Sun-Woo Yoon

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.

Increased Acid Stability of the Hemagglutinin Protein Enhances H5N1 Influenza Virus Growth in the Upper Respiratory Tract but Is Insufficient for Transmission in Ferrets

ABSTRACT Influenza virus entry is mediated by the acidic-pH-induced activation of hemagglutinin (HA) protein. Here, we investigated how a decrease in the HA activation pH (an increase in acid stability) influences the properties of highly pathogenic H5N1 influenza virus in mammalian hosts. We generated isogenic A/Vietnam/1203/2004 (H5N1) (VN1203) viruses containing either wild-type HA protein (activation pH 6.0) or an HA2-K58I point mutation (K to I at position 58) (activation pH 5.5). The VN1203-HA2-K58I virus had replication kinetics similar to those of wild-type VN1203 in MDCK and normal human bronchial epithelial cells and yet had reduced growth in human alveolar A549 cells, which were found to have a higher endosomal pH than MDCK cells. Wild-type and HA2-K58I viruses promoted similar levels of morbidity and mortality in C57BL/6J mice and ferrets, and neither virus transmitted efficiently to naive contact cage-mate ferrets. The acid-stabilizing HA2-K58I mutation, which diminishes H5N1 replication and transmission in ducks, increased the virus load in the ferret nasal cavity early during infection while simultaneously reducing the virus load in the lungs. Overall, a single, acid-stabilizing mutation was found to enhance the growth of an H5N1 influenza virus in the mammalian upper respiratory tract, and yet it was insufficient to enable contact transmission in ferrets in the absence of additional mutations that confer α(2,6) receptor binding specificity and remove a critical N-linked glycosylation site. The information provided here on the contribution of HA acid stability to H5N1 influenza virus fitness and transmissibility in mammals in the background of a non-laboratory-adapted virus provides essential information for the surveillance and assessment of the pandemic potential of currently circulating H5N1 viruses.

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 HA225G and NA315N 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 HA225G and NA315N as potential virulence markers in mammals.

A single dose of whole inactivated H7N9 influenza vaccine confers protection from severe disease but not infection in ferrets

The H7N9 influenza virus caused significant mortality and morbidity in infected humans during an outbreak in China in 2013 stimulating vaccine development efforts. As previous H7-based vaccines have been poorly immunogenic in humans we sought to determine the immunogenic and protective properties of an inactivated whole virus vaccine derived from a 2013 H7N9 virus in ferrets. As whole virus vaccine preparations have been shown to be more immunogenic in humans, but less likely to be used, than split or surface antigen formulations, we vaccinated ferrets with a single dose of 15, 30, or 50 μg of the vaccine and subsequently challenged with wild-type A/Anhui/1/2013 (H7N9) either by direct instillation or by contact with infected animals. Although ferrets vaccinated with higher doses of vaccine had higher serum hemagglutinin inhibition (HI) titers, the titers were still low. During subsequent instillation challenge, however, ferrets vaccinated with 50 μg of vaccine showed no illness and shed significantly less virus than mock vaccinated controls. All vaccinated ferrets had lower virus loads in their lungs as compared to controls. In a separate study where unvaccinated-infected ferrets were placed in the same cage with vaccinated-uninfected ferrets, vaccination did not prevent infection in the contact ferrets, although they showed a trend of lower viral load. Overall, we conclude that inactivated whole-virus H7N9 vaccine was able to reduce the severity of infection and viral load, despite the lack of hemagglutinin-inhibiting antibodies.

Identification and characterization of influenza variants resistant to a viral endonuclease inhibitor

