Human Neuraminidases: Structures and Stereoselective Inhibitors.

This Perspective describes the classification, structures, substrates, mechanisms of action, and implications of human neuraminidases (hNEUs) in various pathologies. Some inhibitors have been developed for each isoform, leading to more precise interactions with hNEUs. Although crystal structure data are available for NEU2, most of the findings are based on NEU1 inhibition, and limited information is available for other hNEUs. Therefore, the synthesis of new compounds would facilitate the enrichment of the arsenal of inhibitors to better understand the roles of hNEUs and their mechanisms of action. Nevertheless, due to the already known inhibitors of human neuraminidase enzymes, a structure-activity relationship is presented along with different approaches to inhibit these enzymes for the development of potent and selective inhibitors. Among the different emerging strategies, one is the inhibition of the dimerization of NEU1 or NEU3, and the second is the inhibition of certain receptors located close to hNEU.

Investigation of genetic variation: Neuraminidase gene of influenza A virus H1N1/pdm09, Shiraz, Iran (2015-2016).

Oseltamivir and antiviral agents are frequently used for the prevention and treatment of influenza infection. However, resistance to oseltamivir has been reported globally due to a mutation in the Influenza virus neuraminidase gene. Such resistance will be detected by genotyping and phenotyping studies of viral isolates. The recent study aimed to determine the genetic mutation of neuraminidase gene in influenza A (H1N1) viruses isolated from children referred to Shiraz tertiary hospitals during 1 year (2015-2016) with influenza-like symptoms. A total of 300 patients were registered and throat samples were taken. The throat swabs were used for viral RNA extraction. Detection of influenza A (H1N1) was performed using the one-step real-time polymerase chain reaction (qRT-PCR) method. From positive isolates for H1N1, 51 random samples were evaluated for neuraminidase gene mutation with the nested PCR-sequencing method. Of 300 cases, 102 (34%) isolates were detected as influenza A (H1N1) pdm09. Based on sequencing results, 2 of the 44 sequenced isolates exhibited H275Y substitution, which presented oseltamivir resistance. In comparison with reference strain, the phylogenetic analysis of sequenced isolates was classified in genogroup 6B. While this result is the first report of emerging oseltamivir-resistant in the southwest of Iran, it is highly recommended to perform these evaluations on the different geographical regions in any prevalence area to plan treatment strategies for influenza.

In Vitro Profiling of Laninamivir-Resistant Substitutions in N3 to N9 Avian Influenza Virus Neuraminidase Subtypes and Their Association with In Vivo Susceptibility.

Laninamivir (LAN) is a long-acting neuraminidase (NA) inhibitor (NAI) with a similar binding profile in the influenza NA enzyme active site as those of other NAIs, oseltamivir (OS), zanamivir (ZAN), and peramivir, and may share common resistance markers with these NAIs. We screened viruses with NA substitutions previously found during OS and ZAN selection in avian influenza viruses (AIVs) of the N3 to N9 subtypes for LAN susceptibility. Of the 72 NA substitutions, 19 conferred resistance to LAN, which ranged from 11.2- to 549.8-fold-decreased inhibitory activity over that of their parental viruses. Ten NA substitutions reduced the susceptibility to all four NAIs, whereas the remaining 26 substitutions yielded susceptibility to one or more NAIs. To determine whether the in vitro susceptibility of multi-NAI-resistant AIVs is associated with in vivo susceptibility, we infected BALB/c mice with recombinant AIVs with R292K (ma81K-N3R292K) or Q136K (ma81K-N8Q136K) NA substitutions, which impart in vitro susceptibility only to LAN or OS, respectively. Both ma81K-N3R292K and ma81K-N8Q136K virus-infected mice exhibited reduced weight loss, mortality, and lung viral titers when treated with their susceptible NAIs, confirming the in vitro susceptibility of these substitutions. Together, LAN resistance profiling of AIVs of a range of NA subtypes improves the understanding of NAI resistance mechanisms. Furthermore, the association of in vitro and in vivo NAI susceptibility indicates that our models are useful tools for monitoring NAI susceptibility of AIVs.IMPORTANCE The chemical structures of neuraminidase inhibitors (NAIs) possess similarities, but slight differences can result in variable susceptibility of avian influenza viruses (AIVs) carrying resistance-associated NA substitutions. Therefore, comprehensive susceptibility profiling of these substitutions in AIVs is critical for understanding the mechanism of antiviral resistance. In this study, we profiled resistance to the anti-influenza drug laninamivir in AIVs with substitutions known to impart resistance to other NAIs. We found 10 substitutions that conferred resistance to all four NAIs tested. On the other hand, we found that the remaining 26 NA substitutions were susceptible to at least one or more NAIs and showed for a small selection that in vitro data predicted in vivo behavior. Therefore, our findings highlight the usefulness of screening resistance markers in NA enzyme inhibition assays and animal models of AIV infections.

