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In 2010, H5N8 highly pathogenic avian influenza (HPAI) viruses of the A/Goose/Guangdong/1/1996 lineage dramatically affected poultry and wild birds in Asia, Europe, and North America. In November 2016, HPAI H5N8 was detected in a commercial layer farm in Tehran province. The diagnosis was based on real-time reverse transcriptase PCR (RRT-PCR) and sequencing of haemaglutinin (HA) and neuraminidase (NA) genes from suspected samples. Genetic and phylogenetic analysis of the HA gene demonstrated that the Iranian HPAI H5N8 viruses belong to the HPAI H5 virus clade 2.3.4.4 and cluster within group B (Gochang-like). In particular, the highest similarity was found with the sequences of the HPAI H5N8 identified in Russia in 2016. To our knowledge, this clade has not been previously detected in Iran. Previous HPAI A (H5) epidemic in Iran occurred in 2015 and involved exclusively viruses of clade 2.3.2.1c. These findings indicate that Iran is at high risk of introduction of HPAI H5 of the A/Goose/Guangdong/1/1996 lineage from East Asia and highlight the need to maintain adequate monitoring activities in target wild and domestic bird species for HPAI early detection. This study is useful for better understanding the genetic and antigenic evolution of H5 HPAI viruses in the region and the world.

Measles virus (MV) remains a leading cause of vaccine-preventable deaths in children. Protection against MV is associated with neutralizing antibodies that preferentially recognize the viral hemagglutinin (MV-H), and to a lesser extent, the fusion protein (MV-F). Although MV is serologically monotypic, 24 genotypes have been identified. Here we report three neutralization epitopes conserved in the more prevalent circulating MV genotypes, two located in the MV-H receptor binding site (RBS) (antigenic site III) and a third in MV-H/MV-F interphase (antigenic site Ia) which are essential for MV multiplication. In contrast, two MV-H neutralization epitopes, showed a genotype-specific neutralization escape due to a single amino acid change, that we mapped in the "noose" antigenic site, or an enhanced neutralization epitope (antigenic site IIa). The monoclonal antibody (mAb) neutralization potency correlated with its binding affinity and was mainly driven by kinetic dissociation rate (k off ). We developed an immunoassay for mAb binding to MV-H in its native hetero-oligomeric structure with MV-F on the surface of a MV productive steady-state persistently infected (p.i.) human cell lines, and a competitive-binding assay with serum from individuals with past infection by different MV genotypes. Binding assays revealed that a broad neutralization epitope, in RBS antigenic site, a genotype specific neutralization epitopes, in noose and IIa sites, were immunogenic in natural infection and vaccination and may elicit long-lasting humoral immunity that might contribute to explain MV immunogenic stability. These results support the design of improved measles vaccines, broad-spectrum prophylactic or therapeutic antibodies and MV-used in oncolytic therapies., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The measles virus hemagglutinin (MeV-H) protein is the main target of protective neutralizing antibodies. Using a panel of monoclonal antibodies (MAbs) that recognize known major antigenic sites in MeV-H, we identified a D4 genotype variant that escapes neutralization by MAbs targeting the neutralizing epitope (NE) antigenic site. By site-directed mutagenesis, L249P was identified as the critical mutation disrupting the NE in this genotype D4 variant. Forty-two available D4 genotype gene sequences were subsequently analyzed and divided into 2 groups according to the presence or absence of the L249P MeV-H mutation. Further analysis of the MeV-N gene sequences of these 2 groups confirmed that they represent clearly definable, sequence-divergent D4 subgenotypes, which we named subgenotypes D4.1 and D4.2. The subgenotype D4.1 MeVs were isolated predominantly in Kenya and Ethiopia, whereas the MAb-resistant subgenotype D4.2 MeVs were isolated predominantly in France and Great Britain, countries with higher vaccine coverage rates. Interestingly, D4.2 subgenotype viruses showed a trend toward diminished susceptibility to neutralization by human sera pooled from approximately 60 to 80 North American donors. Escape from MAb neutralization may be a powerful epidemiological surveillance tool to monitor the evolution of new MeV subgenotypes. IMPORTANCE Measles virus is a paradigmatic RNA virus, as the antigenic composition of the vaccination has not needed to be updated since its discovery. The vaccine confers protection by inducing neutralizing antibodies that interfere with the function of the hemagglutinin protein. Viral strains are indistinguishable serologically, although characteristic nucleotide sequences differentiate 24 genotypes. In this work, we describe a distant evolutionary branch within genotype D4. Designated subgenotype D4.2, this virus is distinguishable by neutralization with vaccine-induced monoclonal antibodies that target the neutralizing epitope (NE). The subgenotype D4.2 viruses have a higher predominance in countries with intermediary levels of vaccine coverage. Our studies demonstrate that subgenotype D4.2 lacks epitopes associated with half of the known antigenic sites, which significantly impacts our understanding of measles virus evolution., (Copyright © 2017 American Society for Microbiology.)

In this study, we analyzed the genome of a H10N5 influenza virus from wild birds. This virus was identified as a novel reassortant virus with internal genes from multiple subtypes and of distinct origins. After sequential passage in mice, mouse-adapted viruses bearing mutations PB2-E627K and HA-G218E were generated. These viruses caused dramatic body weight loss and death, and also replicated in mouse brain, suggesting that the pathogenicity of low pathogenic H10N5 in chickens can be enhanced after passage in mammals. Our data imply that H10N5 viruses might be a potential risk to human health therefore it is important to undertake continued surveillance and biosecurity evaluation of these viruses.

We demonstrate here that the genetic incorporation of the fusogenic peptide HA2 into a CXCR4-targeted protein nanoparticle dramatically reduces the specificity of the interaction between nanoparticles and cell receptors, a factor to be considered when designing tumor-homing drug vehicles displaying endosomal-escape agents. The loss of specificity is concomitant with enhanced cell penetrability.

Orthomyxoviruses are an important family of RNA viruses, which include the various influenza viruses. Despite global efforts to eradicate orthomyxoviral pathogens, these infections remain pervasive. One such orthomyxovirus, infectious salmon anemia virus (ISAV), spreads easily throughout farmed and wild salmonids, constituting a significant economic burden. ISAV entry requires the interplay of the virion-attached hemagglutinin-esterase and fusion glycoproteins. Preventing infections will rely on improved understanding of ISAV entry. Here, we present the crystal structures of ISAV hemagglutinin-esterase unbound and complexed with receptor. Several distinctive features observed in ISAV HE are not seen in any other viral glycoprotein. The structures reveal a unique mode of receptor binding that is dependent on the oligomeric assembly of hemagglutinin-esterase. Importantly, ISAV hemagglutinin-esterase receptor engagement does not initiate conformational rearrangements, suggesting a distinct viral entry mechanism. This work improves our understanding of ISAV pathogenesis and expands our knowledge on the overall diversity of viral glycoprotein-mediated entry mechanisms. Finally, it provides an atomic-resolution model of the primary neutralizing antigen critical for vaccine development.

