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From 2014 to week 07/2020 the Centre for Health Protection in Hong Kong conducted screening for influenza C virus (ICV). A retrospective analysis of ICV detections to week 26/2019 revealed persistent low-level circulation with outbreaks occurring biennially in the winters of 2015 to 2016 and 2017 to 2018 (R. S. Daniels et al., J Virol 94:e01051-20, 2020, https://doi.org/10.1128/JVI.01051-20). Here, we report on an outbreak occurring in 2019 to 2020, reinforcing the observation of biennial seasonality in Hong Kong. All three outbreaks occurred in similar time frames, were subsequently dwarfed by seasonal epidemics of influenza types A and B, and were caused by similar proportions of C/Kanagawa/1/76 (K)-lineage and C/São Paulo/378/82 S1- and S2-sublineage viruses. Ongoing genetic drift was observed in all genes, with some evidence of amino acid substitution in the hemagglutinin-esterase-fusion (HEF) glycoprotein possibly associated with antigenic drift. A total of 61 ICV genomes covering the three outbreaks were analyzed for reassortment, and 9 different reassortant constellations were identified, 1 K-lineage, 4 S1-sublineage, and 4 S2-sublineage, with 6 of these being identified first in the 2019-1920 outbreak (2 S2-lineage and 4 S1-lineage). The roles that virus interference/enhancement, ICV persistent infection, genome evolution, and reassortment might play in the observed seasonality of ICV in Hong Kong are discussed. IMPORTANCE Influenza C virus (ICV) infection of humans is common, with the great majority of people being infected during childhood, though reinfection can occur throughout life. While infection normally results in "cold-like" symptoms, severe disease cases have been reported in recent years. However, knowledge of ICV is limited due to poor systematic surveillance and an inability to propagate the virus in large amounts in the laboratory. Following recent systematic surveillance in Hong Kong SAR, China, and direct ICV gene sequencing from clinical specimens, a 2-year cycle of disease outbreaks (epidemics) has been identified, with gene mixing playing a significant role in ICV evolution. Studies like those reported here are key to developing an understanding of the impact of influenza C virus infection in humans, notably where comorbidities exist and severe respiratory disease can develop.

Historically part of the coronavirus (CoV) family, torovirus (ToV) was recently classified in the new family Tobaniviridae . While reverse genetics systems have been established for various CoVs, none exist for ToVs. Here, we developed a reverse genetics system using an infectious full-length cDNA clone of bovine ToV (BToV) in a bacterial artificial chromosome (BAC). Recombinant BToV harboring genetic markers had the same phenotype as wild-type (wt) BToV. To generate two types of recombinant virus, the hemagglutinin-esterase (HE) gene was edited, as cell-adapted wtBToV generally loses full-length HE (HEf), resulting in soluble HE (HEs). First, recombinant viruses with HEf and hemagglutinin (HA)-tagged HEf or HEs genes were rescued. These exhibited no significant differences in their effect on virus growth in HRT18 cells, suggesting that HE is not essential for viral replication in these cells. Thereafter, we generated a recombinant virus (rEGFP) wherein HE was replaced by the enhanced green fluorescent protein (EGFP) gene. rEGFP expressed EGFP in infected cells but showed significantly lower levels of viral growth than wtBToV. Moreover, rEGFP readily deleted the EGFP gene after one passage. Interestingly, rEGFP variants with two mutations (C1442F and I3562T) in nonstructural proteins (NSPs) that emerged during passage exhibited improved EGFP expression, EGFP gene retention, and viral replication. An rEGFP into which both mutations were introduced displayed a phenotype similar to that of these variants, suggesting that the mutations contributed to EGFP gene acceptance. The current findings provide new insights into BToV, and reverse genetics will help advance the current understanding of this neglected pathogen. IMPORTANCE ToVs are diarrhea-causing pathogens detected in various species, including humans. Through the development of a BAC-based BToV, we introduced the first reverse genetics system for Tobaniviridae . Utilizing this system, recombinant BToVs with a full-length HE gene were generated. Remarkably, although clinical BToVs generally lose the HE gene after a few passages, some recombinant viruses generated in the current study retained the HE gene for up to 20 passages while accumulating mutations in NSPs, which suggested that these mutations may be involved in HE gene retention. The EGFP gene of recombinant viruses was unstable, but rEGFP into which two NSP mutations were introduced exhibited improved EGFP expression, gene retention, and viral replication. These data suggested the existence of an NSP-based acceptance or retention mechanism for exogenous RNA or HE genes. Recombinant BToVs and reverse genetics are powerful tools for understanding fundamental viral processes, pathogenesis, and BToV vaccine development.

Cleavage of the influenza A virus (IAV) hemagglutinin (HA) by host proteases is indispensable for virus replication. Most IAVs possess a monobasic HA cleavage site cleaved by trypsin-like proteases. Previously, the transmembrane protease TMPRSS2 was shown to be essential for proteolytic activation of IAV HA subtypes H1, H2, H7, and H10 in mice. In contrast, additional proteases are involved in activation of certain H3 IAVs, indicating that HAs with monobasic cleavage sites can differ in their sensitivity to host proteases. Here, we investigated the role of TMPRSS2 in proteolytic activation of avian HA subtypes H1 to H11 and H14 to H16 in human and mouse airway cell cultures. Using reassortant viruses carrying representative HAs, we analyzed HA cleavage and multicycle replication in (i) lung cells of TMPRSS2-deficient mice and (ii) Calu-3 cells and primary human bronchial cells subjected to morpholino oligomer-mediated knockdown of TMPRSS2 activity. TMPRSS2 was found to be crucial for activation of H1 to H11, H14, and H15 in airway cells of human and mouse. Only H9 with an R-S-S-R cleavage site and H16 were proteolytically activated in the absence of TMPRSS2 activity, albeit with reduced efficiency. Moreover, a TMPRSS2-orthologous protease from duck supported activation of H1 to H11, H15, and H16 in MDCK cells. Together, our data demonstrate that in human and murine respiratory cells, TMPRSS2 is the major activating protease of almost all IAV HA subtypes with monobasic cleavage sites. Furthermore, our results suggest that TMPRSS2 supports activation of IAV with a monobasic cleavage site in ducks. IMPORTANCE Human infections with avian influenza A viruses upon exposure to infected birds are frequently reported and have received attention as a potential pandemic threat. Cleavage of the envelope glycoprotein hemagglutinin (HA) by host proteases is a prerequisite for membrane fusion and essential for virus infectivity. In this study, we identify the transmembrane protease TMPRSS2 as the major activating protease of avian influenza virus HAs of subtypes H1 to H11, H14 and H15 in human and murine airway cells. Our data demonstrate that inhibition of TMPRSS2 activity may provide a useful approach for the treatment of human infections with avian influenza viruses that should be considered for pandemic preparedness as well. Additionally, we show that a TMPRSS2-orthologous protease from duck can activate avian influenza virus HAs with a monobasic cleavage site and, thus, represents a potential virus-activating protease in waterfowl, the primary reservoir for influenza A viruses.

Influenza vaccines have traditionally been tested in naive mice and ferrets. However, humans are first exposed to influenza viruses within the first few years of their lives. Therefore, there is a pressing need to test influenza virus vaccines in animal models that have been previously exposed to influenza viruses before being vaccinated. In this study, previously described H2 computationally optimized broadly reactive antigen (COBRA) hemagglutinin (HA) vaccines (Z1 and Z5) were tested in influenza virus "preimmune" ferret models. Ferrets were infected with historical, seasonal influenza viruses to establish preimmunity. These preimmune ferrets were then vaccinated with either COBRA H2 HA recombinant proteins or wild-type H2 HA recombinant proteins in a prime-boost regimen. A set of naive preimmune or nonpreimmune ferrets were also vaccinated to control for the effects of the multiple different preimmunities. All of the ferrets were then challenged with a swine H2N3 influenza virus. Ferrets with preexisting immune responses influenced recombinant H2 HA-elicited antibodies following vaccination, as measured by hemagglutination inhibition (HAI) and classical neutralization assays. Having both H3N2 and H1N1 immunological memory regardless of the order of exposure significantly decreased viral nasal wash titers and completely protected all ferrets from both morbidity and mortality, including the mock-vaccinated ferrets in the group. While the vast majority of the preimmune ferrets were protected from both morbidity and mortality across all of the different preimmunities, the Z1 COBRA HA-vaccinated ferrets had significantly higher antibody titers and recognized the highest number of H2 influenza viruses in a classical neutralization assay compared to the other H2 HA vaccines. IMPORTANCE H1N1 and H3N2 influenza viruses have cocirculated in the human population since 1977. Nearly every human alive today has antibodies and memory B and T cells against these two subtypes of influenza viruses. H2N2 influenza viruses caused the 1957 global pandemic and people born after 1968 have never been exposed to H2 influenza viruses. It is quite likely that a future H2 influenza virus could transmit within the human population and start a new global pandemic, since the majority of people alive today are immunologically naive to viruses of this subtype. Therefore, an effective vaccine for H2 influenza viruses should be tested in an animal model with previous exposure to influenza viruses that have circulated in humans. Ferrets were infected with historical influenza A viruses to more accurately mimic the immune responses in people who have preexisting immune responses to seasonal influenza viruses. In this study, preimmune ferrets were vaccinated with wild-type (WT) and COBRA H2 recombinant HA proteins in order to examine the effects that preexisting immunity to seasonal human influenza viruses have on the elicitation of broadly cross-reactive antibodies from heterologous vaccination., (Copyright © 2021 Reneer et al.)