Significance The endonuclease domain within the influenza virus heterotrimeric replication machinery is essential and represents an attractive drug target. It is important to understand the structural basis of potential inhibitor resistance, to design appropriate inhibitors and to prioritize drug candidates that are unlikely to cause the rapid development of clinically-relevant resistance mutations. Using a prototypical endonuclease inhibitor (L-742,001), we used mutagenesis to select for replication competent resistant mutants and studied the structural and functional basis for the observed resistance. These studies confirm that the endonuclease domain is an excellent drug target for treating influenza. They also provide reagents (mutant viruses and constructs) and crucial pharmacophore knowledge that will aid in the development of new drug candidates for urgently needed influenza therapies. The influenza endonuclease is an essential subdomain of the viral RNA polymerase. It processes host pre-mRNAs to serve as primers for viral mRNA and is an attractive target for antiinfluenza drug discovery. Compound L-742,001 is a prototypical endonuclease inhibitor, and we found that repeated passaging of influenza virus in the presence of this drug did not lead to the development of resistant mutant strains. Reduced sensitivity to L-742,001 could only be induced by creating point mutations via a random mutagenesis strategy. These mutations mapped to the endonuclease active site where they can directly impact inhibitor binding. Engineered viruses containing the mutations showed resistance to L-742,001 both in vitro and in vivo, with only a modest reduction in fitness. Introduction of the mutations into a second virus also increased its resistance to the inhibitor. Using the isolated wild-type and mutant endonuclease domains, we used kinetics, inhibitor binding and crystallography to characterize how the two most significant mutations elicit resistance to L-742,001. These studies lay the foundation for the development of a new class of influenza therapeutics with reduced potential for the development of clinical endonuclease inhibitor-resistant influenza strains.

Pandemic Swine H1N1 Influenza Viruses with Almost Undetectable Neuraminidase Activity Are Not Transmitted via Aerosols in Ferrets and Are Inhibited by Human Mucus but Not Swine Mucus

ABSTRACT A balance between the functions of the influenza virus surface proteins hemagglutinin (HA) and neuraminidase (NA) is thought to be important for the transmission of viruses between humans. Here we describe two pandemic H1N1 viruses, A/swine/Virginia/1814-1/2012 and A/swine/Virginia/1814-2/2012 (pH1N1low-1 and -2, respectively), that were isolated from swine symptomatic for influenza. The enzymatic activity of the NA of these viruses was almost undetectable, while the HA binding affinity for α2,6 sialic acids was greater than that of the highly homologous pH1N1 viruses A/swine/Pennsylvania/2436/2012 and A/swine/Minnesota/2499/2012 (pH1N1-1 and -2), which exhibited better-balanced HA and NA activities. The in vitro growth kinetics of pH1N1low and pH1N1 viruses were similar, but aerosol transmission of pH1N1low-1 was abrogated and transmission via direct contact in ferrets was significantly impaired compared to pH1N1-1, which transmitted by direct and aerosol contact. In normal human bronchial epithelial cells, pH1N1low-1 was significantly inhibited by mucus but pH1N1-1 was not. In Madin-Darby canine kidney cell cultures overlaid with human or swine mucus, human mucus inhibited pH1N1low-1 but swine mucus did not. These data show that the interaction between viruses and mucus may be an important factor in viral transmissibility and could be a barrier for interspecies transmission between humans and swine for influenza viruses. IMPORTANCE A balance between the functions of the influenza virus surface proteins hemagglutinin (HA) and neuraminidase (NA) is thought to be important for transmission of viruses from swine to humans. Here we show that a swine virus with extremely functionally mismatched HA and NAs (pH1N1low-1) cannot transmit via aerosol in ferrets, while another highly homologous virus with HA and NAs that are better matched functionally (pH1N1-1) can transmit via aerosol. These viruses show similar growth kinetics in Madin-Darby canine kidney (MDCK) cells, but pH1N1low-1 is significantly inhibited by mucus in normal human bronchial epithelial cells whereas pH1N1-1 is not. Further, human mucus could inhibit these viruses, but swine mucus could not. These data show that the interaction between viruses and mucus may be an important factor in viral transmissibility and could be a species barrier between humans and swine for influenza viruses.

A Single Amino Acid at the Hemagglutinin Cleavage Site Contributes to the Pathogenicity but Not the Transmission of Egyptian Highly Pathogenic H5N1 Influenza Virus in Chickens

ABSTRACT Highly pathogenic avian influenza (HPAI) virus H5N1 has been enzootic in Egypt since 2008. Virus-associated mortality (but not the number of cases) in humans and poultry seems to have decreased over time, but the reason for this remains unknown. We investigated the role of a single amino acid substitution in the hemagglutinin cleavage site on virus pathogenicity and transmission in chickens. The R325G substitution significantly reduced pathogenicity without altering the transmission efficiency of HPAI H5N1 virus.