Human beta1-coronavirus (β1CoV) OC43 emerged relatively recently through a single zoonotic introduction. Like related animal β1CoVs, OC43 uses 9-O-acetylated sialic acid as receptor determinant. β1CoV receptor binding is typically controlled by attachment/fusion spike protein S and receptor-binding/receptor-destroying hemagglutinin-esterase protein HE. We show that following OC43's introduction into humans, HE-mediated receptor binding was selected against and ultimately lost through progressive accumulation of mutations in the HE lectin domain. Consequently, virion-associated receptor-destroying activity toward multivalent glycoconjugates was reduced and altered such that some clustered receptor populations are no longer cleaved. Loss of HE lectin function was also observed for another respiratory human coronavirus, HKU1. This thus appears to be an adaptation to the sialoglycome of the human respiratory tract and for replication in human airways. The findings suggest that the dynamics of virion-glycan interactions contribute to host tropism. Our observations are relevant also to other human respiratory viruses of zoonotic origin, particularly influenza A virus., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Canine distemper virus (CDV) is one of the most serious pathogens found in many species of carnivores, including domestic dogs. In this study, hemagglutinin (H) genes were detected in five domestic Vietnamese dogs with diarrhea, and two CDVs were successfully isolated from dogs positive for H genes. The complete genome of one isolate, CDV/dog/HCM/33/140816, was determined. Phylogenetic analysis showed that all Vietnamese CDVs belonged to the Asia-1 genotype. In addition, the H proteins of Vietnamese CDV strains were the most homologous to those of Chinese CDVs (98.4% to 99.3% identity). These results indicated that the Asia-1 genotype of CDV was the predominant genotype circulating among the domestic dog population in Vietnam and that transboundary transmission of CDV has occurred between Vietnam and China.

Human coronaviruses (HCoVs), including SARS-CoV and MERS-CoV, are zoonotic pathogens that originated in wild animals. HCoVs have large genomes that encode a fixed array of structural and nonstructural components, as well as a variety of accessory proteins that differ in number and sequence even among closely related CoVs. Thus, in addition to recombination and mutation, HCoV genomes evolve through gene gains and losses. In this review we summarize recent findings on the molecular evolution of HCoV genomes, with special attention to recombination and adaptive events that generated new viral species and contributed to host shifts and to HCoV emergence. VIDEO ABSTRACT., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

In September 2014, seven sperm whales were stranded along Italy's Adriatic coastline. Postmortem investigations on 3 female adult whales and 1 male fetus carried by the largest female revealed molecular and immunohistochemical evidence of dolphin morbillivirus infection. A possible role of the virus in the stranding event was considered.

Rapid and accurate detection of measles virus is important for case diagnosis and public health management. This study compared the performance of two monoplex RT-PCR reactions targeting the H and N genes to a duplex RT-PCR targeting both genes simultaneously. The duplex simplified processing without compromising assay performance characteristic., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Wild birds are known to play a major role in the evolution, maintenance, and spread of the avian influenza viruses (AIVs). More specifically, the waterfowl are thought to be the natural reservoirs of AIVs. Here, we conducted a survey in 2015 at the Hongze Lake and characterized 11 H5N1 highly pathogenic AIVs isolated from wild waterfowls which were found to belong to clade 2.3.2.1. In contrast, the 11 variants of H5N1 viruses did not align with the three previously defined monophyletic subclades. Antigenicity analysis revealed that antigenic drift occurred in these H5N1 variants. Hence, current vaccines may fail to confer protection against the H5N1 AIV variants in poultry., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Infections with the influenza C virus causing respiratory symptoms are common, particularly among children. Since isolation and detection of the virus are rarely performed, compared with influenza A and B viruses, the small number of available sequences of the virus makes it difficult to analyze its evolutionary dynamics. Recently, we reported the full genome sequence of 102 strains of the virus. Here, we exploited the data to elucidate the evolutionary characteristics and phylodynamics of the virus compared with influenza A and B viruses. Along with our data, we obtained public sequence data of the hemagglutinin-esterase gene of the virus; the dataset consists of 218 unique sequences of the virus collected from 14 countries between 1947 and 2014. Informatics analyses revealed that (1) multiple lineages have been circulating globally; (2) there have been weak and infrequent selective bottlenecks; (3) the evolutionary rate is low because of weak positive selection and a low capability to induce mutations; and (4) there is no significant positive selection although a few mutations affecting its antigenicity have been induced. The unique evolutionary dynamics of the influenza C virus must be shaped by multiple factors, including virological, immunological, and epidemiological characteristics., Competing Interests: The authors declare no conflict of interest.