In 2014, the Centre for Health Protection in Hong Kong introduced screening for influenza C virus (ICV) as part of its routine surveillance for infectious agents in specimens collected from patients presenting with symptoms of respiratory viral infection, including influenza-like illness (ILI). A retrospective analysis of ICV detections up to week 26 of 2019 revealed persistent low-level circulation, with two outbreaks having occurred in the winters of 2015 to 2016 and 2017 to 2018. These outbreaks occurred at the same time as, and were dwarfed by, seasonal epidemics of influenza types A and B. Gene sequencing studies on stored ICV-positive clinical specimens from the two outbreaks have shown that the hemagglutinin-esterase (HE) genes of the viruses fall into two of the six recognized genetic lineages (represented by C/Kanagawa/1/76 and C/São Paulo/378/82), with there being significant genetic drift compared to earlier circulating viruses within both lineages. The location of a number of encoded amino acid substitutions in hemagglutinin-esterase fusion (HEF) glycoproteins suggests that antigenic drift may also have occurred. Observations of ICV outbreaks in other countries, with some of the infections being associated with severe disease, indicates that ICV infection has the potential to have significant clinical and health care impacts in humans. IMPORTANCE Influenza C virus infection of humans is common, and reinfection can occur throughout life. While symptoms are generally mild, severe disease cases have been reported, but knowledge of the virus is limited, as little systematic surveillance for influenza C virus is conducted and the virus cannot be studied by classical virologic methods because it cannot be readily isolated in laboratories. A combination of systematic surveillance in Hong Kong SAR, China, and new gene sequencing methods has been used in this study to assess influenza C virus evolution and provides evidence for a 2-year cycle of disease outbreaks. The results of studies like that reported here are key to developing an understanding of the impact of influenza C virus infection in humans and how virus evolution might be associated with epidemics., (Copyright © 2020 Daniels et al.)

H7N9 avian influenza viruses (AIVs) continue to evolve and remain a huge threat to human health and the poultry industry. Previously, serially passaging the H7N9 A/Anhui/1/2013 virus in the presence of homologous ferret antiserum resulted in immune escape viruses containing amino acid substitutions alanine to threonine at residues 125 (A125T) and 151 (A151T) and leucine to glutamine at residue 217 (L217Q) in the hemagglutinin (HA) protein. These HA mutations have also been found in field isolates in 2019. To investigate the potential threat of serum escape mutant viruses to humans and poultry, the impact of these HA substitutions, either individually or in combination, on receptor binding, pH of fusion, thermal stability, and virus replication were investigated. Our results showed the serum escape mutant formed large plaques in Madin-Darby canine kidney (MDCK) cells and grew robustly in vitro and in ovo They had a lower pH of fusion and increased thermal stability. Of note, the serum escape mutant completely lost the ability to bind to human-like receptor analogues. Further analysis revealed that N-linked glycosylation, as a result of A125T or A151T substitutions in HA, resulted in reduced receptor-binding avidity toward both human and avian-like receptor analogues, and the A125T+A151T mutations completely abolished human-like receptor binding. The L217Q mutation enhanced the H7N9 acid and thermal stability while the A151T mutation dramatically decreased H7N9 HA thermal stability. To conclude, H7N9 AIVs that contain A125T+A151T+L217Q mutations in the HA protein may pose a reduced pandemic risk but remain a heightened threat for poultry. IMPORTANCE Avian influenza H7N9 viruses have been causing disease outbreaks in poultry and humans. We previously determined that propagation of H7N9 virus in virus-specific antiserum gives rise to mutant viruses carrying mutations A125T+A151T+L217Q in their hemagglutinin protein, enabling the virus to overcome vaccine-induced immunity. As predicted, these immune escape mutations were also observed in the field viruses that likely emerged in the immunized or naturally exposed birds. This study demonstrates that the immune escape mutants also (i) gained greater replication ability in cultured cells and in chicken embryos as well as (ii) increased acid and thermal stability but (iii) lost preferences for binding to human-type receptor while maintaining binding for the avian-like receptor. Therefore, they potentially pose reduced pandemic risk. However, the emergent virus variants containing the indicated mutations remain a significant risk to poultry due to antigenic drift and improved fitness for poultry., (Copyright © 2020 Chang et al.)

Highly pathogenic avian influenza (HPAI) viruses are enzootic in wild birds and poultry and continue to cause human infections with high mortality. To date, more than 850 confirmed human cases of H5N1 virus infection have been reported, of which ∼60% were fatal. Global concern persists that these or similar avian influenza viruses will evolve into viruses that can transmit efficiently between humans, causing a severe influenza pandemic. It was shown previously that a change in receptor specificity is a hallmark for adaptation to humans and evolution toward a transmittable virus. Substantial genetic diversity was detected within the receptor binding site of hemagglutinin of HPAI A/H5N1 viruses, evolved during human infection, as detected by next-generation sequencing. Here, we investigated the functional impact of substitutions that were detected during these human infections. Upon rescue of 21 mutant viruses, most substitutions in the receptor binding site (RBS) resulted in viable virus, but virus replication, entry, and stability were often impeded. None of the tested substitutions individually resulted in a clear switch in receptor preference as measured with modified red blood cells and glycan arrays. Although several combinations of the substitutions can lead to human-type receptor specificity, accumulation of multiple amino acid substitutions within a single hemagglutinin during human infection is rare, thus reducing the risk of virus adaptation to humans. IMPORTANCE H5 viruses continue to be a threat for public health. Because these viruses are immunologically novel to humans, they could spark a pandemic when adapted to transmit between humans. Avian influenza viruses need several adaptive mutations to bind to human-type receptors, increase hemagglutinin (HA) stability, and replicate in human cells. However, knowledge on adaptive mutations during human infections is limited. A previous study showed substantial diversity within the receptor binding site of H5N1 during human infection. We therefore analyzed the observed amino acid changes phenotypically in a diverse set of assays, including virus replication, stability, and receptor specificity. None of the tested substitutions resulted in a clear step toward a human-adapted virus capable of aerosol transmission. It is notable that acquiring human-type receptor specificity needs multiple amino acid mutations, and that variability at key position 226 is not tolerated, reducing the risk of them being acquired naturally., (Copyright © 2020 American Society for Microbiology.)

Measles virus (MeV) is an enveloped RNA virus bearing two envelope glycoproteins, the hemagglutinin (H) and fusion (F) proteins. Upon receptor binding, the H protein triggers conformational changes of the F protein, causing membrane fusion and subsequent virus entry. MeV may persist in the brain, infecting neurons and causing fatal subacute sclerosing panencephalitis (SSPE). Since neurons do not express either of the MeV receptors, signaling lymphocytic activation molecule (SLAM; also called CD150) and nectin-4, how MeV propagates in neurons is unknown. Recent studies have shown that specific substitutions in the F protein found in MeV isolates from SSPE patients are critical for MeV neuropathogenicity by rendering the protein unstable and hyperfusogenic. Recombinant MeVs possessing the F proteins with such substitutions can spread in primary human neurons and in the brains of mice and hamsters and induce cell-cell fusion in cells lacking SLAM and nectin-4. Here, we show that receptor-blind mutant H proteins that have decreased binding affinities to receptors can support membrane fusion mediated by hyperfusogenic mutant F proteins, but not the wild-type F protein, in cells expressing the corresponding receptors. The results suggest that weak interactions of the H protein with certain molecules (putative neuron receptors) trigger hyperfusogenic F proteins in SSPE patients. Notably, where cell-cell contacts are ensured, the weak cis interaction of the H protein with SLAM on the same cell surface also could trigger hyperfusogenic F proteins. Some enveloped viruses may exploit such cis interactions with receptors to infect target cells, especially in cell-to-cell transmission. IMPORTANCE Measles virus (MeV) may persist in the brain, causing incurable subacute sclerosing panencephalitis (SSPE). Because neurons, the main target in SSPE, do not express receptors for wild-type (WT) MeV, how MeV propagates in the brain is a key question for the disease. Recent studies have demonstrated that specific substitutions in the MeV fusion (F) protein are critical for neuropathogenicity. Here, we show that weak cis and trans interactions of the MeV attachment protein with receptors that are not sufficient to trigger the WT MeV F protein can trigger the mutant F proteins from neuropathogenic MeV isolates. Our study not only provides an important clue to understand MeV neuropathogenicity but also reveals a novel viral strategy to expand cell tropism., (Copyright © 2020 American Society for Microbiology.)