Two novel reassortant H11N8 avian influenza viruses occur in wild birds found in East Dongting Lake, China.

During the surveillance of avian influenza viruses in East Dongting Lake, China (2014-2015), two H11N8 avian influenza viruses were detected in the bean goose (Anser fabalis) and the falcated teal (Anas falcata). Phylogenetic analysis showed that these two novel reassortant H11N8 avian influenza viruses contain genes from poultry and wild birds. This is the first report detecting the H11N8 subtype influenza virus from wild birds in Asia. These findings indicate a dissemination of avian influenza virus along the East Asian-Australian flyway. In addition, the interaction between poultry and wild birds was addressed suggesting the need for intensive surveillance of wild bird populations.

Neuraminidase-Inhibiting Antibody Titers Correlate with Protection from Heterologous Influenza Virus Strains of the Same Neuraminidase Subtype.

Immune responses induced by currently licensed inactivated influenza vaccines are mainly directed against the hemagglutinin (HA) glycoprotein, the immunodominant antigen of influenza viruses. The resulting antigenic drift of HA requires frequent updating of the vaccine composition and annual revaccination. On the other hand, the levels of antibodies directed against the neuraminidase (NA) glycoprotein, the second major influenza virus antigen, vary greatly. To investigate the potential of the more conserved NA protein for the induction of subtype-specific protection, vesicular stomatitis virus-based replicons expressing a panel of N1 proteins from prototypic seasonal and pandemic H1N1 strains and human H5N1 and H7N9 isolates were generated. Immunization of mice and ferrets with the replicon carrying the matched N1 protein resulted in robust humoral and cellular immune responses and protected against challenge with the homologous influenza virus with an efficacy similar to that of the matched HA protein, illustrating the potential of the NA protein as a vaccine antigen. The extent of protection after immunization with mismatched N1 proteins correlated with the level of cross-reactive neuraminidase-inhibiting antibody titers. Passive serum transfer experiments in mice confirmed that these functional antibodies determine subtype-specific cross-protection. Our findings illustrate the potential of NA-specific immunity for achieving broader protection against antigenic drift variants or newly emerging viruses carrying the same NA but a different HA subtype.IMPORTANCE Despite the availability of vaccines, annual influenza virus epidemics cause 250,000 to 500,000 deaths worldwide. Currently licensed inactivated vaccines, which are standardized for the amount of the hemagglutinin (HA) antigen, primarily induce strain-specific antibodies, whereas the immune response to the neuraminidase (NA) antigen, which is also present on the viral surface, is usually low. Using NA-expressing single-cycle vesicular stomatitis virus replicons, we show that the NA antigen conferred protection of mice and ferrets against not only the matched influenza virus strains but also viruses carrying NA proteins from other strains of the same subtype. The extent of protection correlated with the level of cross-reactive NA-inhibiting antibodies. This highlights the potential of the NA antigen for the development of more broadly protective influenza vaccines. Such vaccines may also provide partial protection against newly emerging strains with the same NA but a different HA subtype.

Expanding an expanded genome: long-read sequencing of Trypanosoma cruzi.

Although the genome of Trypanosoma cruzi, the causative agent of Chagas disease, was first made available in 2005, with additional strains reported later, the intrinsic genome complexity of this parasite (the abundance of repetitive sequences and genes organized in tandem) has traditionally hindered high-quality genome assembly and annotation. This also limits diverse types of analyses that require high degrees of precision. Long reads generated by third-generation sequencing technologies are particularly suitable to address the challenges associated with T. cruzi's genome since they permit direct determination of the full sequence of large clusters of repetitive sequences without collapsing them. This, in turn, not only allows accurate estimation of gene copy numbers but also circumvents assembly fragmentation. Here, we present the analysis of the genome sequences of two T. cruzi clones: the hybrid TCC (TcVI) and the non-hybrid Dm28c (TcI), determined by PacBio Single Molecular Real-Time (SMRT) technology. The improved assemblies herein obtained permitted us to accurately estimate gene copy numbers, abundance and distribution of repetitive sequences (including satellites and retroelements). We found that the genome of T. cruzi is composed of a 'core compartment' and a 'disruptive compartment' which exhibit opposite GC content and gene composition. Novel tandem and dispersed repetitive sequences were identified, including some located inside coding sequences. Additionally, homologous chromosomes were separately assembled, allowing us to retrieve haplotypes as separate contigs instead of a unique mosaic sequence. Finally, manual annotation of surface multigene families, mucins and trans-sialidases allows now a better overview of these complex groups of genes.

Screening for Neuraminidase Inhibitor Resistance Markers among Avian Influenza Viruses of the N4, N5, N6, and N8 Neuraminidase Subtypes.

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.IMPORTANCE The 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.

Charged amino acid variability related to N-glyco -sylation and epitopes in A/H3N2 influenza: Hem -agglutinin and neuraminidase.

BACKGROUND: The A/H3N2 influenza viruses circulated in humans have been shown to undergo antigenic drift, a process in which amino acid mutations result from nucleotide substitutions. There are few reports regarding the charged amino acid mutations. The purpose of this paper is to explore the relations between charged amino acids, N-glycosylation and epitopes in hemagglutinin (HA) and neuraminidase (NA). METHODS: A total of 700 HA genes (691 NA genes) of A/H3N2 viruses were chronologically analyzed for the mutational variants in amino acid features, N-glycosylation sites and epitopes since its emergence in 1968. RESULTS: It was found that both the number of HA N-glycosylation sites and the electric charge of HA increased gradually up to 2016. The charges of HA and HA1 increased respectively 1.54-fold (+7.0 /+17.8) and 1.08-fold (+8.0/+16.6) and the number of NGS in nearly doubled (7/12). As great diversities occurred in 1990s, involving Epitope A, B and D mutations, the charged amino acids in Epitopes A, B, C and D in HA1 mutated at a high frequency in global circulating strains last decade. The charged amino acid mutations in Epitopes A (T135K) has shown high mutability in strains near years, resulting in a decrease of NGT135-135. Both K158N and K160T not only involved mutations charged in epitope B, but also caused a gain of NYT158-160. Epitope B and its adjacent N-glycosylation site NYT158-160 mutated more frequently, which might be under greater immune pressure than the rest. CONCLUSIONS: The charged amino acid mutations in A/H3N2 Influenza play a significant role in virus evolution, which might cause an important public health issue. Variability related to both the epitopes (A and B) and N-glycosylation is beneficial for understanding the evolutionary mechanisms, disease pathogenesis and vaccine research.

A QSAR classification model for neuraminidase inhibitors of influenza A viruses (H1N1) based on weighted penalized support vector machine.

Descriptor selection is a procedure widely used in chemometrics. The aim is to select the best subset of descriptors relevant to the quantitative structure-activity relationship (QSAR) study being considered. In this paper, a new descriptor selection method for the QSAR classification model is proposed by adding a new weight inside L1-norm. The experimental results from classifying the neuraminidase inhibitors of influenza A viruses (H1N1) demonstrate that the proposed method in the QSAR classification model performs effectively and competitively compared with other existing penalized methods in terms of classification performance and the number of selected descriptors.

Evolution of the neuraminidase gene of seasonal influenza A and B viruses in Thailand between 2010 and 2015.

The neuraminidase inhibitors (NAIs) oseltamivir and zanamivir are commonly used for the treatment and control of influenza A and B virus infection. However, the emergence of new influenza virus strains with reduced susceptibility to NAIs may appear with the use of these antivirals or even naturally. We therefore screened the neuraminidase (NA) sequences of seasonal influenza virus A(H1N1), A(H1N1)pdm09, A(H3N2), and influenza B virus strains identified in Thailand for the presence of substitutions previously reported to reduce susceptibility to NAIs. We initially examined oseltamivir resistance (characterized by the H275Y mutation in the NA gene) in 485 A(H1N1)pdm09 strains circulating in Thailand and found that 0.82% (4/485) had this substitution. To further evaluate the evolution of the NA gene, we also randomly selected 98 A(H1N1)pdm09, 158 A(H3N2), and 69 influenza B virus strains for NA gene amplification and sequencing, which revealed various amino acid mutations in the active site of the NA protein previously shown to be associated with reduced susceptibility to NAIs. Phylogenetic analysis of the influenza virus strains from this study and elsewhere around the world, together with the estimations of nucleotide substitution rates and selection pressure, and the predictions of B-cell epitopes and N-linked glycosylation sites all provided evidence for the ongoing evolution of NA. The overall rates of NA evolution for influenza A viruses were higher than for influenza B virus at the nucleotide level, although influenza B virus possessed more genealogical diversity than that of influenza A viruses. The continual surveillance of the antigenic changes associated with the NA protein will not only contribute to the influenza virus database but may also provide a better understanding of selection pressure exerted by antiviral use.

