Profiling and characterization of influenza virus N1 strains potentially resistant to multiple neuraminidase inhibitors.

UNLABELLED: 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.

Virulence and transmissibility of H1N2 influenza virus in ferrets imply the continuing threat of triple-reassortant swine viruses.

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.

Evaluation of heterosubtypic cross-protection against highly pathogenic H5N1 by active infection with human seasonal influenza A virus or trivalent inactivated vaccine immunization in ferret models.

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.

The homologous tripartite viral RNA polymerase of A/swine/Korea/CT1204/2009(H1N2) influenza virus synergistically drives efficient replication and promotes respiratory droplet transmission in ferrets.

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 (HAD225G) and neuraminidase (NAS315N) 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 HA225G and NA315N 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.

Differential microRNA expression following infection with a mouse-adapted, highly virulent avian H5N2 virus.

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.

Establishment of Vero cell RNA polymerase I-driven reverse genetics for Influenza A virus and its application for pandemic (H1N1) 2009 influenza virus vaccine production.

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.

Prokaryote-expressed M2e protein improves H9N2 influenza vaccine efficacy and protection against lethal influenza A virus in mice.

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.

Comparison of the virulence and transmissibility of canine H3N2 influenza viruses and characterization of their canine adaptation factors.

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.

Avian-derived NS gene segments alter pathogenicity of the A/Puerto Rico/8/34 virus.

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)MLD50), 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)MLD50) as well as relatively high cytokine levels (IL-1α, IL-1ß, 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.

Pathobiological features of a novel, highly pathogenic avian influenza A(H5N8) virus.

The endemicity of highly pathogenic avian influenza (HPAI) A(H5N1) viruses in Asia has led to the generation of reassortant H5 strains with novel gene constellations. A newly emerged HPAI A(H5N8) virus caused poultry outbreaks in the Republic of Korea in 2014. Because newly emerging high-pathogenicity H5 viruses continue to pose public health risks, it is imperative that their pathobiological properties be examined. Here, we characterized A/mallard duck/Korea/W452/2014 (MDk/W452(H5N8)), a representative virus, and evaluated its pathogenic and pandemic potential in various animal models. We found that MDk/W452(H5N8), which originated from the reassortment of wild bird viruses harbored by migratory waterfowl in eastern China, replicated systemically and was lethal in chickens, but appeared to be attenuated, albeit efficiently transmitted, in ducks. Despite predominant attachment to avian-like virus receptors, MDk/W452(H5N8) also exhibited detectable human virus-like receptor binding and replicated in human respiratory tract tissues. In mice, MDk/W452(H5N8) was moderately pathogenic and had limited tissue tropism relative to previous HPAI A(H5N1) viruses. It also induced moderate nasal wash titers in inoculated ferrets; additionally, it was recovered in extrapulmonary tissues and one of three direct-contact ferrets seroconverted without shedding. Moreover, domesticated cats appeared to be more susceptible than dogs to virus infection. With their potential to become established in ducks, continued circulation of A(H5N8) viruses could alter the genetic evolution of pre-existing avian poultry strains. Overall, detailed virological investigation remains a necessity given the capacity of H5 viruses to evolve to cause human illness with few changes in the viral genome.

Emergence of H3N2pM-like and novel reassortant H3N1 swine viruses possessing segments derived from the A (H1N1)pdm09 influenza virus, Korea.

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.

Immunomodulaton and attenuation of lethal influenza A virus infection by oral administration with KIOM-C.

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.

Complete Genome Sequences of Novel Reassortant H1N2 Swine Influenza Viruses Isolated from Pigs in the Republic of Korea.

Novel reassortant swine H1N2 influenza viruses were isolated from pigs in a commercial slaughterhouse in the Republic of Korea. Genome sequence analyses revealed that these isolates contain segments from Eurasian avian-like swine (hemagglutinin [HA]), Korean swine H1N2 (neuraminidase [NA]), and North American H3N2pM-like (remaining genes) viruses. Further characterization is needed to gauge the potential threats of these viruses to public health.

Increased virulence of neuraminidase inhibitor-resistant pandemic H1N1 virus in mice: potential emergence of drug-resistant and virulent variants.

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.

Adjuvant efficacy of mOMV against avian influenza virus infection in mice.

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.