Putative amino acid determinants of the emergence of the 2009 influenza A (H1N1) virus in the human population

The emergence of the unique H1N1 influenza A virus in 2009 resulted in a pandemic that has spread to over 200 countries. The constellation of molecular factors leading to the emergence of this strain is still unclear. Using a computational approach, we identified molecular determinants that may discriminate the hemagglutinin protein of the 2009 human pandemic H1N1 (pH1N1) strain from that of other H1N1 strains. As expected, positions discriminating the pH1N1 from seasonal human strains were located in or near known H1N1 antigenic sites, thus camouflaging the pH1N1 strain from immune recognition. For example, the alteration S145K (an antigenic position) was found as a characteristic of the pH1N1 strain. We also detected positions in the hemagglutinin protein differentiating classical swine viruses from pH1N1. These positions were mostly located in and around the receptor-binding pocket, possibly influencing binding affinity to the human cell. Such alterations may be liable in part for the virus’s efficient infection and adaptation to humans. For instance, 133A and 149 were identified as discriminative positions. Significantly, we showed that the substitutions R133AK and R149K, predicted to be pH1N1 characteristics, each altered virus binding to erythrocytes and conferred virulence to A/swine/NC/18161/02 in mice, reinforcing the computational findings. Our findings provide a structural explanation for the deficient immunity of humans to the pH1N1 strain. Moreover, our analysis points to unique molecular factors that may have facilitated the emergence of this swine variant in humans, in contrast to other swine variants that failed.

Rapid acquisition of polymorphic virulence markers during adaptation of highly pathogenic avian influenza H5N8 virus in the mouse

Emergence of a highly pathogenic avian influenza (HPAI) H5N8 virus in Asia and its spread to Europe and North America has caused great concern for human health. Although the H5N8 virus has been only moderately pathogenic to mammalian hosts, virulence can still increase. We evaluated the pathogenic potential of several H5N8 strains via the mouse-adaptation method. Two H5N8 viruses were sequentially passaged in BALB/c mice and plaque-purified from lung samples. The viruses rapidly obtained high virulence (MLD50, up to 0.5 log10u2009PFU/mL) within 5 passages. Sequence analysis revealed the acquisition of several virulence markers, including the novel marker P708S in PB1 gene. Combinations of markers synergistically enhanced viral replication and polymerase activity in human cell lines and virulence and multiorgan dissemination in mice. These results suggest that H5N8 viruses can rapidly acquire virulence markers in mammalian hosts; thus, rapid spread as well as repeated viral introduction into the hosts may significantly increase the risk of human infection and elevate pandemic potential.

Egg-adaptive mutations in H3N2v vaccine virus enhance egg-based production without loss of antigenicity or immunogenicity

The recently detected zoonotic H3N2 variant influenza A (H3N2v) viruses have caused 343 documented cases of human infection linked to contact with swine. An effective vaccine is needed for these viruses, which may acquire transmissibility among humans. However, viruses isolated from human cases do not replicate well in embryonated chicken eggs, posing an obstacle to egg-based vaccine production. To address this issue, we sought to identify egg-adaptive mutations in surface proteins that increase the yield of candidate vaccine viruses (CVVs) in eggs while preserving their immunizing effectiveness. After serial passage of a representative H3N2v isolate (A/Indiana/08/2011), we identified several egg-adaptive combinations of HA mutations and assessed the egg-based replication, antigenicity, and immunogenicity of A/Puerto Rico/8/34 (H1N1, PR8)-based 6+2 reverse genetics CVVs carrying these mutations. Here we demonstrate that the respective combined HA substitutions G1861V+N2461K, N1651K+G1861V, T1281N+N1651K+R762G, and T1281N+N1651K+I102M, all identified after egg passage, enhanced the replication of the CVVs in eggs without substantially affecting their antigenicity or immunogenicity. The mutations were stable, and the mutant viruses acquired no additional substitutions during six subsequent egg passages. We found two crucial mutations, G186V, which was previously defined, and N246K, which in combination improved virus yield in eggs without significantly impacting antigenicity or immunogenicity. This combination of egg-adaptive mutations appears to most effectively generate high egg-based yields of influenza A/Indiana/08/2011-like CVVs.