Outcomes and Susceptibility to Neuraminidase Inhibitors in Individuals Infected With Different Influenza B Lineages: The Influenza Resistance Information Study.

BACKGROUND: Little is known about how influenza infections caused by B/Victoria and B/Yamagata virus lineages compare with respect to disease course and susceptibility to antiviral therapy. METHODS: Data from patients with influenza B infections from the first 5 years (2009-2013) of the prospective Influenza Resistance Information Study (IRIS, NCT00884117) were evaluated. Cultured viruses were phenotypically tested for neuraminidase inhibitor (NAI) sensitivity, and sequenced to determine virus lineage (B/Victoria or B/Yamagata). Differences in clinical outcomes (viral clearance and symptom resolution) between virus lineages were assessed using Kaplan-Meier analysis. RESULTS: In all, 914 patients were positive for influenza B by reverse transcriptase polymerase chain reaction ( RT-PCR: B/Victoria, 586; B/Yamagata, 289; not subtyped, 39); 474 were treated with antivirals. No phenotypic resistance to oseltamivir or zanamivir was found in B/Victoria or B/Yamagata viruses. Of 15 predefined resistance mutations, 2 were detected by neuraminidase sequencing: I221T had reduced sensitivity to oseltamivir, and I221V was sensitive to NAI inhibition. No consistent differences between virus lineages in times to viral clearance or to symptom or fever resolution were found in adults and adolescents or in children. CONCLUSIONS: Influenza B virus lineage had no notable effect on disease outcomes or antiviral susceptibility in this population.

Comparability of neuraminidase inhibition antibody titers measured by enzyme-linked lectin assay (ELLA) for the analysis of influenza vaccine immunogenicity.

Neuraminidase-inhibition (NI) antibody titers can be used to evaluate the immunogenicity of inactivated influenza vaccines and have provided evidence of serologic cross-reactivity between seasonal and pandemic H1N1 viruses. The traditional thiobarbituric acid assay is impractical for large serologic analyses, and therefore many laboratories use an enzyme-linked lectin assay (ELLA) to determine serum NI antibody titers. The comparability of ELLA NI antibody titers when measured in different laboratories was unknown. Here we report a study conducted through the Consortium for the Standardisation of Influenza SeroEpidemiology (CONSISE) to evaluate the variability of the ELLA. NI antibody titers of a set of 12 samples were measured against both N1 and N2 neuraminidase antigens in 3 independent assays by each of 23 laboratories. For a sample repeated in the same assay, ≥96% of N1 and N2 assays had less than a 4-fold difference in titer. Comparison of the titers measured in assays conducted on 3 different days in the same laboratory showed that a four-fold difference in titer was uncommon. Titers of the same sera measured in different laboratories spanned 3 to 6 two-fold dilutions (i.e., 8-64 fold difference in titer), with an average percent geometric coefficient of variation (%GCV) of 112 and 82% against N1 and N2 antigens, respectively. The difference in titer as indicated by fold range and %GCV was improved by normalizing the NI titers to a standard that was included in each assay. This study identified background signal and the amount of antigen in the assay as critical factors that influence titer, providing important information toward development of a consensus ELLA protocol.

NanI Sialidase, CcpA, and CodY Work Together To Regulate Epsilon Toxin Production by Clostridium perfringens Type D Strain CN3718.

UNLABELLED: Clostridium perfringens type D strains are usually associated with diseases of livestock, and their virulence requires the production of epsilon toxin (ETX). We previously showed (J. Li, S. Sayeed, S. Robertson, J. Chen, and B. A. McClane, PLoS Pathog 7:e1002429, 2011, http://dx.doi.org/10.1371/journal.ppat.1002429) that BMC202, a nanI null mutant of type D strain CN3718, produces less ETX than wild-type CN3718 does. The current study proved that the lower ETX production by strain BMC202 is due to nanI gene disruption, since both genetic and physical (NanI or sialic acid) complementation increased ETX production by BMC202. Furthermore, a sialidase inhibitor that interfered with NanI activity also reduced ETX production by wild-type CN3718. The NanI effect on ETX production was shown to involve reductions in codY and ccpA gene transcription levels in BMC202 versus wild-type CN3718. Similar to CodY, CcpA was found to positively control ETX production. A double codY ccpA null mutant produced even less ETX than a codY or ccpA single null mutant. CcpA bound directly to sequences upstream of the etx or codY start codon, and bioinformatics identified putative CcpA-binding cre sites immediately upstream of both the codY and etx start codons, suggesting possible direct CcpA regulatory effects. A ccpA mutation also decreased codY transcription, suggesting that CcpA effects on ETX production can be both direct and indirect, including effects on codY transcription. Collectively, these results suggest that NanI, CcpA, and CodY work together to regulate ETX production, with NanI-generated sialic acid from the intestines possibly signaling type D strains to upregulate their ETX production and induce disease. IMPORTANCE: Clostridium perfringens NanI was previously shown to increase ETX binding to, and cytotoxicity for, MDCK host cells. The current study demonstrates that NanI also regulates ETX production via increased transcription of genes encoding the CodY and CcpA global regulators. Results obtained using single ccpA or codY null mutants and a ccpA codY double null mutant showed that codY and ccpA regulate ETX production independently of one another but that ccpA also affects codY transcription. Electrophoretic mobility shift assays and bioinformatic analyses suggest that both CodY and CcpA may directly regulate etx transcription. Collectively, results of this study suggest that sialic acid generated by NanI from intestinal sources signals ETX-producing C. perfringens strains, via CcpA and CodY, to upregulate ETX production and cause disease.

Novel Reassortant Human-Like H3N2 and H3N1 Influenza A Viruses Detected in Pigs Are Virulent and Antigenically Distinct from Swine Viruses Endemic to the United States.

UNLABELLED: Human-like swine H3 influenza A viruses (IAV) were detected by the USDA surveillance system. We characterized two novel swine human-like H3N2 and H3N1 viruses with hemagglutinin (HA) genes similar to those in human seasonal H3 strains and internal genes closely related to those of 2009 H1N1 pandemic viruses. The H3N2 neuraminidase (NA) was of the contemporary human N2 lineage, while the H3N1 NA was of the classical swine N1 lineage. Both viruses were antigenically distant from swine H3 viruses that circulate in the United States and from swine vaccine strains and also showed antigenic drift from human seasonal H3N2 viruses. Their pathogenicity and transmission in pigs were compared to those of a human H3N2 virus with a common HA ancestry. Both swine human-like H3 viruses efficiently infected pigs and were transmitted to indirect contacts, whereas the human H3N2 virus did so much less efficiently. To evaluate the role of genes from the swine isolates in their pathogenesis, reverse genetics-generated reassortants between the swine human-like H3N1 virus and the seasonal human H3N2 virus were tested in pigs. The contribution of the gene segments to virulence was complex, with the swine HA and internal genes showing effects in vivo. The experimental infections indicate that these novel H3 viruses are virulent and can sustain onward transmission in pigs, and the naturally occurring mutations in the HA were associated with antigenic divergence from H3 IAV from humans and swine. Consequently, these viruses could have a significant impact on the swine industry if they were to cause more widespread outbreaks, and the potential risk of these emerging swine IAV to humans should be considered. IMPORTANCE: Pigs are important hosts in the evolution of influenza A viruses (IAV). Human-to-swine transmissions of IAV have resulted in the circulation of reassortant viruses containing human-origin genes in pigs, greatly contributing to the diversity of IAV in swine worldwide. New human-like H3N2 and H3N1 viruses that contain a mix of human and swine gene segments were recently detected by the USDA surveillance system. The human-like viruses efficiently infected pigs and resulted in onward airborne transmission, likely due to the multiple changes identified between human and swine H3 viruses. The human-like swine viruses are distinct from contemporary U.S. H3 swine viruses and from the strains used in swine vaccines, which could have a significant impact on the swine industry due to a lack of population immunity. Additionally, public health experts should consider an appropriate assessment of the risk of these emerging swine H3 viruses for the human population.

Detection and Characterization of Clade 1 Reassortant H5N1 Viruses Isolated from Human Cases in Vietnam during 2013.