Camelpox and camel contagious ecthyma are infectious viral diseases of camelids caused by camelpox virus (CMLV) and camel contagious ecthyma virus (CCEV), respectively. Even though, in Ethiopia, pox disease has been creating significant economic losses in camel production, little is known on the responsible pathogens and their genetic diversity. Thus, the present study aimed at isolation, identification and genetic characterization of the causative viruses. Accordingly, clinical case observations, infectious virus isolation, and molecular and phylogenetic analysis of poxviruses infecting camels in three regions and six districts in the country, Afar (Chifra), Oromia (Arero, Miyu and Yabello) and Somali (Gursum and Jijiga) between 2011 and 2014 were undertaken. The full hemagglutinin (HA) and partial A-type inclusion protein (ATIP) genes of CMLV and full major envelope protein (B2L) gene of CCEV of Ethiopian isolates were sequenced, analyzed and compared among each other and to foreign isolates. The viral isolation confirmed the presence of infectious poxviruses. The preliminary screening by PCR showed 27 CMLVs and 20 CCEVs. The sequence analyses showed that the HA and ATIP gene sequences are highly conserved within the local isolates of CMLVs, and formed a single cluster together with isolates from Somalia and Syria. Unlike CMLVs, the B2L gene analysis of Ethiopian CCEV showed few genetic variations. The phylogenetic analysis revealed three clusters of CCEV in Ethiopia with the isolates clustering according to their geographical origins. To our knowledge, this is the first report indicating the existence of CCEV in Ethiopia where camel contagious ecthyma was misdiagnosed as camelpox. Additionally, this study has also disclosed the existence of co-infections with CMLV and CCEV. A comprehensive characterization of poxviruses affecting camels in Ethiopia and the full genome sequencing of representative isolates are recommended to better understand the dynamics of pox diseases of camels and to assist in the implementation of more efficient control measures., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Plasmids expressing interferon (IFN) have recently been shown to function as adjuvants in Atlantic salmon when co-injected with a DNA vaccine encoding hemagglutinin-esterase (HE) from infectious salmon anemia virus (ISAV). In this work we have compared the antibody kinetics and the systemic Mx/ISG15 response of fish vaccinated with HE-plasmid using either IFNa plasmid (pIFNa) or pIFNc as adjuvants over a longer time period, i.e. 22 weeks post vaccination (pv). The results showed that the antibody response against ISAV with pIFNa as adjuvant arose earlier (7 weeks pv) than with pIFNc as adjuvant (10 weeks pv), peaked at week 10 and declined at week 22. The antibody response with pIFNc as adjuvant peaked at 16 weeks and kept at this level 22 weeks pv. Fish injected with pIFNc alone expressed high levels of Mx and ISG15 in liver throughout the 22 week period. In contrast, fish injected with pIFNc together with HE-plasmid expressed high levels of Mx and ISG15 in liver for the first 10 weeks, but at week 16 this response was absent in two of three fish at week 16 and was absent in all tested fish at week 22 pv. This suggests that cells expressing HE and IFNc are intact at week 10 pv, but are eliminated by adaptive immune responses after week 10 due to recognition of HE. The longevity of the Mx/ISG15 response in pIFNc treated fish is likely due to the fact that IFNc is a self-antigen of salmon and is not attacked by the adaptive immune system., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Canine distemper virus (CDV) is a highly contagious pathogen for domestic dogs and several wild carnivore species. In Brazil, natural infection of CDV in dogs is very high due to the large non-vaccinated dog population, a scenario that calls for new studies on the molecular epidemiology. This study investigates the phylodynamics and amino-acid signatures of CDV epidemic in South America by analyzing a large dataset compiled from publicly available sequences and also by collecting new samples from Brazil. A population of 175 dogs with canine distemper (CD) signs was sampled, from which 89 were positive for CDV, generating 42 new CDV sequences. Phylogenetic analysis of the new and publicly available sequences revealed that Brazilian sequences mainly clustered in South America 1 (SA1) clade, which has its origin estimated to the late 1980's. The reconstruction of the demographic history in SA1 clade showed an epidemic expanding until the recent years, doubling in size every nine years. SA1 clade epidemic distinguished from the world CDV epidemic by the emergence of the R580Q strain, a very rare and potentially detrimental substitution in the viral genome. The R580Q substitution was estimated to have happened in one single evolutionary step in the epidemic history in SA1 clade, emerging shortly after introduction to the continent. Moreover, a high prevalence (11.9%) of the Y549H mutation was observed among the domestic dogs sampled here. This finding was associated (p<0.05) with outcome-death and higher frequency in mixed-breed dogs, the later being an indicator of a continuous exchange of CDV strains circulating among wild carnivores and domestic dogs. The results reported here highlight the diversity of the worldwide CDV epidemic and reveal local features that can be valuable for combating the disease., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Modified Vaccinia Ankara (MVA) has proven its efficacy as a recombinant vector vaccine for numerous pathogens including influenza virus. The present study aimed at evaluating a recombinant MVA candidate vaccine against low pathogenic avian influenza virus subtype H9N2 in the chicken model. As the high genetic and antigenic diversity of H9N2 viruses increases vaccine design complexity, one strategy to widen the range of vaccine coverage is to use an ancestor sequence. We therefore generated a recombinant MVA encoding for the gene sequence of an ancestral hemagglutinin H9 protein (a computationally derived amino acid sequence of the node of the H9N2 G1 lineage strains was obtained using the ANCESCON program). We analyzed the genetics and the growth properties of the MVA vector virus confirming suitability for use under biosafety level 1 and tested its efficacy when applied either as an intra-muscular (IM) or an oral vaccine in specific pathogen free chickens challenged with A/chicken/Tunisia/12/2010(H9N2). Two control groups were studied in parallel (unvaccinated and inoculated birds; unvaccinated and non-inoculated birds). IM vaccinated birds seroconverted as early as four days post vaccination and neutralizing antibodies were detected against A/chicken/Tunisia/12/2010(H9N2) in all the birds before challenge. The role of local mucosal immunity is unclear here as no antibodies were detected in eye drop or aerosol vaccinated birds. Clinical signs were not detected in any of the infected birds even in absence of vaccination. Virus replication was observed in both vaccinated and unvaccinated chickens, suggesting the MVA-ancestral H9 vaccine may not stop virus spread in the field. However vaccinated birds showed less histological damage, fewer influenza-positive cells and shorter virus shedding than their unvaccinated counterparts., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Hemagglutinin-esterases (HEs) are bimodular envelope proteins of orthomyxoviruses, toroviruses, and coronaviruses with a carbohydrate-binding "lectin" domain appended to a receptor-destroying sialate-O-acetylesterase ("esterase"). In concert, these domains facilitate dynamic virion attachment to cell-surface sialoglycans. Most HEs (type I) target 9-O-acetylated sialic acids (9-O-Ac-Sias), but one group of coronaviruses switched to using 4-O-Ac-Sias instead (type II). This specificity shift required quasisynchronous adaptations in the Sia-binding sites of both lectin and esterase domains. Previously, a partially disordered crystal structure of a type II HE revealed how the shift in lectin ligand specificity was achieved. How the switch in esterase substrate specificity was realized remained unresolved, however. Here, we present a complete structure of a type II HE with a receptor analog in the catalytic site and identify the mutations underlying the 9-O- to 4-O-Ac-Sia substrate switch. We show that (i) common principles pertaining to the stereochemistry of protein-carbohydrate interactions were at the core of the transition in lectin ligand and esterase substrate specificity; (ii) in consequence, the switch in O-Ac-Sia specificity could be readily accomplished via convergent intramolecular coevolution with only modest architectural changes in lectin and esterase domains; and (iii) a single, inconspicuous Ala-to-Ser substitution in the catalytic site was key to the emergence of the type II HEs. Our findings provide fundamental insights into how proteins "see" sugars and how this affects protein and virus evolution.

Unlabelled: Imported measles virus (MV) outbreaks are maintained by poor vaccine responders and unvaccinated people. A convenient but more immunogenic vaccination strategy would enhance vaccine performance, contributing to measles eradication efforts. We report here the generation of alternative pediatric vaccines against MV with increased expression of the H protein in the background of the current MV vaccine strain. We generated two recombinants: MVvac2-H2, with increased full-length H expression resulting in a 3-fold increase in H incorporation into virions, and MVvac2-Hsol, vectoring a truncated, soluble form of the H protein that is secreted into the supernatants of infected cells. Replication fitness was conserved despite the duplication of the H cistron for both vectors. The modification to the envelope of MVvac2-H2 conferred upon this virus a measurable level of resistance to in vitro neutralization by MV polyclonal immune sera without altering its thermostability. Most interestingly, both recombinant MVs with enhanced H expression were significantly more immunogenic than their parental strain in outbred mice, while MVvac2-H2 additionally proved more immunogenic after a single, human-range dose in genetically modified MV-susceptible mice., Importance: Measles incidence was reduced drastically following the introduction of attenuated vaccines, but progress toward the eradication of this virus has stalled, and MV still threatens unvaccinated populations. Due to the contributions of primary vaccine failures and too-young-to-be-vaccinated infants to this problem, more immunogenic measles vaccines are highly desirable. We generated two experimental MV vaccines based on a current vaccine's genome but with enriched production of the H protein, the main MV antigen in provoking immunity. One vaccine incorporated H at higher rates in the viral envelope, and the other secreted a soluble H protein from infected cells. The increased expression of H by these vectors improved neutralizing responses induced in two small-animal models of MV immunogenicity. The enhanced immunogenicity of these vectors, mainly from the MV that incorporates additional H, suggests their value as potential alternative pediatric MV vaccines., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

The local and global transmission dynamics of influenza B virus is not completely understood mainly because of limited epidemiological and sequence data for influenza B virus. Here we report epidemiological and molecular characteristics of influenza B viruses from 2010 to 2013 in Leyte Island, Philippines. Phylogenetic analyses showed global dissemination of the virus among both neighboring and distant areas. The analyses also suggest that southeast Asia is not a distributor of influenza B virus and can introduce the virus from other areas. Furthermore, we found evidence on the local persistence of the virus over years in the Philippines. Taken together, both local persistence and global dissemination play a significant role in the circulation of influenza B virus., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Peste des Petits Ruminants (PPR) is an acute, highly contagious, and febrile viral disease that affects both domestic and wild small ruminants. The disease has become a major obstacle to the development of sustainable Agriculture. Hemagglutinin (H), the envelope glycoprotein of Peste des Petits Ruminants Virus (PPRV), plays a crucial role in regulating viral adsorption and entry, thus determining pathogenicity, and release of newly produced viral particles. In order to accurately understand the epidemic of the disease and the interactions between the virus and host, we launch the work. Here, we examined H gene from all four lineages of the PPRV to investigate evolutionary and epidemiologic dynamics of PPRV by the Bayesian method. In addition, we predicted positive selection sites due to selective pressures. Finally, we studied the interaction between H protein and SLAM receptor based on homology model of the complex. Phylogenetic analysis suggested that H gene can also be used to investigate evolutionary and epidemiologic dynamics of PPRV. Positive selection analysis identified four positive selection sites in H gene, in which only one common site (aa246) was detected by two methods, suggesting strong operation structural and/or functional constraint of changes on the H protein. This target site may be of interest for future mutagenesis studies. The results of homology modeling showed PPRVHv-shSLAM binding interface and MVH-maSLAM binding interface were consistent, wherein the groove in the B4 blade and B5 of the head domain of PPRVHv bound to the AGFCC' β-sheets of the membrane-distal ectodomain of shSLAM. The binding regions could provide insight on the nature of the protein for epitope vaccine design, novel drug discovery, and rational drug design against PPRV.

Limited information is available regarding epidemiology of mumps in India. Mumps vaccine is not included in the Universal Immunization Program of India. The complete genome sequences of Indian mumps virus (MuV) isolates are not available, hence this study was performed. Five isolates from bilateral parotitis and pancreatitis patients from Maharashtra, a MuV isolate from unilateral parotitis patient from Tamil Nadu, and a MuV isolate from encephalitis patient from Uttar Pradesh were genotyped by the standard protocol of the World Health Organization and subsequently complete genomes were sequenced. Indian MuV genomes were compared with published MuV genomes, including reference genotypes and eight vaccine strains for the genetic differences. The SH gene analysis revealed that five MuV isolates belonged to genotype C and two belonged to genotype G strains. The percent nucleotide divergence (PND) was 1.1% amongst five MuV genotype C strains and 2.2% amongst two MuV genotype G strains. A comparison with widely used mumps Jeryl Lynn vaccine strain revealed that Indian mumps isolates had 54, 54, 53, 49, 49, 38, and 49 amino acid substitutions in Chennai-2012, Kushinagar-2013, Pune-2008, Osmanabad-2012a, Osmanabad-2012b, Pune-1986 and Pune-2012, respectively. This study reports the complete genome sequences of Indian MuV strains obtained in years 1986, 2008, 2012 and 2013 that may be useful for further studies in India and globally., (Copyright © 2016 Elsevier B.V. All rights reserved.)

A desirable vaccine against respiratory syncytial virus (RSV) should induce neutralizing antibodies without eliciting abnormal T cell responses to avoid vaccine-enhanced pathology. In an approach to deliver RSV neutralizing epitopes without RSV-specific T cell antigens, we genetically engineered chimeric influenza virus expressing RSV F262-276 neutralizing epitopes in the globular head domain as a chimeric hemagglutinin (HA) protein. Immunization of mice with formalin-inactivated recombinant chimeric influenza/RSV F262-276 was able to induce RSV protective neutralizing antibodies and lower lung viral loads after challenge. Formalin-inactivated RSV immune mice showed high levels of pulmonary inflammatory cytokines, macrophages, IL-4-producing T cells, and extensive histopathology. However, RSV-specific T cell responses and enhancement of pulmonary histopathology were not observed after RSV infection of inactivated chimeric influenza/RSV F262-276. This study provides evidence that an inactivated vaccine platform of chimeric influenza/RSV virus can be developed into a safe RSV vaccine candidate without priming RSV-specific T cells and immunopathology., From the Clinical Editor: Respiratory syncytial virus (RSV) is a major cause of respiratory tract illness and morbidity in children. Hence, there is a need to develop an effective vaccine against this virus. In this article, the authors engineered chimeric influenza virus to express RSV neutralizing epitopes. The positive findings in in-vivo experiments provide a beginning for future clinical trials and perhaps eventual product realization., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Introduction: It is increasingly difficult to differentiate measles viruses (MeVs) relating to certain outbreaks on the basis of the nucleoprotein (N) gene sequence only, as the diversity of circulating MeV strains has decreased. We studied genomic regions that could provide better molecular discrimination between epidemiologically linked and unlinked MeV variants identified in Sweden during 2013-2014., Methods: The hemagglutinin (H) gene and hypervariable region between the fusion and matrix genes (MF-HVR) from 53 MeV-positive samples were amplified and sequenced. Data on phylogenetic clustering of MeVs on the basis of N, H, and MF-HVR sequences were compared to epidemiological data., Results: MeVs were genotyped: 27 were B3, and 26 were D8. One genotype B3 cluster based on the N gene sequence contained epidemiologically unrelated viruses from 4 outbreaks, whereas analysis of H and MF-HVR sequences separated them into phylogenetic clusters consistent with the epidemiological data. Similarly, the single cluster of viruses with a genotype D8 N gene could be divided into the 5 outbreak groups on the basis of the phylogeny of MF-HVR sequences., Conclusions: A detailed picture of MeV circulation with more-defined links between outbreaks was obtained by sequencing the H gene and MF-HVR. Further identification and better genetic characterization of MeVs internationally is essential in identifying sources and routes of MeV spread within and beyond Europe in the elimination end game., (© The Author 2015. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.)

Influenza viruses that express reporter proteins are useful tools, but are often attenuated. Recently, we found that an influenza virus encoding the Venus fluorescent protein acquired two mutations in its PB2 and HA proteins upon mouse adaptation. Here, we demonstrate that the enhanced viral replication and virulence in mice of this Venus-expressing influenza virus are primarily conferred by the PB2-E712D mutation, with only a minor contribution by the HA-T380A mutation.

During 2011-2013, dolphin morbillivirus was molecularly identified in 4 stranded fin whales from the Mediterranean Sea. Nucleoprotein, phosphoprotein, and hemagglutinin gene sequences of the identified strain were highly homologous with those of a morbillivirus that caused a 2006-2007 epidemic in the Mediterranean. Dolphin morbillivirus represents a serious threat for fin whales.

In Egypt, two distinct lineages of H5N1 highly pathogenic avian influenza (HPAI) viruses, "classic 2.2.1.2" and "variant 2.2.1.1" strains, have evolved. The underlying host immune responses counteracting these viruses in chickens remain not well understood. In the present study, the cytokine responses to a classic strain (C121) and those to a variant strain (V1063) were compared in naïve and vaccinated chickens. In naïve chickens, the C121 replicated more efficiently than the V1063. Both the C121 and the V1063 increased interferon (IFN)-γ and interleukin (IL)-10 gene expression at 48 h post inoculation (hpi) in the lung and spleen but the levels of these cytokines were lower in chickens infected with the C121 than those infected with the V1063. In contrast, in chickens vaccinated with inactivated C121-based vaccine, the C121 replicated less than the V1063. Both challenge with the C121 and that with the V1063 did not increase IFN-γ gene expression at 48 hpi; rather, the C121 increased IL-4 gene expression in the lung accompanied with lower viral titer and higher HI titers. These results suggested that the pathogenicity of HPAI viruses correlated with IFN-γ-producing helper and/or cytotoxic T cell responses in naïve chickens, whereas vaccine efficacy to HPAI viruses correlated with IL-4 producing helper T cell responses in the lung in vaccinated chickens. It implies that IL-4 in the lung, in addition to the traditional serum HI titers, could be used to screen novel vaccine strategies, such as strains, adjuvant, prime/boost protocols, against HPAI in chickens., (Copyright © 2015 Elsevier B.V. All rights reserved.)

