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Objective: To investigate the variations on hemagglutinin (H) gene of measles virus (MV) in Zhejiang province, and to analyze the differences with strains circulated both at home and abroad., Methods: In total, 33 MV strains isolated in Zhejiang province between 1999 and 2011 were collected.RNA of the isolated MV strains was extracted and the complete sequences on H gene were amplified using RT-PCR assay. The products were compared with the Chinese vaccine strain Shanghai-191, which was downloaded from GenBank, and other 95 different MV strains from all over the world., Results: 33 MV strains, isolated from the throat swab specimens collected from MV patients in Zhejiang province during 1999 to 2001, were used to conduct phylogenetic analysis with MV strains circulated in other areas of China during 1993 to 2007. The phylogenetic tree based on H gene sequences showed that all the Zhejiang MV strains located in H1a cluster, and no apparent time series and geographic restrictions were observed. Compared to the referenced vaccine strain Shanghai-191, the average variation rate on nucleotides and amino acids, and the evolutionary rate of H1a viruses from China during 2003 to 2011 were separately 5.15%, 4.44% and 5.81%, which were higher than the rates of H1a viruses during 1965 to 1993 (4.75%, 3.86% and 5.30%), and the rates of viruses during 1994 to 2002 (4.80%, 4.08% and 5.37%).However, the dn/ds ratios of strains within the three time periods were 0.19,0.21 and 0.23 respectively, which indicated that no evidence of positive selection was found on H1a MV strains during 1993 to 2011. A 24 stable amino acid variation sites on H gene was found between H1a viruses during 2003 to 2011 and the vaccine strain Shanghai-191. The largest variation occurred between vaccine and H1a strains, with 0.053 of the p-distance and 26-28 of amino acid mutations.However, only 15 stable amino acid variations were found between vaccine strain and genotype B3 or D4 strains.In addition, significant differences were found between H1a viruses and genotype B or D viruses, with 0.074 and 0.071 of p-distance and 27-33 of amino acid differences., Conclusion: Significant differences were found on H gene between MV strains subtype H1a and vaccine strains and other genotype strains. The variations were enlarged with the time coursing; therefore, the surveillance on variation of Chinese MV strains should be taken into account.

Background: There are little data about adverse effects and immunogenicity of flu vaccine in Asian pregnant women., Methods: This prospective trial (NCT01514708) enrolled 46 pregnant women who received a single intramuscular dose of trivalent flu vaccine (AdimFlu-S®) containing 15 mcg of hemagglutinin for each strain/0.5 mL from influenza A (H1N1), influenza A (H3N2), and influenza B after the first trimester. Blood samples were collected at day 0 and 28 after vaccination, and at delivery. Cord blood was also collected. Hemagglutination inhibition (HAI) assays were performed to determine seroprotection and seroconversion rates and fold increase in the HAI geometric mean titer (GMT)., Results: Twenty-eight days after vaccination the seroprotection rate against H1N1, H3N2, and influenza B was 91.3%, 84.8% and 56.5%, respectively. The GMT fold increase was 12.8, 8.4, and 4.6 for H1N1, H3N2, and influenza B, respectively. At delivery, both the seroprotection rate (86.4%, 68.2%, and 47.7%) and GMT fold increase (9.4, 5.7 and 3.8) were slightly lower than day 28. The seroprotection rate and GMT fold increase in maternal and cord blood samples were comparable. No significant adverse effects were detected., Conclusions: Trivalent flu vaccine induces a strong immune response in pregnant women and their infants without adverse effects., Trial Registration: Clinical Trials. gov NCT01514708.

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.

Canine distemper virus (CDV; Paramyxoviridae, Morbillivirus) is the etiologic agent of a multisystemic infectious disease affecting all terrestrial carnivore families with high incidence and mortality in domestic dogs. Sequence analysis of the hemagglutinin (H) gene has been widely employed to characterize field strains, permitting the identification of nine CDV lineages worldwide. Recently, it has been established that the sequences of the fusion protein signal-peptide (Fsp) coding region are extremely variable, suggesting that analysis of its sequence might be useful for strain characterization studies. However, the divergence of Fsp sequences among worldwide strains and its phylogenetic resolution has not yet been evaluated. We constructed datasets containing the Fsp-coding region and H gene sequences of the same strains belonging to eight CDV lineages. Both datasets were used to evaluate their phylogenetic resolution. The phylogenetic analysis revealed that both datasets clustered the same strains into eight different branches, corresponding to CDV lineages. The inter-lineage amino acid divergence was fourfold greater for the Fsp peptide than for the H protein. The likelihood mapping revealed that both datasets display strong phylogenetic signals in the region of well-resolved topologies. These features indicate that Fsp-coding region sequence analysis is suitable for evolutionary studies as it allows for straightforward identification of CDV lineages.

One H5N8 and three H5N5 highly pathogenic avian influenza (HPAI) viruses which derived their HA genes from the Asian H5N1 lineage were isolated from poultry during 2009-2010 in mainland China. Pathogenicity studies showed that these viruses were all highly virulent to chickens, while they varied from moderate to high virulence in mice and from mild to intermediate virulence in mallards. Phylogenetic analyses showed that these viruses were reassortants bearing the H5N1 backbone while acquiring PB1, NP and NA genes from unidentified non-H5N1 viruses, and had developed into three distinct genotypes (B-D). Molecular characterization indicated that all these viruses might resist to antiviral agents. Our findings highlight the emergence and development of HPAI H5 viruses of other NA subtypes in H5N1 endemic areas and their potential threat to poultry industry and public health., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Annual H3N2 subtype influenza outbreaks in Guangdong, China are a severe public health issue and require ongoing monitoring of emerging viral variants. The variation and evolution of haemagglutinin (HA) and neuraminidase (NA) genes of influenza isolates from Guangdong during 2007-2011 and others from GenBank were analysed using Lasergene 7.1 and MEGA 5.05, and serological analysis of antigens was determined by haemagglutination inhibition (HI). Susceptibility to antiviral drugs was correlated with genetic mutations. Phylogenetic analysis and alignment of HA and NA genes were performed on 18 Guangdong isolates and 26 global reference strains. The non-synonymous (dN) evolutionary rate of HA1 was 3.13 times that of HA2. Compared with the A/Perth/16/2009 vaccine HA gene, homologies of Guangdong isolates were between 98.8-99.7% and 98.0-98·4% in 2009 and 2010, respectively. Amino-acid substitutions were found in five epitopes of HA1 from Guangdong isolates between 2007 and 2011, especially in epitopes B (N160K) and D (K174R/N). The K189E/N/Q and T228A mutations in the receptor-binding site (RBS) occurred in the 2010 strains, which affected the antigenicity of HA1. The antigenicity of the epidemic H3N2 isolates in 2010 was somewhat different from that of A/Perth/16/2009. The Guangdong H3N2 isolates were determined to be oseltamivir-resistant with IC50 of 0.396 ± 0.085 nmol/l (n=17) and zanamivir-resistant with IC50 of 0.477 ± 0.149 nmol/l (n=18). