17 (MA) virus grew to greater titers in the ferret upper respiratory tract compared with the P0 and intermediate viruses and only the P17 (MA) virus was transmissible between ferrets via both direct and aerosol contact. To our knowledge, this is the first study to demonstrate ferret-to-ferret transmission of influenza B virus and to delineate factors that may affect its transmission.

An increasing number of outbreaks of avian influenza H5N1 and H9N2 viruses in poultry have caused serious economic losses and raised concerns for human health due to the risk of zoonotic transmission. However, licensed H5N1 and H9N2 vaccines for animals and humans have not been developed. Thus, to develop a dual H5N1 and H9N2 live-attenuated influenza vaccine (LAIV), the HA and NA genes from a virulent mouse-adapted avian H5N2 (A/WB/Korea/ma81/06) virus and a recently isolated chicken H9N2 (A/CK/Korea/116/06) virus, respectively, were introduced into the A/Puerto Rico/8/34 backbone expressing truncated NS1 proteins (NS1-73, NS1-86, NS1-101, NS1-122) but still possessing a full-length NS gene. Two H5N2/NS1-LAIV viruses (H5N2/NS1-86 and H5N2/NS1-101) were highly attenuated compared with the full-length and remaining H5N2/NS-LAIV viruses in a mouse model. Furthermore, viruses containing NS1 modifications were found to induce more IFN-β activation than viruses with full-length NS1 proteins and were correspondingly attenuated in mice. Intranasal vaccination with a single dose (10(4.0) PFU/ml) of these viruses completely protected mice from a lethal challenge with the homologous A/WB/Korea/ma81/06 (H5N2), heterologous highly pathogenic A/EM/Korea/W149/06 (H5N1), and heterosubtypic highly virulent mouse-adapted H9N2 viruses. This study clearly demonstrates that the modified H5N2/NS1-LAIV viruses attenuated through the introduction of mutations in the NS1 coding region display characteristics that are desirable for live attenuated vaccines and hold potential as vaccine candidates for mammalian hosts.

Background MicroRNAs (miRNAs) are known to regulate various biological processes, including expression of cellular gene and virus-induced inflammation. Recently, studies have indicated that some miRNAs could regulate influenza virus replication. Due to differential sensitivities of influenza A virus strains to different species (avian and mammalian), variations in host responses may be observed. Therefore, we investigated and compared the differences in global host miRNA expression in mouse lungs infected with wild type low pathogenicity A/Aquatic bird/Korea/w81/2005 (H5N2) (w81) or mouse-adapted virulent A/Aquatic bird /Korea/ma81/2007 (H5N2) (ma81) virus. Results Although the mice infected with ma81 exhibited much greater mortality than w81-infected mice, the parental w81 virus induced a higher number of differentially expressed miRNAs compared to the ma81 virus. Between these 2 viruses, a total of 27 and 20 miRNAs were commonly expressed at 1 dpi and 3 dpi, respectively. It is noteworthy that only 9 miRNAs (miR-100-5p, miR-130a-5p, miR-146b-3p, miR-147-3p, miR-151-5p, miR-155-3p, miR-223-3p, miR-301a-3p, and miR-495-3p) were significantly upregulated in both lungs infected with either wild type w81 or the mouse-adapted ma81 strain at both time points. Notably, expression levels of miR-147-3p, miR-151-5p, miR-155-3p, and miR-223-3p were higher in the lungs of mice infected with the ma81 virus than those infected with the w81 virus. To identify potential roles of these miRNAs in regulating influenza virus replication, each group of mice was intranasally treated with each inhibitor of specifically targeting 4 miRNAs, and then challenged with 5 mouse lethal dose 50% (MLD50) of the virulent ma81 virus on the following day. Although the specific miRNA inhibitors could not completely attenuate mortality or reduce viral replication, the miR-151-5p- and miR-223-3p-inhibitors reduced mortality of inoculated mice to 70% and substantially delayed death. Conclusions Our results suggest that the mammalian adaptation of avian influenza A virus results in a different miRNA expression pattern in lungs of virus-infected mice compared with its parental strain, and use of specific miRNA inhibitors to target genes associated with the immune response or cell death may affect virulence and virus replication. Electronic supplementary material The online version of this article (doi:10.1186/s12866-014-0252-0) contains supplementary material, which is available to authorized users.

The threat of highly pathogenic avian influenza (HPAI) H5N1 viruses to cause the next pandemic remains a major concern. Here, we evaluated the cross-protection induced by natural infection of human seasonal influenza strains or immunization with trivalent inactivated influenza vaccine (TIV) against HPAI H5N1 (A/Vietnam/1203/2004) virus in ferrets. Groups were treated with PBS (group A), infected with H1N1 (group B) or H3N2 (group C) virus, or immunized with TIV (group D). Twelve weeks after the last treatment, serological assays revealed that groups B and C, but not group D, sustained moderate immunogenicity against homologous viruses; cross-reactivity against the H5N1 virus was not detected in any group. Following challenge with A/Vietnam/1203/2004 (H5N1) virus, only groups B and C exhibited attenuated viral loads leading to 100 % survival. Our data suggest that natural infection with human seasonal strains could potentially provide better heterosubtypic protection against HPAI H5N1 virus infection compared to TIV immunization.

We previously reported that influenza A/swine/Korea/1204/2009(H1N2) virus was virulent and transmissible in ferrets in which the respiratory-droplet-transmissible virus (CT-Sw/1204) had acquired simultaneous hemagglutinin (HA D225G ) and neuraminidase (NA S315N ) mutations. Incorporating these mutations into the nonpathogenic A/swine/Korea/1130/2009(H1N2, Sw/1130) virus consequently altered pathogenicity and growth in animal models but could not establish efficient transmission or noticeable disease. We therefore exploited various reassortants of these two viruses to better understand and identify other viral factors responsible for pathogenicity, transmissibility, or both. We found that possession of the CT-Sw/1204 tripartite viral polymerase enhanced replicative ability and pathogenicity in mice more significantly than did expression of individual polymerase subunit proteins. In ferrets, homologous expression of viral RNA polymerase complex genes in the context of the mutant Sw/1130 carrying the HA 225G and NA 315N modifications induced optimal replication in the upper nasal and lower respiratory tracts and also promoted efficient aerosol transmission to respiratory droplet contact ferrets. These data show that the synergistic function of the tripartite polymerase gene complex of CT-Sw/1204 is critically important for virulence and transmission independent of the surface glycoproteins. Sequence comparison results reveal putative differences that are likely to be responsible for variation in disease. Our findings may help elucidate previously undefined viral factors that could expand the host range and disease severity induced by triple-reassortant swine viruses, including the A(H1N1)pdm09 virus, and therefore further justify the ongoing development of novel antiviral drugs targeting the viral polymerase complex subunits.

Pandemic H1N1 2009 (A[H1N1]pdm09) variants associated with oseltamivir resistance have emerged with a histidine-to-tyrosine substitution in the neuraminidase(NA) at position 274 (H274Y). To determine whether the H274Y variant has increased virulence potential, A(H1N1)pdm09 virus, with or without the H274Y mutation, was adapted by serial lung-to-lung passages in mice. The mouse-adapted H274Y (maCA04H274Y) variants showed increased growth properties and virulence in vitro and in vivo while maintaining high NA inhibitor resistance. Interestingly, most maCA04H274Y and maCA04 viruses acquired common mutations in HA (S183P and D222G) and NP (D101G), while only maCA04H274Y viruses had consensus additional K153E mutation in the HA gene, suggesting a potential association with the H274Y substitution. Collectively, our findings highlight the potential emergence of A(H1N1)pdm09 drug-resistant variants with increased virulence and the need for rapid development of novel antiviral drugs.