The hemagglutinin (HA), a glycoprotein on the surface of influenza A virus (IAV), initiates the virus life cycle by binding to terminal sialic acid (SA) residues on host cells. The HA gradually accumulates amino acid substitutions that allow IAV to escape immunity through a mechanism known as antigenic drift. We recently confirmed that a small set of amino acid residues are largely responsible for driving antigenic drift in swine-origin H3 IAV. All identified residues are located adjacent to the HA receptor binding site (RBS), suggesting that substitutions associated with antigenic drift may also influence receptor binding. Among those substitutions, residue 145 was shown to be a major determinant of antigenic evolution. To determine whether there are functional constraints to substitutions near the RBS and their impact on receptor binding and antigenic properties, we carried out site-directed mutagenesis experiments at the single-amino-acid level. We generated a panel of viruses carrying substitutions at residue 145 representing all 20 amino acids. Despite limited amino acid usage in nature, most substitutions at residue 145 were well tolerated without having a major impact on virus replication in vitro All substitution mutants retained receptor binding specificity, but the substitutions frequently led to decreased receptor binding. Glycan microarray analysis showed that substitutions at residue 145 modulate binding to a broad range of glycans. Furthermore, antigenic characterization identified specific substitutions at residue 145 that altered antibody recognition. This work provides a better understanding of the functional effects of amino acid substitutions near the RBS and the interplay between receptor binding and antigenic drift. IMPORTANCE The complex and continuous antigenic evolution of IAVs remains a major hurdle for vaccine selection and effective vaccination. On the hemagglutinin (HA) of the H3N2 IAVs, the amino acid substitution N 145 K causes significant antigenic changes. We show that amino acid 145 displays remarkable amino acid plasticity in vitro , tolerating multiple amino acid substitutions, many of which have not yet been observed in nature. Mutant viruses carrying substitutions at residue 145 showed no major impairment in virus replication in the presence of lower receptor binding avidity. However, their antigenic characterization confirmed the impact of the 145 K substitution in antibody immunodominance. We provide a better understanding of the functional effects of amino acid substitutions implicated in antigenic drift and its consequences for receptor binding and antigenicity. The mutation analyses presented in this report represent a significant data set to aid and test the ability of computational approaches to predict binding of glycans and in antigenic cartography analyses., (This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.)

Influenza A viruses in swine (IAV-S) circulating in the United States of America are phylogenetically and antigenically distinct. A human H3 hemagglutinin (HA) was introduced into the IAV-S gene pool in the late 1990s, sustained continued circulation, and evolved into five monophyletic genetic clades, H3 clades IV-A to -E, after 2009. Across these phylogenetic clades, distinct antigenic clusters were identified, with three clusters (cyan, red, and green antigenic cluster) among the most frequently detected antigenic phenotypes (Abente EJ, Santos J, Lewis NS, Gauger PC, Stratton J, et al. J Virol 90:8266-8280, 2016, https://doi.org/10.1128/JVI.01002-16). Although it was demonstrated that antigenic diversity of H3N2 IAV-S was associated with changes at a few amino acid positions in the head of the HA, the implications of this diversity for vaccine efficacy were not tested. Using antigenically representative H3N2 viruses, we compared whole inactivated virus (WIV) and live-attenuated influenza virus (LAIV) vaccines for protection against challenge with antigenically distinct H3N2 viruses in pigs. WIV provided partial protection against antigenically distinct viruses but did not prevent virus replication in the upper respiratory tract. In contrast, LAIV provided complete protection from disease and virus was not detected after challenge with antigenically distinct viruses. IMPORTANCE Due to the rapid evolution of the influenza A virus, vaccines require continuous strain updates. Additionally, the platform used to deliver the vaccine can have an impact on the breadth of protection. Currently, there are various vaccine platforms available to prevent influenza A virus infection in swine, and we experimentally tested two: adjuvanted-whole inactivated virus and live-attenuated virus. When challenged with an antigenically distinct virus, adjuvanted-whole inactivated virus provided partial protection, while live-attenuated virus provided effective protection. Additional strategies are required to broaden the protective properties of inactivated virus vaccines, given the dynamic antigenic landscape of cocirculating strains in North America, whereas live-attenuated vaccines may require less frequent strain updates, based on demonstrated cross-protection. Enhancing vaccine efficacy to control influenza infections in swine will help reduce the impact they have on swine production and reduce the risk of swine-to-human transmission.

Upon infection, morbilliviruses such as measles virus, rinderpest virus, and canine distemper virus (CDV) initially target immune cells via the signaling lymphocyte activation molecule (SLAM) before spreading to respiratory epithelia through the adherens junction protein nectin-4. However, the roles of these receptors in transmission from infected to naive hosts have not yet been formally tested. To experimentally addressing this question, we established a model of CDV contact transmission between ferrets. We show here that transmission of wild-type CDV sometimes precedes the onset of clinical disease. In contrast, transmission was not observed in most animals infected with SLAM- or nectin-4-blind CDVs, even though all animals infected with the nectin-4-blind virus developed sustained viremia. There was an unexpected case of transmission of a nectin-4-blind virus, possibly due to biting. Another unprecedented event was transient viremia in an infection with a SLAM-blind virus. We identified three compensatory mutations within or near the SLAM-binding surface of the attachment protein. A recombinant CDV expressing the mutated attachment protein regained the ability to infect ferret lymphocytes in vitro , but its replication was not as efficient as that of wild-type CDV. Ferrets infected with this virus developed transient viremia and fever, but there was no transmission to naive contacts. Our study supports the importance of epithelial cell infection and of sequential CDV H protein interactions first with SLAM and then nectin-4 receptors for transmission to naive hosts. It also highlights the in vivo selection pressure on the H protein interactions with SLAM. IMPORTANCE Morbilliviruses such as measles virus, rinderpest virus, and canine distemper virus (CDV) are highly contagious. Despite extensive knowledge of how morbilliviruses interact with their receptors, little is known about how those interactions influence viral transmission to naive hosts. In a ferret model of CDV contact transmission, we showed that sequential use of the signaling lymphocytic activation molecule (SLAM) and nectin-4 receptors is essential for transmission. In one animal infected with a SLAM-blind CDV, we documented mild viremia due to the acquisition of three compensatory mutations within or near the SLAM-binding surface. The interaction, however, was not sufficient to cause disease or sustain transmission to naive contacts. This work confirms the sequential roles of SLAM and nectin-4 in morbillivirus transmission and highlights the selective pressure directed toward productive interactions with SLAM., (Copyright © 2018 American Society for Microbiology.)

Measles virus (MV) usually causes acute infection but in rare cases persists in the brain, resulting in subacute sclerosing panencephalitis (SSPE). Since human neurons, an important target affected in the disease, do not express the known MV receptors (signaling lymphocyte activation molecule [SLAM] and nectin 4), how MV infects neurons and spreads between them is unknown. Recent studies have shown that many virus strains isolated from SSPE patients possess substitutions in the extracellular domain of the fusion (F) protein which confer enhanced fusion activity. Hyperfusogenic viruses with such mutations, unlike the wild-type MV, can induce cell-cell fusion even in SLAM- and nectin 4-negative cells and spread efficiently in human primary neurons and the brains of animal models. We show here that a hyperfusogenic mutant MV, IC323-F(T461I)-EGFP (IC323 with a fusion-enhancing T461I substitution in the F protein and expressing enhanced green fluorescent protein), but not the wild-type MV, spreads in differentiated NT2 cells, a widely used human neuron model. Confocal time-lapse imaging revealed the cell-to-cell spread of IC323-F(T461I)-EGFP between NT2 neurons without syncytium formation. The production of virus particles was strongly suppressed in NT2 neurons, also supporting cell-to-cell viral transmission. The spread of IC323-F(T461I)-EGFP was inhibited by a fusion inhibitor peptide as well as by some but not all of the anti-hemagglutinin antibodies which neutralize SLAM- or nectin-4-dependent MV infection, suggesting the presence of a distinct neuronal receptor. Our results indicate that MV spreads in a cell-to-cell manner between human neurons without causing syncytium formation and that the spread is dependent on the hyperfusogenic F protein, the hemagglutinin, and the putative neuronal receptor for MV. IMPORTANCE Measles virus (MV), in rare cases, persists in the human central nervous system (CNS) and causes subacute sclerosing panencephalitis (SSPE) several years after acute infection. This neurological complication is almost always fatal, and there is currently no effective treatment for it. Mechanisms by which MV invades the CNS and causes the disease remain to be elucidated. We have previously shown that fusion-enhancing substitutions in the fusion protein of MVs isolated from SSPE patients contribute to MV spread in neurons. In this study, we demonstrate that MV bearing the hyperfusogenic mutant fusion protein spreads between human neurons in a cell-to-cell manner. Spread of the virus was inhibited by a fusion inhibitor peptide and antibodies against the MV hemagglutinin, indicating that both the hemagglutinin and hyperfusogenic fusion protein play important roles in MV spread between human neurons. The findings help us better understand the disease process of SSPE., (Copyright © 2018 American Society for Microbiology.)