In June 2009, the World Health Organization (WHO) issued a pandemic alert concerning the spread of an influenza A (H1N1) virus that showed distinctive genetic characteristics vis-à-vis both seasonal influenza strains and vaccine strains. The main mutation occurred in the gene coding for hemagglutinin (HA). Mathematical models were developed to calculate the transmissibility of the virus; the results indicated a significant overlap with the transmissibility of previous pandemic strains and seasonal strains. The remarkable feature of A/(H1N1)pdm09, compared with seasonal strains, is its high fatality rate and its higher incidence among younger people. Data provided by the WHO on the number of deaths caused by A/(H1N1)pdm09 only include laboratory-confirmed cases. Some authors suggest that these data could underestimate the magnitude of the event, as laboratory confirmation is not obtained in all cases. It is important to bear in mind that the A/(H1N1)pdm09 virus is still circulating in the population. It is therefore essential to maintain its epidemiological and virological surveillance.

Coordination and cross talks of MAPK pathways are critical for signaling efficiency, but their mechanisms are not well understood. Slt2, the MAP kinase of cell wall integrity pathway (CWI), is activated by heat stress even in the absence of upstream components of this pathway, suggesting a supplementary input for Slt2 activation. Here, we identify a new interaction of Ste11 and Mkk1, mediated by Nst1 that connects the high-osmolarity glycerol and pheromone pathways directly to CWI pathway in response to heat and pheromone. We suggest that Ser(407) and Thr(411) are novel residues of Mkk1 activated by these MAPK pathways., (© 2015 Federation of European Biochemical Societies.)

In an earlier study on respiratory infections in Singapore military recruits, four influenza C virus (FLUCV) infections were detected out of the 1354 samples collected. All four isolates were detected in 2006, and their whole genome was completely sequenced and analysed. Phylogenetic analysis of the hemagglutinin esterase fusion (HEF) gene revealed that all four Singapore isolates belonged to the C/Japan-Kanagawa/1/76-related lineage. However, the genes of the four FLUCV isolates had origins from several different lineages, and the genome composition resembles that of the C/Japan-Miyagi/9/96-like strains that had been circulating in Japan between 1996 and 2000., (© 2015 The Authors. Influenza and Other Respiratory Viruses Published by John Wiley & Sons Ltd.)

Influenza virus is a class I enveloped virus which is initially endocytosed into a host respiratory epithelial cell. Subsequent reduction of the pH to the 5-6 range triggers a structural change of the viral hemagglutinin II (HA2) protein, fusion of the viral and endosomal membranes, and release of the viral nucleocapsid into the cytoplasm. HA2 contains fusion peptide (FP), soluble ectodomain (SE), transmembrane (TM), and intraviral domains with respective lengths of ∼ 25, ∼ 160, ∼ 25, and ∼ 10 residues. The present work provides a straightforward protocol for producing and purifying mg quantities of full-length HA2 from expression in bacteria. Biophysical and structural comparisons are made between full-length HA2 and shorter constructs including SHA2 ≡ SE, FHA2 ≡ FP+SE, and SHA2-TM ≡ SE+TM constructs. The constructs are helical in detergent at pH 7.4 and the dominant trimer species. The proteins are highly thermostable in decylmaltoside detergent with Tm>90 °C for HA2 with stabilization provided by the SE, FP, and TM domains. The proteins are likely in a trimer-of-hairpins structure, the final protein state during fusion. All constructs induce fusion of negatively-charged vesicles at pH 5.0 with much less fusion at pH 7.4. Attractive protein/vesicle electrostatics play a role in fusion, as the proteins are positively-charged at pH 5.0 and negatively-charged at pH 7.4 and the pH-dependence of fusion is reversed for positively-charged vesicles. Comparison of fusion between constructs supports significant contributions to fusion from the SE and the FP with little effect from the TM., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The WHO Collaborating Centre for Reference and Research on Influenza in Melbourne is part of the World Health Organization's (WHO) Global Influenza Surveillance and Response System. In 2014 the Centre received a total of 5,374 influenza samples from laboratories primarily in the Asia-Pacific region. Viruses were characterised by their antigenic, genetic and antiviral drug resistance properties. Of the viruses successfully analysed 52% were A(H1N1)pdm09 viruses. The majority of these were antigenically and genetically similar to the WHO recommended reference strain for the 2014 Southern Hemisphere influenza vaccine. Results for A(H3N2) and B/Yamagata viruses suggested that circulating viruses of this subtype and lineage, respectively, had undergone antigenic and/or genetic changes, consistent with the decision by WHO to change recommended strains for the 2015 Southern Hemisphere vaccine. A small number of A(H1N1)pdm09 and B/Victoria viruses had highly reduced inhibition to the neuraminidase inhibitors oseltamivir and zanamivir. The Centre also undertook primary isolation of vaccine candidate viruses directly into eggs. A total of 38 viruses were successfully isolated in eggs, of which 1 (B/Phuket/3073/2013) was included in the 2015 Southern Hemisphere influenza vaccine.