Highly pathogenic avian influenza H5N1 viruses are found chiefly in birds and have caused severe disease and death in infected humans. Development of influenza vaccines capable of inducing heterosubtypic immunity against a broad range of influenza viruses is the best option for the preparedness, since vaccination remains the principal method in controlling influenza viral infections. Here, a mOMV-adjuvanted recombinant H5N2 (rH5N2) whole virus antigen vaccine with A/Environment/Korea/W149/06(H5N1)-derived H5 HA and A/Chicken/Korea/ma116/04(H9N2)-derived N2 NA in the backbone of A/Puerto Rico/8/34(H1N1) was prepared and generated by reverse genetics. Groups of mice were vaccinated by a prime-boost regime with the rH5N2 vaccine (1.75 μg of HA with/without 10 μg mOMV or aluminum hydroxide adjuvant for comparison). At two weeks post-immunizations, vaccinated mice were challenged with lethal doses of 10(3.5) EID50/ml of H5N1 or H9N2 avian influenza viruses, and were monitored for 15 days. Both mOMV- and alum-adjuvant vaccine groups had high survival rates after H5N1 infection and low levels of body weight changes compared to control groups. Interestingly, the mOMV-adjuvanted group induced better cross-reactive antibody responses serologically and promoted cross-protectivity against H5N1 and H9N2 virus challenges. Our results suggest that mOMV could be used as a vaccine adjuvant in the development of effective vaccines used to control influenza A virus transmission.

Background Influenza vaccines are prepared annually based on global epidemiological surveillance data. However, since there is no method by which to predict the influenza strain that will cause the next pandemic, the demand to develop new vaccination strategies with broad cross-reactivity against influenza viruses are clearly important. The ectodomain of the influenza M2 protein (M2e) is an attractive target for developing a vaccine with broad cross-reactivity. For these reasons, we investigated the efficacy of an inactivated H9N2 virus vaccine (a-H9N2) mixed with M2e (1xM2e or 4xM2e) proteins expressed in Escherichia coli, which contains the consensus of sequence the extracellular domain of matrix 2 (M2e) of A/chicken/Vietnam/27262/09 (H5N1) avian influenza virus, and investigated its humoral immune response and cross-protection against influenza A viruses. Results Mice were intramuscularly immunized with a-H9N2, 1xM2e alone, 4xM2e alone, a-H9N2/1xM2e, or a-H9N2/4xM2e. Three weeks post-vaccination, mice were challenged with lethal homologous (A/ chicken /Korea/ma163/04, H9N2) or heterosubtypic virus (A/Philippines/2/82, H3N2 and A/aquatic bird/Korea/maW81/05, H5N2). Our studies demonstrate that the survival of mice immunized with a-H9N2/1xM2e or with a-H9N2/4xM2e (100% survival) was significantly higher than that of mouse-adapted H9N2 virus-infected mice vaccinated with 1xM2e alone or with 4xM2e alone (0% survival). We also evaluated the protective efficacy of the M2e + vaccine against infection with mouse-adapted H5N2 influenza virus. Protection from death in the control group (0% survival) was similar to that of the 1×M2e alone and 4xM2e alone-vaccinated groups (0% survival). Only 40% of mice vaccinated with vaccine alone survived challenge with H5N2, while the a-H9N2/1×M2e and a-H9N2/4×M2e groups showed 80% and 100% survival following mouse-adapted H5N2 challenge, respectively. We also examined cross-protection against human H3N2 virus and found that the a-H9N2/1×M2e group displayed partial cross-protection against H3N2 (40% survival), whereas vaccine alone, 1×M2e alone, 4×M2e alone, or H9N2/1×M2e groups showed incomplete protection (0% survival) in response to challenge with a lethal dose of human H3N2 virus. Conclusions Taken together, these results suggest that prokaryote-expressed M2e protein improved inactivated H9N2 virus vaccine efficacy and achieved cross-protection against lethal influenza A virus infection in mice.