Highly pathogenic avian influenza (HPAI) H5N1 is endemic in Vietnamese poultry and has caused sporadic human infection in Vietnam since 2003. Human infections with HPAI H5N1 are of concern due to a high mortality rate and the potential for the emergence of pandemic viruses with sustained human-to-human transmission. Viruses isolated from humans in southern Vietnam have been classified as clade 1 with a single genome constellation (VN3) since their earliest detection in 2003. This is consistent with detection of this clade/genotype in poultry viruses endemic to the Mekong River Delta and surrounding regions. Comparison of H5N1 viruses detected in humans from southern Vietnamese provinces during 2012 and 2013 revealed the emergence of a 2013 reassortant virus with clade 1.1.2 hemagglutinin (HA) and neuraminidase (NA) surface protein genes but internal genes derived from clade 2.3.2.1a viruses (A/Hubei/1/2010-like; VN12). Closer analysis revealed mutations in multiple genes of this novel genotype (referred to as VN49) previously associated with increased virulence in animal models and other markers of adaptation to mammalian hosts. Despite the changes identified between the 2012 and 2013 genotypes analyzed, their virulence in a ferret model was similar. Antigenically, the 2013 viruses were less cross-reactive with ferret antiserum produced to the clade 1 progenitor virus, A/Vietnam/1203/2004, but reacted with antiserum produced against a new clade 1.1.2 WHO candidate vaccine virus (A/Cambodia/W0526301/2012) with comparable hemagglutination inhibition titers as the homologous antigen. Together, these results indicate changes to both surface and internal protein genes of H5N1 viruses circulating in southern Vietnam compared to 2012 and earlier viruses.

Optimization of an enzyme-linked lectin assay suitable for rapid antigenic characterization of the neuraminidase of human influenza A(H3N2) viruses.

Antibodies to neuraminidase (NA), the second most abundant surface protein of the influenza virus, contribute to protection against influenza virus infection. Although traditional and miniaturized thiobarbituric acid (TBA) neuraminidase inhibition (NI) assays have been successfully used to characterize the antigenic properties of NA, these methods are cumbersome and not easily amendable to rapid screening. An additional difficulty of the NI assay is the interference by hemagglutinin (HA)-specific antibodies. To prevent interference of HA-specific antibodies, most NI assays are performed with recombinant viruses containing a mismatched HA. However, generation of these viruses is time consuming and unsuitable for large-scale surveillance. The feasibility of using the recently developed enzyme-linked lectin assay (ELLA) to evaluate the antigenic relatedness of NA of wild type A(H3N2) viruses was assessed. Rather than using recombinant viruses, wild type A(H3N2) viruses were used as antigen with ferret sera elicited against recombinant viruses with a mismatched HA. In this study, details of the critical steps that are needed to modify and optimize the NI ELLA in a format that is reproducible, highly sensitive, and useful for influenza virus surveillance to monitor antigenic drift of NA are provided.

Progressive antigenic drift and phylogeny of human influenza A(H3N2) virus over five consecutive seasons (2009-2013) in Hangzhou, China.

Vaccine efficacy (VE) can be affected by progressive antigenic drift or any new reassortment of influenza viruses. To effectively track the evolution of human influenza A(H3N2) virus circulating in Hangzhou, China, a total of 65 clinical specimens were selected randomly from outpatients infected by A(H3N2) viruses during the study period from November 2009 to December 2013. The results of reduced VE and antigenic drift of the correspondent epitopes (C-D-E to A-B) suggest that the current vaccine provides suboptimal protection against the A(H3N2) strains circulating recently. Phylogenetic analysis of the entire HA and NA sequences demonstrated that these two genes underwent independent evolutionary pathways during recent seasons. The H3-based phylogenetic tree showed that a special strain A/Hangzhou/A289/2012 fell in a cluster among viruses with reduced VE predominantly circulating in 2013. Our findings underscore a possible early warning for the circulation of A(H3N2) variants with antigenic drift during the previous seasons.

A synchronized global sweep of the internal genes of modern avian influenza virus.