The yield of heterologous proteins is often limited by several bottlenecks in the secretory pathway of yeast Saccharomyces cerevisiae. It was shown earlier that synthesis of measles virus hemagglutinin (MeH) is inefficient mostly due to a bottleneck in the translocation of viral protein precursors into the endoplasmic reticulum (ER) of yeast cells. Here we report that heat shock with subsequent induction of MeH expression at 37°C improved translocation of MeH precursors when applied at higher cell densities. The amount of MeH glycoprotein increased by about 3-fold after heat shock in the late-log phases of both glucose and ethanol growth. The same temperature conditions increased both secretion titer and yield of another heterologous protein human GRP78/BiP by about 50%. Furthermore, heat shock at the late-log glucose growth phase also improved endogenous invertase yield by approximately 2.7-fold. In contrast, a transfer of yeast culture to lower temperature at diauxic shift followed by protein expression at 20°C almost totally inhibited translocation of MeH precursors. The difference in amounts of MeH glycoprotein under expression at 37°C and 20°C was about 80-fold, while amounts of unglycosylated MeH polypeptides were similar under both conditions. Comparative proteomic analysis revealed that besides over-expressed ER-resident chaperone Kar2, an increased expression of several cytosolic proteins (such as Hsp104, Hsp90 and eEF1A) may contribute to improved translocation of MeH., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Unlabelled: A previous screening of more than 50,000 compounds led to the identification of a pool of bioactive small molecules with inhibitory effect on the influenza A virus. One of these compounds, now widely known as nucleozin, is a small molecule that targets the influenza A virus nucleoprotein. Here we identify and characterize two structurally different novel fusion inhibitors of the influenza A virus group 1 hemagglutinin (HA), FA-583 and FA-617, with low nanomolar activities. Escape mutants that are highly resistant to each of these compounds were generated, and both were found to carry mutations localized in close proximity to the B-loop of the hemagglutinin 2 protein, which plays a crucial role in the virion-host cell fusion process. Recombinant virus, generated through reverse genetics, confirmed the resistance phenotype. In addition, the proposed binding pockets predicted by molecular docking studies are in accordance with the resistance-bearing mutation sites. We show through mechanistic studies that FA-583 and FA-617 act as fusion inhibitors by prohibiting the low-pH-induced conformational change of hemagglutinin. Our study has offered concrete biological and mechanistic explorations for the strategic development of novel fusion inhibitors of influenza A viruses., Importance: Here we report two structurally distinctive novel fusion inhibitors of influenza A virus that act by interfering with the structural change of HA at acidic pH, a process necessary for successful entry of the virus. Mutational and molecular docking studies have identified their binding pockets situated in close proximity to the B-loop region of hemagglutinin 2. The reduced sensitivity of FA-583- or FA-617-associated mutants to another compound suggests a close proximity and even partial overlap of their binding sites on hemagglutinin. Amino acid sequence alignments and crystal structure analyses of group 1 and group 2 hemagglutinins have shed light on the possible binding mode of these two compounds. This report offers new lead compounds for the design of fusion inhibitors for influenza A viruses and further shows that analysis by forward chemical genetics is a highly effective approach for the identification of novel compounds that can perturb the infectivity of viruses and to probe new druggable targets or druggable domains in various viruses., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Subtype H3 influenza A viruses (IAVs) are abundant in wild waterfowl and also infect humans, pigs, horses, dogs, and seals. In Minnesota, turkeys are important and frequent hosts of IAV from wild waterfowl and from pigs. Over 48 yr of surveillance history, 11 hemagglutinin (HA) subtypes of IAV from waterfowl, as well as two HA subtypes from swine, H1 and H3, have infected turkeys in Minnesota. However, there have only been two cases of avian-origin H3 IAV infections in turkeys during this 48-yr period. The first avian-origin IAV infection was detected in seven breeder and commercial flocks in 1982 and was caused by a mixed H3H4/N2 infection. In 2013, an avian-origin H3H9/N2 outbreak occurred in five flocks of turkeys between 15 and 56 wk of age. Phylogenetic analysis of the HA gene segment from the 2013 isolate indicated that the virus was related to a wild bird lineage H3 IAV. A meta-analysis of historical H3 infections in domesticated poultry demonstrated that avian-origin H3 infections have occurred in chickens and ducks but were rare in turkeys. H9N2 virus was subsequently selected during the egg cultivation of the 2013 H3H9/N2 mixed virus. A growth curve analysis suggested that passage 3 of A/Turkey/Minnesota/13-20710-2/2013(mixed) had a slightly lower replication rate than a similar avian-origin H3N2. The challenge studies indicated that the infectious dose of avian-origin H3N2 for turkey poults was greater than 10(6) 50% egg infective dose. Considered together, these data suggest that avian-origin H3 introductions to turkeys are rare events.

In Infectious salmon anaemia virus (ISAV), deletions in the highly polymorphic region (HPR) in the near membrane domain of the haemagglutinin-esterase (HE) stalk, influence viral fusion. It is suspected that selected mutations in the associated Fusion (F) protein may also be important in regulating fusion activity. To better understand the underlying mechanisms involved in ISAV fusion, several mutated F proteins were generated from the Scottish Nevis and Norwegian SK779/06 HPR0. Co-transfection with constructs encoding HE and F were performed, fusion activity assessed by content mixing assay and the degree of proteolytic cleavage by western blot. Substitutions in Nevis F demonstrated that K276 was the most likely cleavage site in the protein. Furthermore, amino acid substitutions at three sites and two insertions, all slightly upstream of K276, increased fusion activity. Co-expression with HE harbouring a full-length HPR produced high fusion activities when trypsin and low pH were applied. In comparison, under normal culture conditions, groups containing a mutated HE with an HPR deletion were able to generate moderate fusion levels, while those with a full length HPR HE could not induce fusion. This suggested that HPR length may influence how the HE primes the F protein and promotes fusion activation by an ubiquitous host protease and/or facilitate subsequent post-cleavage refolding steps. Variations in fusion activity through accumulated mutations on surface glycoproteins have also been reported in other orthomyxoviruses and paramyxoviruses. This may in part contribute to the different virulence and tissue tropism reported for HPR0 and HPR deleted ISAV genotypes.