The inhibitors carbobenzoxy (Z)-d-Phe-l-Phe-Gly (fusion inhibitor peptide [FIP]) and 4-nitro-2-phenylacetyl amino-benzamide (AS-48) have similar efficacies in blocking membrane fusion and syncytium formation mediated by measles virus (MeV). Other homologues, such as Z-d-Phe, are less effective but may act through the same mechanism. In an attempt to map the site of action of these inhibitors, we generated mutant viruses that were resistant to the inhibitory effects of Z-d-Phe-l-Phe-Gly. These 10 mutations were localized to the heptad repeat B (HRB) region of the fusion protein, and no changes were observed in the viral hemagglutinin, which is the receptor attachment protein. Mutations were validated in a luciferase-based membrane fusion assay, using transfected fusion and hemagglutinin expression plasmids or with syncytium-based assays in Vero, Vero-SLAM, and Vero-Nectin 4 cell lines. The changes I452T, D458N, D458G/V459A, N462K, N462H, G464E, and I483R conferred resistance to both FIP and AS-48 without compromising membrane fusion. The inhibitors did not block hemagglutinin protein-mediated binding to the target cell. Edmonston vaccine/laboratory and IC323 wild-type strains were equally affected by the inhibitors. Escape mutations were mapped upon a three-dimensional (3D) structure modeled from the published crystal structure of parainfluenzavirus 5 fusion protein. The most effective mutations were situated in a region located near the base of the globular head and its junction with the alpha-helical stalk of the prefusion protein. We hypothesize that the fusion inhibitors could interfere with the structural changes that occur between the prefusion and postfusion conformations of the fusion protein. IMPORTANCE Due to lapses in vaccination worldwide that have caused localized outbreaks, measles virus (MeV) has regained importance as a pathogen. Antiviral agents against measles virus are not commercially available but could be useful in conjunction with MeV eradication vaccine programs and as a safeguard in oncolytic viral therapy. Three decades ago, the small hydrophobic peptide Z-d-Phe-l-Phe-Gly (FIP) was shown to block MeV infections and syncytium formation in monkey kidney cell lines. The exact mechanism of its action has yet to be determined, but it does appear to have properties similar to those of another chemical inhibitor, AS-48, which appears to interfere with the conformational change in the viral F protein that is required to elicit membrane fusion. Escape mutations were used to map the site of action for FIP. Knowledge gained from these studies could help in the design of new inhibitors against morbilliviruses and provide additional knowledge concerning the mechanism of virus-mediated membrane fusion., (Copyright © 2017 American Society for Microbiology.)

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.)

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.)

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.)

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_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_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.)

Unlabelled: Measles virus (MeV), a morbillivirus within the paramyxovirus family, expresses two envelope glycoproteins. The attachment (H) protein mediates receptor binding, followed by triggering of the fusion (F) protein, which leads to merger of the viral envelope with target cell membranes. Receptor binding by members of related paramyxovirus genera rearranges the head domains of the attachment proteins, liberating an F-contact domain within the attachment protein helical stalk. However, morbillivirus glycoproteins first assemble intracellularly prior to receptor binding, raising the question of whether alternative protein-protein interfaces are involved or whether an entirely distinct triggering principle is employed. To test these possibilities, we generated headless H stem mutants of progressively shorter length. Conformationally restricted H stems remained capable of intracellular assembly with a standard F protein and a soluble MeV F mutant. Proteolytic maturation of F, but not the altered biochemical conditions at the cell surface, reduces the strength of glycoprotein interaction, readying the complexes for triggering. F mutants stabilized in the prefusion conformation interact with H intracellularly and at the cell surface, while destabilized F mutants interact only intracellularly, prior to F maturation. These results showcase an MeV entry machinery that functionally varies conserved motifs of the proposed paramyxovirus infection pathway. Intracellular and plasma membrane-resident MeV glycoprotein complexes employ the same protein-protein interface. F maturation prepares for complex separation after triggering, and the H head domains in prereceptor-bound conformation prevent premature stalk rearrangements and F activation. Intracellular preassembly affects MeV fusion profiles and may contribute to the high cell-to-cell fusion activity characteristic of the morbillivirus genus., Importance: Paramyxoviruses of the morbillivirus genus, such as measles, are highly contagious, major human and animal pathogens. MeV envelope glycoproteins preassemble intracellularly into tightly associated hetero-oligomers. To address whether preassembly reflects a unique measles virus entry strategy, we characterized the protein-protein interface of intracellular and surface-exposed fusion complexes and investigated the effect of the attachment protein head domains, glycoprotein maturation, and altered biochemical conditions at the cell surface on measles virus fusion complexes. Our results demonstrate that measles virus functionally varies conserved elements of the paramyxovirus entry pathway, providing a possible explanation for the high cell-to-cell fusion activity of morbilliviruses. Insight gained from these data affects the design of effective broad-spectrum paramyxovirus entry inhibitors., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

The evolution of oseltamivir resistance mutations during selection through serial passages in animals is still poorly described. Herein, we assessed the evolution of neuraminidase (NA) and hemagglutinin (HA) genes of influenza A/WSN/33 (H1N1) and A/Victoria/3/75 (H3N2) viruses recovered from the lungs of experimentally infected BALB/c mice receiving suboptimal doses (0.05 and 1 mg/kg of body weight/day) of oseltamivir over two generations. The traditional phenotypic and genotypic methods as well as deep-sequencing analysis were used to characterize the potential selection of mutations and population dynamics of oseltamivir-resistant variants. No oseltamivir-resistant NA or HA changes were detected in the recovered A/WSN/33 viruses. However, we observed a positive selection of the I222T NA substitution in the recovered A/Victoria/3/75 viruses, with a frequency increasing over time and with an oseltamivir concentration from 4% in the initial pretherapy inoculum up to 28% after two lung passages. Although the presence of mixed I222T viral populations in mouse lungs only led to a minimal increase in oseltamivir 50% enzyme-inhibitory concentrations (IC50s) (by a mean of 5.7-fold) compared to that of the baseline virus, the expressed recombinant A/Victoria/3/75 I222T NA protein displayed a 16-fold increase in the oseltamivir IC50 level compared to that of the recombinant wild type (WT). In conclusion, the combination of serial in vivo passages under neuraminidase inhibitor (NAI) pressure and temporal deep-sequencing analysis enabled, for the first time, the identification and selection of the oseltamivir-resistant I222T NA mutation in an influenza H3N2 virus. Additional in vivo selection experiments with other antivirals and drug combinations might provide important information on the evolution of antiviral resistance in influenza viruses., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