Zoonotic infectious diseases such as influenza continue to pose a grave threat to human health. However, the factors that mediate the emergence of RNA viruses such as influenza A virus (IAV) are still incompletely understood. Phylogenetic inference is crucial to reconstructing the origins and tracing the flow of IAV within and between hosts. Here we show that explicitly allowing IAV host lineages to have independent rates of molecular evolution is necessary for reliable phylogenetic inference of IAV and that methods that do not do so, including 'relaxed' molecular clock models, can be positively misleading. A phylogenomic analysis using a host-specific local clock model recovers extremely consistent evolutionary histories across all genomic segments and demonstrates that the equine H7N7 lineage is a sister clade to strains from birds--as well as those from humans, swine and the equine H3N8 lineage--sharing an ancestor with them in the mid to late 1800s. Moreover, major western and eastern hemisphere avian influenza lineages inferred for each gene coalesce in the late 1800s. On the basis of these phylogenies and the synchrony of these key nodes, we infer that the internal genes of avian influenza virus (AIV) underwent a global selective sweep beginning in the late 1800s, a process that continued throughout the twentieth century and up to the present. The resulting western hemispheric AIV lineage subsequently contributed most of the genomic segments to the 1918 pandemic virus and, independently, the 1963 equine H3N8 panzootic lineage. This approach provides a clear resolution of evolutionary patterns and processes in IAV, including the flow of viral genes and genomes within and between host lineages.

Epidemiological surveillance of low pathogenic avian influenza virus (LPAIV) from poultry in Guangxi Province, Southern China.

Low pathogenic avian influenza virus (LPAIV) usually causes mild disease or asymptomatic infection in poultry. However, some LPAIV strains can be transmitted to humans and cause severe infection. Genetic rearrangement and recombination of even low pathogenic influenza may generate a novel virus with increased virulence, posing a substantial risk to public health. Southern China is regarded as the world "influenza epicenter", due to a rash of outbreaks of influenza in recent years. In this study, we conducted an epidemiological survey of LPAIV at different live bird markets (LBMs) in Guangxi province, Southern China. From January 2009 to December 2011, we collected 3,121 cotton swab samples of larynx, trachea and cloaca from the poultry at LBMs in Guangxi. Virus isolation, hemagglutination inhibition (HI) assay, and RT-PCR were used to detect and subtype LPAIV in the collected samples. Of the 3,121 samples, 336 samples (10.8%) were LPAIV positive, including 54 (1.7%) in chicken and 282 (9.1%) in duck. The identified LPAIV were H3N1, H3N2, H6N1, H6N2, H6N5, H6N6, H6N8, and H9N2, which are combinations of seven HA subtypes (H1, H3, H4, H6, H9, H10 and H11) and five NA subtypes (N1, N2, N5, N6 and N8). The H3 and H9 subtypes are predominant in the identified LPAIVs. Among the 336 cases, 29 types of mixed infection of different HA subtypes were identified in 87 of the cases (25.9%). The mixed infections may provide opportunities for genetic recombination. Our results suggest that the LPAIV epidemiology in poultry in the Guangxi province in southern China is complicated and highlights the need for further epidemiological and genetic studies of LPAIV in this area.

Evidence for the circulation and inter-hemispheric movement of the H14 subtype influenza A virus.

Three H14 influenza A virus (IAV) isolates recovered in 2010 during routine virus surveillance along the Mississippi Migratory Bird Flyway in Wisconsin, U.S.A. raised questions about the natural history of these rare viruses. These were the first H14 IAV isolates recovered in the Western Hemisphere and the only H14 IAV isolates recovered since the original four isolates in 1982 in Asia. Full length genomic sequencing of the 2010 H14 isolates demonstrated the hemagglutinin (HA) gene from the 1982 and 2010 H14 isolates showed 89.6% nucleotide and 95.6% amino acid similarity and phylogenetic analysis of these viruses placed them with strong support within the H14 subtype lineage. The level of genomic divergence observed between the 1982 and 2010 viruses provides evidence that the H14 HA segment was circulating undetected in hosts and was not maintained in environmental stasis. Further, the evolutionary relationship observed between 1982 H14 and the closely related H4 subtype HA segments were similar to contemporary comparisons suggesting limited adaptive divergence between these sister subtypes. The nonstructural (NS) segment of one 2010 isolate was placed in a NS clade isolated infrequently over the last several decades that includes the NS segment from a previously reported 1982 H14 isolate indicating the existence of an unidentified pool of genomic diversity. An additional neuraminidase reassortment event indicated a recent inter-hemispheric gene flow from Asia into the center of North America. These results demonstrate temporal and spatial gaps in the understanding of IAV natural history. Additionally, the reassortment history of these viruses raises concern for the inter-continental spread of IAVs and the efficacy of current IAV surveillance efforts in detecting genomic diversity of viruses circulating in wild birds.