Unlabelled: In 1999, after circulation for a few months in poultry in Italy, low-pathogenic (LP) avian influenza (AI) H7N1 virus mutated into a highly pathogenic (HP) form by acquisition of a unique multibasic cleavage site (mCS), PEIPKGSRVRR*GLF (asterisk indicates the cleavage site), in the hemagglutinin (HA) and additional alterations with hitherto unknown biological function. To elucidate these virulence-determining alterations, recombinant H7N1 viruses carrying specific mutations in the HA of LPAI A/chicken/Italy/473/1999 virus (Lp) and HPAI A/chicken/Italy/445/1999 virus (Hp) were generated. Hp with a monobasic CS or carrying the HA of Lp induced only mild or no disease in chickens, thus resembling Lp. Conversely, Lp with the HA of Hp was as virulent and transmissible as Hp. While Lp with a multibasic cleavage site (Lp&#95;CS445) was less virulent than Hp, full virulence was exhibited when HA2 was replaced by that of Hp. In HA2, three amino acid differences consistently detected between LP and HP H7N1 viruses were successively introduced into Lp&#95;CS445. Q450L in the HA2 stem domain increased virulence and transmission but was detrimental to replication in cell culture, probably due to low-pH activation of HA. A436T and/or K536R restored viral replication in vitro and in vivo. Viruses possessing A436T and K536R were observed early in the HPAI outbreak but were later superseded by viruses carrying all three mutations. Together, besides the mCS, stepwise mutations in HA2 increased the fitness of the Italian H7N1 virus in vivo. The shift toward higher virulence in the field was most likely gradual with rapid optimization., Importance: In 1999, after 9 months of circulation of low-pathogenic (LP) avian influenza virus (AIV), a devastating highly pathogenic (HP) H7N1 AIV emerged in poultry, marking the largest epidemic of AIV reported in a Western country. The HPAIV possessed a unique multibasic cleavage site (mCS) complying with the minimum motif for HPAIV. The main finding in this report is the identification of three mutations in the HA2 domain that are required for replication and stability, as well as for virulence, transmission, and tropism of H7N1 in chickens. In addition to the mCS, Q450L was required for full virulence and transmissibility of the virus. Nonetheless, it was detrimental to virus replication and required A436T and/or K536R to restore replication, systemic spread, and stability. These results are important for better understanding of the evolution of highly pathogenic avian influenza viruses from low-pathogenic precursors., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Unlabelled: Paramyxoviruses include several insidious and ubiquitous pathogens of humans and animals, with measles virus (MeV) being a prominent one. The MeV membrane fusion apparatus consists of a receptor binding protein (hemagglutinin [H]) tetramer and a fusion (F) protein trimer. Four globular MeV H heads are connected to a tetrameric stalk through flexible linkers. We sought here to characterize the function of a 17-residue H-head segment proximal to the stalk that was unresolved in all five MeV H-head crystal or cocrystal structures. In particular, we assessed whether its primary sequence and length are critical for proper protein oligomerization and intracellular transport or for membrane fusion triggering. Extensive alanine substitutions had no effect on fusion triggering, suggesting that sequence identity is not critical for this function. Excessive shortening of this segment reduced or completely abrogated fusion trigger function, while length compensation restored it. We then characterized the mechanism of function loss. Mutated H proteins were efficiently transported to the cell surface, but certain alterations enhancing linker flexibility resulted in accumulation of high-molecular-weight H oligomers. Some oligomers had reduced fusion trigger capacity, while others retained this function. Thus, length and rigidity of the unresolved head segment favor proper H tetramerization and counteract interactions between subunits from different tetramers. The structurally unresolved H-head segment, together with the top of the stalk, may act as a leash to provide the right degree of freedom for the heads of individual tetramers to adopt a triggering-permissive conformation while avoiding improper contacts with heads of neighboring tetramers., Importance: Understanding the molecular mechanism of membrane fusion triggering may allow development of new antiviral strategies. The fusion apparatus of paramyxoviruses consists of a receptor binding tetramer and a fusion protein trimer. Structural analyses of the receptor binding hemagglutinin-neuraminidases of certain paramyxoviruses suggest that fusion triggering is preceded by relocation of its head domains, facilitated by flexible linkers. Having noted a structurally unresolved 17-residue segment linking the globular heads to the tetrameric stalk of the measles virus hemagglutinin (H), we asked whether and how it may facilitate membrane fusion triggering. We conclude that, together with the top of the stalk, the flexible linker keeps H heads on a leash long enough to adopt a triggering-permissive conformation but short enough to limit roaming and improper contacts with heads of neighboring tetramers. All morbillivirus H-protein heads appear to be connected to their stalks through a "leash," suggesting a conserved triggering mechanism., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Unlabelled: The molecular interaction between viral RNA and the cytosolic sensor RIG-I represents the initial trigger in the development of an effective immune response against infection with RNA viruses, resulting in innate immune activation and subsequent induction of adaptive responses. In the present study, the adjuvant properties of a sequence-optimized 5'-triphosphate-containing RNA (5'pppRNA) RIG-I agonist (termed M8) were examined in combination with influenza virus-like particles (VLP) (M8-VLP) expressing H5N1 influenza virus hemagglutinin (HA) and neuraminidase (NA) as immunogens. In combination with VLP, M8 increased the antibody response to VLP immunization, provided VLP antigen sparing, and protected mice from a lethal challenge with H5N1 influenza virus. M8-VLP immunization also led to long-term protective responses against influenza virus infection in mice. M8 adjuvantation of VLP increased endpoint and antibody titers and inhibited influenza virus replication in lungs compared with approved or experimental adjuvants alum, AddaVax, and poly(I·C). Uniquely, immunization with M8-VLP stimulated a TH1-biased CD4 T cell response, as determined by increased TH1 cytokine levels in CD4 T cells and increased IgG2 levels in sera. Collectively, these data demonstrate that a sequence-optimized, RIG-I-specific agonist is a potent adjuvant that can be utilized to increase the efficacy of influenza VLP vaccination and dramatically improve humoral and cellular mediated protective responses against influenza virus challenge., Importance: The development of novel adjuvants to increase vaccine immunogenicity is an important goal that seeks to improve vaccine efficacy and ultimately prevent infections that endanger human health. This proof-of-principle study investigated the adjuvant properties of a sequence-optimized 5'pppRNA agonist (M8) with enhanced capacity to stimulate antiviral and inflammatory gene networks using influenza virus-like particles (VLP) expressing HA and NA as immunogens. Vaccination with VLP in combination with M8 increased anti-influenza virus antibody titers and protected animals from lethal influenza virus challenge, highlighting the potential clinical use of M8 as an adjuvant in vaccine development. Altogether, the results describe a novel immunostimulatory agonist targeted to the cytosolic RIG-I sensor as an attractive vaccine adjuvant candidate that can be used to increase vaccine efficacy, a pressing issue in children and the elderly population., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

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.

The aquatic orthomyxovirus infectious salmon anaemia virus (ISAV) causes a severe disease in farmed Atlantic salmon, Salmo salar L. Although some ISA outbreaks are caused by horizontal transmission of virus between farms, the source and reservoir of the virus is largely unknown and a wild host has been hypothesized. Atlantic salmon are farmed in open net-pens, allowing transmission of pathogens from wild fish and the surrounding environment to the farmed fish. In this study, a large number of fish species were investigated for ISAV host potential. For orthomyxoviruses, a specific receptor binding is the first requirement for infection; thus, the fish species were investigated for the presence of the ISAV receptor. The receptor was found to be widely distributed across the fish species. All salmonids expressed the receptor. However, only some of the cod-like and perch-like fish did, and all flat fish were negative. In the majority of the positive species, the receptor was found on endothelial cells and/or on red blood cells. The study forms a basis for further investigations and opens up the possibility for screening species to determine whether a wild host of ISAV exists., (© 2014 John Wiley & Sons Ltd.)

Forensic microbial specimens, including bacteria and viruses, are collected at biocrime and bioterrorism scenes. Although it is preferable that the pathogens in these samples are alive and kept in a steady state, the samples may be stored for prolonged periods before analysis. Therefore, it is important to understand the effects of storage conditions on the pathogens contained within such samples. To evaluate the capacity to preserve viable virus and the viral genome, influenza virus was added to the transport medium of the Universal Viral Transport system and stored for over 3 months at various temperatures, after which virus titrations and quantitative analysis of the influenza hemagglutinin gene were performed. Although viable viruses became undetectable 29 days after the medium was stored at room temperature, viruses in the medium stored at 4°C were viable even after 99 days. A quantitative PCR analysis indicated that the hemagglutinin gene was maintained for 99 days at both 4°C and room temperature. Therefore, long-term storage at 4°C has little effect on viable virus and viral genes, so the Universal Viral Transport system can be useful for microbial forensics. This study provides important information for the handling of forensic virus specimens., (Copyright © 2015 Elsevier Ltd and Faculty of Forensic and Legal Medicine. All rights reserved.)

In June 2013, the first human infection by avian influenza A(H6N1) virus was reported in Taiwan. This incident raised the concern for possible human epidemics and pandemics from H6 viruses. In this study, we performed structural and functional investigation on the hemagglutinin (HA) proteins of the human-infecting A/Taiwan/2/2013(H6N1) (TW H6) virus and an avian A/chicken/Guangdong/S1311/2010(H6N6) (GD H6) virus that transmitted efficiently in guinea pigs. Our results revealed that in the presence of HA1 Q226, the triad of HA1 S137, E190 and G228 in GD H6 HA allows the binding to both avian- and human-like receptors with a slight preference for avian receptors. Its conservation among the majority of H6 HAs provides an explanation for the broader host range of this subtype. Furthermore, the triad of N137, V190 and S228 in TW H6 HA may alleviate the requirement for a hydrophobic residue at HA1 226 of H2 and H3 HAs when binding to human-like receptors. Consequently, TW H6 HA has a slight preference for human receptors, thus may represent an intermediate towards a complete human adaptation. Importantly, the triad observed in TW H6 HA is detected in 74% H6 viruses isolated from Taiwan in the past 14 years, suggesting an elevated threat of H6 viruses from this region to human health. The novel roles of the triad at HA1 137, 190 and 228 of H6 HA in binding to receptors revealed here may also be used by other HA subtypes to achieve human adaptation, which needs to be further tested in laboratory and closely monitored in field surveillance.