The constant threat of newly emerging influenza viruses with pandemic potential requires the need for prompt vaccine production. Here, we utilized the Vero cell polymerase I (PolI) promoter, rather than the commonly used human PolI promoter, in an established reverse-genetics system to rescue viable influenza viruses in Vero cells, an approved cell line for human vaccine production. The Vero PolI promoter was more efficient in Vero cells and demonstrated enhanced transcription levels and virus rescue rates commensurate with that of the human RNA PolI promoter in 293T cells. These results appeared to be associated with more efficient generation of A(H1N1)pdm09- and H5N1-derived vaccine seed viruses in Vero cells, whilst the rescue rates in 293T cells were comparable. Our study provides an alternative means for improving vaccine preparation by using a novel reverse-genetics system for generating influenza A viruses.

Herbal medicine is used to treat many conditions such as asthma, eczema, premenstrual syndrome, rheumatoid arthritis, migraine, headaches, menopausal symptoms, chronic fatigue, irritable bowel syndrome, cancer, and viral infections such as influenza. In this study, we investigated the antiviral effect of KIOM-C for the treatment of influenza A virus infection. Our results show that oral administration of KIOM-C conferred a survival benefit to mice infected with the 2009 pandemic H1N1 [A(H1N1)pdm09] virus, and resulted in a 10- to 100-fold attenuation of viral replication in ferrets in a dose-dependent manner. Additionally, oral administration of KIOM-C increased the production of antiviral cytokines, including IFN-γ and TNF-α, and decreased levels of pro-inflammatory cytokines (IL-6) and chemokines (KC, MCP-1) in the Bronchoalveolar lavage fluid (BALF) of A(H1N1)pdm-infected mice. These results indicate that KIOM-C can promote clearance of influenza virus in the respiratory tracts of mice and ferrets by modulating cytokine production in hosts. Taken together, our results suggest that KIOM-C is a potential therapeutic compound mixture for the treatment of influenza virus infection in humans.

Efficient worldwide swine surveillance for influenza A viruses is urgently needed; the emergence of a novel reassortant pandemic H1N1 (pH1N1) virus in 2009 demonstrated that swine can be the direct source of pandemic influenza and that the pandemic potential of viruses prevalent in swine populations must be monitored. We used the ferret model to assess the pathogenicity and transmissibility of predominant Korean triple-reassortant swine (TRSw) H1N2 and H3N2 influenza viruses genetically related to North American strains. Although most of the TRSw viruses were moderately pathogenic, one [A/Swine/Korea/1204/2009; Sw/1204 (H1N2)] was virulent in ferrets, causing death within 10 d of inoculation, and was efficiently transmitted to naive contact ferrets via respiratory droplets. Although molecular analysis did not reveal known virulence markers, the Sw/1204 virus acquired mutations in hemagglutinin (HA) (Asp-225-Gly) and neuraminidase (NA) (Ser-315-Asn) proteins during the single ferret passage. The contact-Sw/1204 virus became more virulent in mice, replicated efficiently in vitro, extensively infected human lung tissues ex vivo, and maintained its ability to replicate and transmit in swine. Reverse-genetics studies further indicated that the HA 225G and NA 315N substitutions contributed substantially in altering virulence and transmissibility. These findings support the continuing threat of some field TRSw viruses to human and animal health, reviving concerns on the capacity of pigs to create future pandemic viruses. Apart from warranting continued and enhanced global surveillance, this study also provides evidence on the emerging roles of HA 225G and NA 315N as potential virulence markers in mammals.