The hemagglutinin (H) gene of canine distemper virus (CDV) encodes the receptor-binding protein. This protein, together with the fusion (F) protein, is pivotal for infectivity since it contributes to the fusion of the viral envelope with the host cell membrane. Of the two receptors currently known for CDV (nectin-4 and the signaling lymphocyte activation molecule [SLAM]), SLAM is considered the most relevant for host susceptibility. To investigate how evolution might have impacted the host-CDV interaction, we examined the functional properties of a series of missense single nucleotide polymorphisms (SNPs) naturally accumulating within the H-gene sequences during the transition between two distinct but related strains. The two strains, a wild-type strain and a consensus strain, were part of a single continental outbreak in European wildlife and occurred in distinct geographical areas 2 years apart. The deduced amino acid sequence of the two H genes differed at 5 residues. A panel of mutants carrying all the combinations of the SNPs was obtained by site-directed mutagenesis. The selected mutant, wild type, and consensus H proteins were functionally evaluated according to their surface expression, SLAM binding, fusion protein interaction, and cell fusion efficiencies. The results highlight that the most detrimental functional effects are associated with specific sets of SNPs. Strikingly, an efficient compensational system driven by additional SNPs appears to come into play, virtually neutralizing the negative functional effects. This system seems to contribute to the maintenance of the tightly regulated function of the H-gene-encoded attachment protein. Importance: To investigate how evolution might have impacted the host-canine distemper virus (CDV) interaction, we examined the functional properties of naturally occurring single nucleotide polymorphisms (SNPs) in the hemagglutinin gene of two related but distinct strains of CDV. The hemagglutinin gene encodes the attachment protein, which is pivotal for infection. Our results show that few SNPs have a relevant detrimental impact and they generally appear in specific combinations (molecular signatures). These drastic negative changes are neutralized by compensatory mutations, which contribute to maintenance of an overall constant bioactivity of the attachment protein. This compensational mechanism might reflect the reaction of the CDV machinery to the changes occurring in the virus following antigenic variations critical for virulence., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

We detected three avian influenza hemagglutinin (HA) subtypes (H7, H9, and H5) and two neuraminidase (NA) subtypes (N9 and N2), as well as H7N9-related H9N9 reassortant intermediates, cocirculating among poultry in Huzhou, China, during April 2013. The results of our study reveal not only that Huzhou is one of the geographic origins of the novel H7N9 virus but also that cocirculation poses a potential threat to humans in the future.

Influenza viruses are a major public health threat worldwide, and options for antiviral therapy are limited by the emergence of drug-resistant virus strains. The influenza virus glycoprotein hemagglutinin (HA) plays critical roles in the early stage of virus infection, including receptor binding and membrane fusion, making it a potential target for the development of anti-influenza drugs. Using pseudotype virus-based high-throughput screens, we have identified several new small molecules capable of inhibiting influenza virus entry. We prioritized two novel inhibitors, MBX2329 and MBX2546, with aminoalkyl phenol ether and sulfonamide scaffolds, respectively, that specifically inhibit HA-mediated viral entry. The two compounds (i) are potent (50% inhibitory concentration [IC50] of 0.3 to 5.9 μM); (ii) are selective (50% cytotoxicity concentration [CC(50)] of >100 μM), with selectivity index (SI) values of >20 to 200 for different influenza virus strains; (iii) inhibit a wide spectrum of influenza A viruses, which includes the 2009 pandemic influenza virus A/H1N1/2009, highly pathogenic avian influenza (HPAI) virus A/H5N1, and oseltamivir-resistant A/H1N1 strains; (iv) exhibit large volumes of synergy with oseltamivir (36 and 331 μM(2) % at 95% confidence); and (v) have chemically tractable structures. Mechanism-of-action studies suggest that both MBX2329 and MBX2546 bind to HA in a nonoverlapping manner. Additional results from HA-mediated hemolysis of chicken red blood cells (cRBCs), competition assays with monoclonal antibody (MAb) C179, and mutational analysis suggest that the compounds bind in the stem region of the HA trimer and inhibit HA-mediated fusion. Therefore, MBX2329 and MBX2546 represent new starting points for chemical optimization and have the potential to provide valuable future therapeutic options and research tools to study the HA-mediated entry process.

DNA vaccines formulated with the cationic lipid-based adjuvant Vaxfectin induce protective immunity in macaques after intradermal (i.d.) or intramuscular (i.m.) delivery of 0.5 to 1 mg of codon-optimized DNA encoding the hemagglutinin (H) and fusion (F) proteins of measles virus (MeV). To characterize the effect of Vaxfectin at lower doses of H+F DNA, rhesus macaques were vaccinated twice with 20 μg of DNA plus Vaxfectin i.d., 100 μg of DNA plus Vaxfectin i.d., 100 μg of DNA plus Vaxfectin i.m. or 100 μg of DNA plus phosphate-buffered saline (PBS) i.m. using a needleless Biojector device. The levels of neutralizing (P = 0.036) and binding (P = 0.0001) antibodies were higher after 20 or 100 μg of DNA plus Vaxfectin than after 100 μg of DNA plus PBS. Gamma interferon (IFN-γ)-producing T cells were induced more rapidly than antibody, but were not improved with Vaxfectin. At 18 months after vaccination, monkeys were challenged with wild-type MeV. None developed rash or viremia, but all showed evidence of infection. Antibody levels increased, and IFN-γ- and interleukin-17-producing T cells, including cells specific for the nucleoprotein absent from the vaccine, were induced. At 3 months after challenge, MeV RNA was detected in the leukocytes of two monkeys. The levels of antibody peaked 2 to 4 weeks after challenge and then declined in vaccinated animals reflecting low numbers of bone marrow-resident plasma cells. Therefore, Vaxfectin was dose sparing and substantially improved the antibody response to the H+F DNA vaccine. This immune response led to protection from disease (rash/viremia) but not from infection. Antibody responses after challenge were more transient in vaccinated animals than in an unvaccinated animal.

A canine distemper virus (CDV) strain, CYN07-dV, associated with a lethal outbreak in monkeys, used human signaling lymphocyte activation molecule as a receptor only poorly but readily adapted to use it following a P541S substitution in the hemagglutinin protein. Since CYN07-dV had an intrinsic ability to use human nectin-4, the adapted virus became able to use both human immune and epithelial cell receptors, as well as monkey and canine ones, suggesting that CDV can potentially infect humans.

Cell entry of enveloped viruses is initiated by attachment to the virus receptor followed by fusion between the virus and host cell membranes. Measles virus (MV) attachment to its receptor is mediated by the hemagglutinin (H), which is thought to produce conformational changes in the membrane fusion protein (F) that trigger insertion of its fusion peptide into the target cell membrane. Here, we uncoupled receptor attachment and the fusion-helper function of H by introducing Y481A, R533A, S548L, and F549S mutations into the viral attachment protein that made it blind to its normal receptors. An artificial receptor attachment protein specific for Her2/neu was incorporated into the membranes of pseudotyped lentivirus particles as a separate transmembrane protein along with the F protein. Surprisingly, these particles entered efficiently into Her2/neu-positive SK-OV-3 as well as CHO-Her2 cells. Cell entry was independent of endocytosis but strictly dependent on the presence of H. H-specific monoclonal antibodies, as well as a mutation in H interfering with H/F cooperation, blocked cell entry. The particles mediated stable and specific transfer of reporter genes into Her2/neu-positive human tumor cells also in vivo, while exhibiting improved infectivity and higher titers than Her2/neu-targeted vectors displaying the targeting domain on H. Extending the current model of MV cell entry, the data suggest that receptor binding of H is not required for its fusion-helper function but that particle-cell contact in general may be sufficient to induce the conformational changes in the H/F complex and activate membrane fusion.

Most betacoronaviruses possess an hemagglutinin-esterase (HE) protein, which appears to play a role in binding to or release from the target cell. Since this HE protein possesses an acetyl-esterase activity that removes acetyl groups from O-acetylated sialic acid, a role as a receptor-destroying enzyme has been postulated. However, the precise function of HE and of its enzymatic activity remains poorly understood. Making use of neutralizing antibody and of molecular clones of recombinant human coronavirus OC43 (HCoV-OC43), our results suggest that the HE protein of this HCoV could be associated with infection of target cells and, most notably, is important in the production of infectious viral particles. Indeed, after transfecting BHK-21 cells with various cDNA infectious clones of HCoV-OC43, either lacking the HE protein or bearing an HE protein with a nonfunctional acetyl-esterase enzymatic activity, we were reproducibly unable to detect recombinant infectious viruses compared to the reference infectious HCoV-OC43 clone pBAC-OC43(FL). Complementation experiments, using BHK-21 cells expressing wild-type HE, either transiently or in a stable ectopic expression, demonstrate that this protein plays a very significant role in the production of infectious recombinant coronaviral particles that can subsequently more efficiently infect susceptible epithelial and neuronal cells. Even though the S protein is the main viral factor influencing coronavirus infection of susceptible cells, our results taken together indicate that a functionally active HE protein enhances the infectious properties of HCoV-OC43 and contributes to efficient virus dissemination in cell culture.