Segment 5, ORF 1 of the infectious salmon anemia virus (ISAV) genome, encodes for the ISAV F protein, which is responsible for viral-host endosomal membrane fusion during a productive ISAV infection. The entry machinery of ISAV is composed of a complex of the ISAV F and ISAV hemagglutinin esterase (HE) proteins in an unknown stoichiometry prior to receptor engagement by ISAV HE. Following binding of the receptor to ISAV HE, dissociation of the ISAV F protein from HE, and subsequent endocytosis, the ISAV F protein resolves into a fusion-competent oligomeric state. Here, we present a 2.1 Å crystal structure of the fusion core of the ISAV F protein determined at low pH. This structure has allowed us to unambiguously demonstrate that the ISAV entry machinery exhibits typical class I viral fusion protein architecture. Furthermore, we have determined stabilizing factors that accommodate the pH-dependent mode of ISAV transmission, and our structure has allowed the identification of a central coil that is conserved across numerous and varied post-fusion viral glycoprotein structures. We then discuss a mechanistic model of ISAV fusion that parallels the paramyxoviral class I fusion strategy wherein attachment and fusion are relegated to separate proteins in a similar fashion to ISAV fusion., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

To wished to characterize the hemagglutinin (H) gene of the measles virus epidemic strain H1a in Liaoning Province (China) from 1997-2014 to provide a basis for the control and elimination of measles. All 63 measles virus strains were the H1a genotype. Fragments of the H gene (1854 nucleotides) and nucleoprotein (N) gene (450 nucleotides) were amplified by reverse transcription-polymerase chain reaction (RT-PCR) and the PCR products sequenced and analyzed. Phylogenetic-trees were constructed with reference strains of the genotype-H measles virus downloaded from GenBank, including Chinese measles virus strains isolated in 1993-1994 and the vaccine reference strains S-191 and C-47. Sixty-three strains of the measles virus in 1997-2014 belonged to genotype H1a. The mean evolutionary rate for gene N-450 was higher than that for the H gene. All 63 strains of the measles virus were mutated from: serine (Ser S) to asparagine (Asn N) in the 240th amino acid; arginine (Arg R) to glycine (Gly G) in the 243th; and tyrosine (Tyr Y) to Asn N in the 481th amino acid. All measles virus strains in cluster 2 were mutated from proline (Pro P) to leucine (Leu L) in the 397th amino acid. The other neutralization sites showed no apparent difference when comparing the nucleotides/amino acids of the H gene of S191 vaccine strains.

We studied the molecular evolution of the haemagglutinin (H) gene (full length) in all genotypes (24 genotypes, 297 strains) of measles virus (MeV). The gene's evolutionary timescale was estimated by the Bayesian Markov chain Monte Carlo (MCMC) method. We also analysed positive selection sites. The MCMC tree indicated that the MeV H gene diverged from the rinderpest virus (same genus) about 250 years ago and that 24 MeV genotypes formed 3 lineages dating back to a 1915 ancestor (95% highest posterior density [HPD] 1882-1941) with relatively rapid evolution (mean rate: 9.02 × 10(-4) substitutions/site/year). The 3 lineages diverged in 1915 (lineage 1, 95% HPD 1882-1941), 1954 (lineage 2, 95% HPD 1937-1969), and 1940 (lineage 3, 95% HPD 1927-1952). These 24 genotypes may have diverged and emerged between the 1940s and 1990 s. Selective pressure analysis identified many negative selection sites on the H protein but only a few positive selection sites, suggesting strongly operated structural and/or functional constraint of changes on the H protein. Based on the molecular evolution of H gene, an ancestor MeV of the 24 genotypes emerged about 100 years ago and the structure of H protein has been well conserved.

Reasons for Performing Study: The protection induced by an equine influenza (EI) vaccine strain depends on its antigenic relatedness to the challenge virus. Although the World Organisation for Animal Health (OIE) recommend that both Florida sublineage clade 1 (Fc1) and clade 2 (Fc2) viruses should be included in EI vaccines, Japanese EI vaccines have not, thus far, been updated to include a Fc2 virus., Objectives: To evaluate the efficacy of antibodies raised against Japanese EI vaccine strains in the neutralisation of recent Fc2 viruses., Study Design: Antigenic analysis., Methods: Virus neutralisation tests were performed using antisera from experimentally infected horses and from horses that had received a primary course of the currently available vaccines., Results: Antiserum raised against the Japanese EI vaccine strain, A/equine/La Plata/1993, exhibited poor cross-neutralising activity against the Fc2 viruses isolated recently in Ireland and the UK, which have the substitution of alanine to valine at position 144 in antigenic site A of the haemagglutinin gene. In contrast, the antiserum exhibited good cross-neutralising activity against the Fc2 viruses without the substitution. This finding was supported in experiments with antisera collected from vaccinated horses., Conclusions: This suggests that the efficacy of the Japanese EI vaccine for some of the recent Fc2 viruses is suboptimal and that vaccines should be updated in accordance with the OIE recommendations., (© 2014 EVJ Ltd.)

Sialic acids (Sias), 9-carbon-backbone sugars, are among the most complex and versatile molecules of life. As terminal residues of glycans on proteins and lipids, Sias are key elements of glycotopes of both cellular and microbial lectins and thus act as important molecular tags in cell recognition and signaling events. Their functions in such interactions can be regulated by post-synthetic modifications, the most common of which is differential Sia-O-acetylation (O-Ac-Sias). The biology of O-Ac-Sias remains mostly unexplored, largely because of limitations associated with their specific in situ detection. Here, we show that dual-function hemagglutinin-esterase envelope proteins of nidoviruses distinguish between a variety of closely related O-Ac-Sias. By using soluble forms of hemagglutinin-esterases as lectins and sialate-O-acetylesterases, we demonstrate differential expression of distinct O-Ac-sialoglycan populations in an organ-, tissue- and cell-specific fashion. Our findings indicate that programmed Sia-O-acetylation/de-O-acetylation may be critical to key aspects of cell development, homeostasis, and/or function., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Large outbreaks of measles occurred in the Philippines in 2010 and 2011. Genetic analysis was performed to identify the genotype of measles virus (MeV) that was responsible for the large outbreaks., Methods: A total of 114 representative MeVs that were detected in the Philippines from 2008 to 2011 were analyzed by sequencing the C-terminal region of nucleocapsid (N) gene and partial hemagglutinin (H) gene and by inferring the phylogenetic trees., Results: Genetic analysis showed that genotype D9 was the predominant circulating strain during the 4-year study period. Genotype D9 was detected in 23 samples (92%) by N gene sequencing and 93 samples (94%) by H gene analysis. Sporadic cases of genotype G3 MeV were identified in 2 samples (8%) by N gene sequencing and 6 samples (6%) by H gene analysis. Genotype G3 MeV was detected mainly in Panay Island in 2009 and 2010. Molecular clock analysis of N gene showed that the recent genotype D9 viruses that caused the big outbreaks in 2010 and 2011 diverged from a common ancestor in 2005 in one of the neighboring Southeast Asian countries, where D9 was endemic. These big outbreaks of measles resulted in a spillover and were associated with genotype D9 MeV importation to Japan and the USA., Conclusion: Genotype D9 MeV became endemic and caused two big outbreaks in the Philippines in 2010 and 2011. Genotype G3 MeV was detected sporadically with limited geographic distribution. This study highlights the importance of genetic analysis not only in helping with the assessment of measles elimination program in the country but also in elucidating the transmission dynamics of measles virus.