Gene mutations and reassortment are key mechanisms by which influenza A virus acquires virulence factors. To evaluate the role of the viral polymerase replication machinery in producing virulent pandemic (H1N1) 2009 influenza viruses, we generated various polymerase point mutants (PB2, 627K/701N; PB1, expression of PB1-F2 protein; and PA, 97I) and reassortant viruses with various sources of influenza viruses by reverse genetics. Although the point mutations produced no significant change in pathogenicity, reassortment between the pandemic A/California/04/09 (CA04, H1N1) and current human and animal influenza viruses produced variants possessing a broad spectrum of pathogenicity in the mouse model. Although most polymerase reassortants had attenuated pathogenicity (including those containing seasonal human H3N2 and high-pathogenicity H5N1 virus segments) compared to that of the parental CA04 (H1N1) virus, some recombinants had significantly enhanced virulence. Unexpectedly, one of the five highly virulent reassortants contained a A/Swine/Korea/JNS06/04(H3N2)-like PB2 gene with no known virulence factors; the other four had mammalian-passaged avian-like genes encoding PB2 featuring 627K, PA featuring 97I, or both. Overall, the reassorted polymerase complexes were only moderately compatible for virus rescue, probably because of disrupted molecular interactions involving viral or host proteins. Although we observed close cooperation between PB2 and PB1 from similar virus origins, we found that PA appears to be crucial in maintaining viral gene functions in the context of the CA04 (H1N1) virus. These observations provide helpful insights into the pathogenic potential of reassortant influenza viruses composed of the pandemic (H1N1) 2009 influenza virus and prevailing human or animal influenza viruses that could emerge in the future.

As the pandemic (H1N1) 2009 influenza virus continues to infect human populations globally, reports on epidemiologically linked animal infections are also on the rise. Since December 2009, pandemic (H1N1) 2009-like viruses have been isolated in pigs from different swine farms of South Korea. Genetic and phylogenetic analyses of viral segments demonstrated several events of human-to-swine transmission with no apparent signs of reassortment. These events were also supported by serological surveillance in pig sera collected from April to December, suggesting that reverse transmission probably started between June and July with a drastic increase in prevalence the following months. Although molecular characterization indicates that the swine isolates are generally stable, some viruses are genetically evolving, most notably in their surface proteins. Animal studies (ferrets and mice) reveal that swine pandemic isolates epitomize biological properties attributed to the currently circulating human pandemic viruses, including replication kinetics and efficient transmission, indicating their potential to return to circulation among humans. Overall, these results indicate widespread human-to-animal transmission of pandemic (H1N1) 2009 influenza viruses in South Korea. With the significant role of pigs in the ecology of influenza viruses, these transmission events should be closely monitored and minimized to prevent the risk of generating viruses with greater human health concerns.

The current pandemic (H1N1) 2009 virus remains transmissible among humans worldwide with cases of reverse zoonosis, providing opportunities to produce more pathogenic variants which could pose greater human health concerns. To investigate whether recent seasonal human or swine H1N1 vaccines could induce cross-reactive immune responses against infection with the pandemic (H1N1) 2009 virus, mice, ferrets or mini-pigs were administered with various regimens (once or twice) and antigen content (1.77, 3.5 or 7.5 microg HA) of a-Brsibane/59/07, a-CAN01/04 or RgCA/04/09xPR8 vaccine. Receipt of a-CAN01/04 (2-doses) but not a-Brisbane/59/07 induced detectable but modest (20-40 units) cross-reactive serum antibody against CA/04/09 by hemagglutinin inhibition (HI) assays in mice. Only double administration (7.5 microg HA) of both vaccine in ferrets could elicit cross-reactivity (30-60 HI titers). Similar antigen content of a-CAN01/04 in mini-pigs also caused a modest approximately 30 HI titers (twice vaccinated). However, vaccine-induced antibody titers could not suppress active virus replication in the lungs (mice) or virus shedding (ferrets and pigs) of immunized hosts intranasally challenged with CA/04/09. Furthermore, neither ferrets nor swine could abrogate aerosol transmission of the virus into naïve contact animals. Altogether, these results suggest that neither recent human nor animal H1N1 vaccine could provide complete protectivity in all animal models. Thus, this study warrants the need for strain-specific vaccines that could yield the optimal protection desired for humans and/or animals.