A vaccine that would protect young infants against measles could facilitate elimination efforts and decrease morbidity and mortality in developing countries. However, immaturity of the immune system is an important obstacle to the development of such a vaccine. In this study, DNA vaccines expressing the measles virus (MeV) hemagglutinin (H) protein or H and fusion (F) proteins, previously shown to protect juvenile macaques, were used to immunize groups of 4 newborn rhesus macaques. Monkeys were inoculated intradermally with 200 μg of each DNA at birth and at 10 months of age. As controls, 2 newborn macaques were similarly vaccinated with DNA encoding the influenza virus H5, and 4 received one dose of the current live attenuated MeV vaccine (LAV) intramuscularly. All monkeys were monitored for development of MeV-specific neutralizing and binding IgG antibody and cytotoxic T lymphocyte (CTL) responses. These responses were poor compared to the responses induced by LAV. At 18 months of age, all monkeys were challenged intratracheally with a wild-type strain of MeV. Monkeys that received the DNA vaccine encoding H and F, but not H alone, were primed for an MeV-specific CD8(+) CTL response but not for production of antibody. LAV-vaccinated monkeys were protected from rash and viremia, while DNA-vaccinated monkeys developed rashes, similar to control monkeys, but had 10-fold lower levels of viremia. We conclude that vaccination of infant macaques with DNA encoding MeV H and F provided only partial protection from MeV infection.

Influenza A virus transmission by direct contact is not well characterized. Here, we describe a mouse model for investigation of factors regulating contact-dependent transmission. Strains within the H3N2 but not H1N1 subtype of influenza virus were transmissible, and reverse-engineered viruses representing hybrids of these subtypes showed that the viral hemagglutinin is a determinant of the transmissible phenotype. Transmission to contact mice occurred within the first 6 to 54 h after cohousing with directly infected index mice, and the proportion of contacts infected within this period was reduced if the index mice had been preinfected with a heterologous subtype virus. A threshold level of virus present in the saliva of the index mice was identified, above which the likelihood of transmission was greatly increased. There was no correlation with transmission and viral loads in the nose or lung. This model could be useful for preclinical evaluation of antiviral and vaccine efficacy in combating contact-dependent transmission of influenza.

Adaptation of avian influenza viruses (AIVs) from waterfowl to domestic poultry with a deletion in the neuraminidase (NA) stalk has already been reported. The way the virus undergoes this evolution, however, is thus far unclear. We address this question using pyrosequencing of duck and turkey low-pathogenicity AIVs. Ducks and turkeys were sampled at the very beginning of an H6N1 outbreak, and turkeys were swabbed again 8 days later. NA stalk deletions were evidenced in turkeys by Sanger sequencing. To further investigate viral evolution, 454 pyrosequencing was performed: for each set of samples, up to 41,500 reads of ca. 400 bp were generated and aligned. Genetic polymorphisms between duck and turkey viruses were tracked on the whole genome. NA deletion was detected in less than 2% of reads in duck feces but in 100% of reads in turkey tracheal specimens collected at the same time. Further variations in length were observed in NA from turkeys 8 days later. Similarly, minority mutants emerged on the hemagglutinin (HA) gene, with substitutions mostly in the receptor binding site on the globular head. These critical changes suggest a strong evolutionary pressure in turkeys. The increasing performances of next-generation sequencing technologies should enable us to monitor the genomic diversity of avian influenza viruses and early emergence of potentially pathogenic variants within bird flocks. The present study, based on 454 pyrosequencing, suggests that NA deletion, an example of AIV adaptation from waterfowl to domestic poultry, occurs by selection rather than de novo emergence of viral mutants.

A major difference between vaccine and wild-type strains of measles virus (MV) in vitro is the wider cell specificity of vaccine strains, resulting from the receptor usage of the hemagglutinin (H) protein. Wild-type H proteins recognize the signaling lymphocyte activation molecule (SLAM) (CD150), which is expressed on certain cells of the immune system, whereas vaccine H proteins recognize CD46, which is ubiquitously expressed on all nucleated human and monkey cells, in addition to SLAM. To examine the effect of the H protein on the tropism and attenuation of MV, we generated enhanced green fluorescent protein (EGFP)-expressing recombinant wild-type MV strains bearing the Edmonston vaccine H protein (MV-EdH) and compared them to EGFP-expressing wild-type MV strains. In vitro, MV-EdH replicated in SLAM(+) as well as CD46(+) cells, including primary cell cultures from cynomolgus monkey tissues, whereas the wild-type MV replicated only in SLAM(+) cells. However, in macaques, both wild-type MV and MV-EdH strains infected lymphoid and respiratory organs, and widespread infection of MV-EdH was not observed. Flow cytometric analysis indicated that SLAM(+) lymphocyte cells were infected preferentially with both strains. Interestingly, EGFP expression of MV-EdH in tissues and lymphocytes was significantly weaker than that of the wild-type MV. Taken together, these results indicate that the CD46-binding activity of the vaccine H protein is important for determining the cell specificity of MV in vitro but not the tropism in vivo. They also suggest that the vaccine H protein attenuates MV growth in vivo.

Continuing outbreaks of H5N1 highly pathogenic (HP) avian influenza virus (AIV) infections of wild birds and poultry worldwide emphasize the need for global surveillance of wild birds. To support the future surveillance activities, we developed a SYBR green-based, real-time reverse transcriptase PCR (rRT-PCR) for detecting nucleoprotein (NP) genes and subtyping 16 hemagglutinin (HA) and 9 neuraminidase (NA) genes simultaneously. Primers were improved by focusing on Eurasian or North American lineage genes; the number of mixed-base positions per primer was set to five or fewer, and the concentration of each primer set was optimized empirically. Also, 30 cycles of amplification of 1:10 dilutions of cDNAs from cultured viruses effectively reduced minor cross- or nonspecific reactions. Under these conditions, 346 HA and 345 NA genes of 349 AIVs were detected, with average sensitivities of NP, HA, and NA genes of 10(1.5), 10(2.3), and 10(3.1) 50% egg infective doses, respectively. Utility of rRT-PCR for subtyping AIVs was compared with that of current standard serological tests by using 104 recent migratory duck virus isolates. As a result, all HA genes and 99% of the NA genes were genetically subtyped, while only 45% of HA genes and 74% of NA genes were serologically subtyped. Additionally, direct subtyping of AIVs in fecal samples was possible by 40 cycles of amplification: approximately 70% of HA and NA genes of NP gene-positive samples were successfully subtyped. This validation study indicates that rRT-PCR with optimized primers and reaction conditions is a powerful tool for subtyping varied AIVs in clinical and cultured samples.

With improved measles virus (MV) control, the genetic variability of the MV-nucleoprotein hypervariable region (NP-HVR) decreases. Thus, it becomes increasingly difficult to determine the origin of a virus using only this part of the genome. During outbreaks in Europe and Africa, we found MV strains with identical NP-HVR sequences. However, these strains showed considerable diversity within a larger sequencing window based on concatenated MV phosphoprotein and hemagglutinin genes (P/H pseudogenes). In Belarus, Germany, Russia, and the Democratic Republic of Congo, the P/H pseudogenes provided insights into chains of transmission, whereas identical NP-HVR provided none. In Russia, for instance, the P/H pseudogene identified temporal clusters rather than geographical clusters, demonstrating the circulation and importation of independent variants rather than large local outbreaks lasting for several years, as suggested by NP-HVR. Thus, by extending the sequencing window for molecular epidemiology, a more refined picture of MV circulation was obtained with more clearly defined links between outbreaks and transmission chains. Our results also suggested that in contrast to the P gene, the H gene acquired fixed substitutions that continued to be found in subsequent outbreaks, possibly with consequences for its antigenicity. Thus, a longer sequencing window has true benefits both for the epidemiological surveillance of measles and for the better monitoring of viral evolution.

Measles virus (MV) vaccine effectively protects seronegative individuals against infection. However, inhibition of vaccine-induced seroconversion by maternal antibodies after vaccination remains a problem, as it leaves infants susceptible to MV infection. In cotton rats, passive transfer of MV-specific IgG mimics maternal antibodies and inhibits vaccine-induced seroconversion. Here, we report that immunization in the presence of passively transferred IgG inhibits the secretion of neutralizing antibodies but not the generation of MV-specific B cells. This finding suggested that MV-specific B cells require an additional stimulus to mature into antibody-secreting plasma cells. In order to provide such a stimulus, we generated a recombinant Newcastle disease virus (NDV) expressing the MV hemagglutinin (NDV-H). In contrast to MV, NDV-H induced high levels of type I interferon in plasmacytoid dendritic cells and in lung tissue. In cotton rats immunized with NDV-H, neutralizing antibodies were also generated in the presence of passively transferred antibodies. In the latter case, however, the level and kinetics of antibody generation were reduced. In vitro, alpha interferon stimulated the activation of MV-specific B cells from MV-immune spleen cells. NDV infection (which induces alpha interferon) had the same effect, and stimulation could be abrogated by antibodies neutralizing alpha interferon, but not interleukin 6 (IL-6). In vivo, coapplication of UV-inactivated MV with NDV led to increased MV-specific antibody production in the presence and absence of passively transferred antibodies. These data indicate that MV-specific B cells are being generated after immunization in the presence of maternal antibodies and that the provision of alpha interferon as an additional signal leads to antibody secretion.

The hemagglutinin (HA) surface glycoprotein promotes influenza virus entry and is the key protective antigen in natural immunity and vaccines. The HA protein is a trimeric envelope glycoprotein consisting of a globular receptor-binding domain (HA-RBD) that is inserted into a membrane fusion-mediating stalk domain. Similar to other class I viral fusion proteins, the fusogenic stalk domain spontaneously refolds into its postfusion conformation when expressed in isolation, consistent with this domain being trapped in a metastable conformation. Using X-ray crystallography, we show that the influenza virus HA-RBD refolds spontaneously into its native, immunogenic structure even when expressed in an unglycosylated form in Escherichia coli. In the 2.10-Å structure of the HA-RBD, the receptor-binding pocket is intact and its conformational epitopes are preserved. Recombinant HA-RBD is immunogenic and protective in ferrets, and the protein also binds with specificity to sera from influenza virus-infected humans. Overall, the data provide a structural basis for the rapid production of influenza vaccines in E. coli. From an evolutionary standpoint, the ability of the HA-RBD to refold spontaneously into its native conformation suggests that influenza virus acquired this domain as an insertion into an ancestral membrane-fusion domain. The insertion of independently folding domains into fusogenic stalk domains may be a common feature of class I viral fusion proteins.

Measles virus (MV) entry requires at least 2 viral proteins, the hemagglutinin (H) and fusion (F) proteins. We describe the rescue and characterization of a measles virus with a specific mutation in the stalk region of H (I98A) that is able to bind normally to cells but infects at a lower rate than the wild type due to a reduction in fusion triggering. The mutant H protein binds to F more avidly than the parent H protein does, and the corresponding virus is more sensitive to inhibition by fusion-inhibitory peptide. We show that after binding of MV to its receptor, H-F dissociation is required for productive infection.

An enzyme-linked immunosorbent assay (ELISA) for the detection of IgG antibodies against the pandemic H1N1 2009 influenza A virus, employing a recombinant hemagglutinin protein of the virus, was compared to the hemagglutination inhibition (HI) test using 783 serum samples. The results showed a concordance of 98.4%, suggesting the utility of the ELISA in serosurveillance. Two hundred sixty-nine (100%) serum samples with an HI titer of > or =20 were ELISA reactive.

Morbillivirus cell entry is controlled by hemagglutinin (H), an envelope-anchored viral glycoprotein determining interaction with multiple host cell surface receptors. Subsequent to virus-receptor attachment, H is thought to transduce a signal triggering the viral fusion glycoprotein, which in turn drives virus-cell fusion activity. Cell entry through the universal morbillivirus receptor CD150/SLAM was reported to depend on two nearby microdomains located within the hemagglutinin. Here, we provide evidence that three key residues in the virulent canine distemper virus A75/17 H protein (Y525, D526, and R529), clustering at the rim of a large recessed groove created by beta-propeller blades 4 and 5, control SLAM-binding activity without drastically modulating protein surface expression or SLAM-independent F triggering.

The members of Betacoronavirus phylocluster A possess two types of surface projections, one comprised of the spike protein (S) and the other of hemagglutinin-esterase (HE). Purportedly, these viruses bind to O-acetylated sialic acids (O-Ac-Sias) primarily through S, with HE serving merely as receptor-destroying enzyme. Here, we show that, in apparent contrast to human and ungulate host range variants of Betacoronavirus-1, murine coronaviruses actually bind to O-Ac-Sias via HE exclusively. Apparently, expansion of group A betacoronaviruses into new hosts and niches was accompanied by changes in HE ligand and substrate preference and in the roles of HE and S in Sia receptor usage.

The continuous circulation of the highly pathogenic avian influenza (HPAI) H5N1 virus has been a cause of great concern. The possibility of this virus acquiring specificity for the human influenza A virus receptor, alpha2,6-linked sialic acids (SA), and being able to transmit efficiently among humans is a constant threat to human health. Different studies have described amino acid substitutions in hemagglutinin (HA) of clinical HPAI H5N1 isolates or that were introduced experimentally that resulted in an increased, but not exclusive, binding of these virus strains to alpha2,6-linked SA. We introduced all previously described amino acid substitutions and combinations thereof into a single genetic background, influenza virus A/Indonesia/5/05 HA, and tested the receptor specificity of these 27 mutant viruses. The attachment pattern to ferret and human tissues of the upper and lower respiratory tract of viruses with alpha2,6-linked SA receptor preference was then determined and compared to the attachment pattern of a human influenza A virus (H3N2). At least three mutant viruses showed an attachment pattern to the human respiratory tract similar to that of the human H3N2 virus. Next, the replication efficiencies of these mutant viruses and the effects of three different neuraminidases on virus replication were determined. These data show that influenza virus A/Indonesia/5/05 potentially requires only a single amino acid substitution to acquire human receptor specificity, while at the same time remaining replication competent, thus suggesting that the pandemic threat posed by HPAI H5N1 is far from diminished.

Influenza epidemics arise through the accumulation of viral genetic changes, culminating in a novel antigenic type that is able to escape host immunity. Following an outbreak of the A/Fujian/411/2002-like strains in Asia, including China, Japan, and South Korea, in 2002, Australia and New Zealand experienced substantial outbreaks of the same strains in 2003, and subsequently worldwide outbreaks occurred in the 2003-2004 season. The emergence of A/Fujian/411/2002-like strains coincided with a higher level of influenza-like illness in South Korea than what is seen at the peak of a normal season, and there was at least a year's difference between South Korea and the United States. Genetic evolution of human influenza A/H3N2 viruses was monitored by sequence analysis of hemagglutinin (HA) genes collected in Asia, including 269 (164 new) HA genes isolated in South Korea from 1999 to 2007. The Fujian-like influenza strains were disseminated with rapid sequence variation across the antigenic sites of the HA1 domain, which sharply distinguished between the A/Moscow/10/1999-like and A/Fujian/411/2002-like strains. This fast variation, equivalent to approximately 10 amino acid changes within a year, occurred in Asia and would be the main cause of the disappearance of the reassortants, although the reassortant and nonreassortant Fujian-like strains circulated simultaneously in Asia.

A new class of N-(1-thia-4-azaspiro[4.5]decan-4-yl)carboxamide inhibitors of influenza virus hemagglutinin (HA)-mediated membrane fusion that has a narrow and defined structure-activity relationship was identified. In Madin-Darby canine kidney (MDCK) cells infected with different strains of human influenza virus A/H3N2, the lead compound, 4c, displayed a 50% effective concentration of 3 to 23 muM and an antiviral selectivity index of 10. No activity was observed for A/H1N1, A/H5N1, A/H7N2, and B viruses. The activity of 4c was reduced considerably when added 30 min or later postinfection, indicating that 4c inhibits an early step in virus replication. 4c and its congeners inhibited influenza A/H3N2 virus-induced erythrocyte hemolysis at low pH. 4c-resistant virus mutants, selected in MDCK cells, contained either a single D112N change in the HA2 subunit of the viral HA or a combination of three substitutions, i.e., R220S (in HA1) and E57K (in HA2) and an A-T substitution at position 43 or 96 of HA2. The mutants showed efficiency for receptor binding and replication similar to that of wild-type virus yet displayed an increased pH of erythrocyte hemolysis. In polykaryon assays with cells expressing single-mutant HA proteins, the E57K, A96T, and D112N mutations resulted in 4c resistance, and the HA proteins containing R220S, A96T, and D112N mutations displayed an increased fusion pH. Molecular modeling identified a binding cavity for 4c involving arginine-54 and glutamic acid-57 in the HA2 subunit. Our studies with the new fusion inhibitor 4c confirm the importance of this HA region in the development of influenza virus fusion inhibitors.

A new oligochromatographic assay, Speed-Oligo Novel Influenza A H1N1, was designed and optimized for the specific detection of the 2009 influenza A H1N1 virus. The assay is based on a PCR method coupled to detection of PCR products by means of a dipstick device. The target sequence is a 103-bp fragment within the hemagglutinin gene. The analytical sensitivity of the new assay was measured with serial dilutions of a plasmid that contained the target sequence, and we determined that down to one copy per reaction of the plasmid was reliably detected. Diagnostic performance was assessed with 103 RNAs from suspected cases (40 positive and 63 negative results) previously analyzed with a reference real-time PCR technique. All positive cases were confirmed, and no false-positive results were detected with the new assay. No cross-reactions were observed when other viral strains or clinical samples with other respiratory viruses were tested. According to these results, this new assay has 100% sensitivity and specificity. The turnaround time for the whole procedure was 140 min. The assay may be especially useful for the specific detection of 2009 H1N1 virus in laboratories not equipped with real-time PCR instruments.

Influenza viruses attach to cells via a sialic acid moiety (sialic acid receptor) that is alpha2-3 linked or alpha2-6 linked to galactose (alpha2-3SAL or alpha2-6SAL); sialic acid acts as a receptor for the virus. Using lectin staining, we demonstrated that the alpha2-6SAL configuration is predominant in the respiratory tract of ferrets, including trachea, bronchus, and lung alveolus tissues. Recombinant wild-type (rWT) influenza A/Solomon Island/3/06 (SI06) (H1N1) viruses were constructed to assess the impact of the hemagglutinin (HA) variations (amino acids 190 or 226) identified in natural variants on virus replication in the upper and lower respiratory tract of ferrets, as well as virus antigenicity and immunogenicity. A single amino acid change at residue 226 (from Gln to Arg) in the HA of SI06 resulted in the complete loss of binding to alpha2-6SAL and a concomitant loss of the virus's ability to replicate in the lower respiratory tract of ferrets. In contrast, the virus with Gln226 in the HA protein has a receptor binding preference for alpha2-6SAL and replicates efficiently in the lungs. There was a good correlation between viral replication in the lungs of ferrets and disease symptoms. In addition, we also showed that the 190 and 226 residues affected viral antigenicity and immunogenicity. Our data emphasize the necessity of thoroughly assessing wild-type influenza viruses for their suitability as reference strains and for carefully selecting the HA antigen for vaccine production during annual influenza vaccine evaluation processes.

The patterns and dynamics of evolution in acutely infecting viruses within individual hosts are largely unknown. To this end, we investigated the intrahost variation of canine influenza virus (CIV) during the course of experimental infections in naïve and partially immune dogs and in naturally infected dogs. Tracing sequence diversity in the gene encoding domain 1 of the hemagglutinin (HA1) protein over the time course of infection provided information on the patterns and processes of intrahost viral evolution and revealed some of the effects of partial host immunity. Viral populations sampled on any given day were generally characterized by mean pairwise genetic diversities between 0.1 and 0.2% and by mutational spectra that changed considerably on different days. Some observed mutations may have affected antigenicity or host range, including reversions of CIV host-associated mutations. Patterns of sequence diversity differed between naïve and vaccinated dogs, with some presumably antigenic mutations transiently reaching high frequency in the latter. CIV populations are therefore characterized by the rapid generation and clearance of genetic diversity. Potentially advantageous mutations arise readily during the course of single infections and may give rise to antigenic escape or host range variants.

Measles remains a major cause of child mortality, in part due to an inability to vaccinate young infants with the current live attenuated virus vaccine (LAV). To explore new approaches to infant vaccination, chimeric Venezuelan equine encephalitis/Sindbis virus (VEE/SIN) replicon particles were used to express the hemagglutinin (H) and fusion (F) proteins of measles virus (MV). Juvenile rhesus macaques vaccinated intradermally with a single dose of VEE/SIN expressing H or H and F proteins (VEE/SIN-H or VEE/SIN-H+F, respectively) developed high titers of MV-specific neutralizing antibody and gamma-interferon (IFN-gamma)-producing T cells. Infant macaques vaccinated with two doses of VEE/SIN-H+F also developed neutralizing antibody and IFN-gamma-producing T cells. Control animals were vaccinated with LAV or with a formalin-inactivated measles vaccine (FIMV). Neutralizing antibody remained above the protective level for more than 1 year after vaccination with VEE/SIN-H, VEE/SIN-H+F, or LAV. When challenged with wild-type MV 12 to 17 months after vaccination, all vaccinated juvenile and infant monkeys vaccinated with VEE/SIN-H, VEE/SIN-H+F, and LAV were protected from rash and viremia, while FIMV-vaccinated monkeys were not. Antibody was boosted by challenge in all groups. T-cell responses to challenge were biphasic, with peaks at 7 to 25 days and at 90 to 110 days in all groups, except for the LAV group. Recrudescent T-cell activity coincided with the presence of MV RNA in peripheral blood mononuclear cells. We conclude that VEE/SIN expressing H or H and F induces durable immune responses that protect from measles and offers a promising new approach for measles vaccination. The viral and immunological factors associated with long-term control of MV replication require further investigation.

In this study, we show that the highly pathogenic H5N1 avian influenza virus (AIV) (A/crow/Kyoto/53/04 and A/chicken/Egypt/CL6/07) induced apoptosis in duck embryonic fibroblasts (DEF). In contrast, apoptosis was reduced among cells infected with low-pathogenic AIVs (A/duck/HK/342/78 [H5N2], A/duck/HK/820/80 [H5N3], A/wigeon/Osaka/1/01 [H7N7], and A/turkey/Wisconsin/1/66 [H9N2]). Thus, we investigated the molecular mechanisms of apoptosis induced by H5N1-AIV infection. Caspase-dependent and -independent pathways contributed to the cytopathic effects. We further showed that, in the induction of apoptosis, the hemagglutinin of H5N1-AIV played a major role and its cleavage sequence was not critical. We also observed outer membrane permeabilization and loss of the transmembrane potential of the mitochondria of infected DEF, indicating that mitochondrial dysfunction was caused by the H5N1-AIV infection. We then analyzed Ca(2+) dynamics in the infected cells and demonstrated an increase in the concentration of Ca(2+) in the cytosol ([Ca(2+)](i)) and mitochondria ([Ca(2+)](m)) after H5N1-AIV infection. Regardless, gene expression important for regulating Ca(2+) efflux from the endoplasmic reticulum did not significantly change after H5N1-AIV infection. These results suggest that extracellular Ca(2+) may enter H5N1-AIV-infected cells. Indeed, EGTA, which chelates extracellular free Ca(2+), significantly reduced the [Ca(2+)](i), [Ca(2+)](m), and apoptosis induced by H5N1-AIV infection. In conclusion, we identified a novel mechanism for influenza A virus-mediated cell death, which involved the acceleration of extracellular Ca(2+) influx, leading to mitochondrial dysfunction and apoptosis. These findings may be useful for understanding the pathogenesis of H5N1-AIV in avian species as well as the impact of Ca(2+) homeostasis on influenza A virus infection.

This paper reports on the development of a one-step, real-time reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay targeting the hemagglutinin (HA) gene for the rapid molecular-based detection of pandemic (H1N1) 2009 virus. The detection limit of the pandemic (H1N1) 2009 virus HA-specific RT-LAMP assay was same as that of the currently used real-time reverse transcription-PCR method. The assay detected the pandemic (H1N1) 2009 virus HA gene in 136 RNA samples extracted from nasopharyngeal swab specimens from Japanese and Vietnamese patients. No cross-reactive amplification with the RNA of other seasonal influenza viruses was observed, and the detection of specific viral genome targets in clinical specimens was achieved in less than 40 min. The sensitivity and specificity of the pandemic (H1N1) 2009 virus HA-specific RT-LAMP assay obtained in this study were 97.8% and 100%, respectively. Use of the (H1N1) 2009 virus HA-specific RT-LAMP assay will enable the faster and easier diagnosis of pandemic (H1N1) 2009 virus infection, especially in resource-limited situations in developing countries.

Attenuated live vaccines of measles virus (MV) have been developed from clinical isolates by serial propagation in heterologous cells, mainly chicken embryonic cells. The safety and effectiveness of these vaccines have been well established. However, the molecular mechanism of their attenuation remains a subject of investigation. The CAM-70 MV vaccine strain was developed from the Tanabe strain by serial propagation in chicken embryonic cells. In the present study, we assessed the contribution of each gene in the CAM-70 strain to efficient growth in chicken embryonic fibroblasts (CEF). We used a cloned MV IC323 based on the wild-type IC-B strain and generated a series of IC323s that possess one or more of the CAM-70 genes. Then, we examined the infection of CEF and CEF expressing human signaling lymphocyte activation molecule with the recombinant MVs. Our results demonstrated that MV needs to adapt to CEF at both the entry and postentry steps and that the CAM-70 matrix protein gene plays an important role in adaptation to CEF at the early stage of the virus replication cycle. The CAM-70 large protein gene was responsible for the efficient transcription and replication in CEF, and the CAM-70 hemagglutinin and fusion protein genes were responsible for efficient entry. Investigations focusing on these genes might elucidate unknown molecular mechanisms underlying the attenuation of MV.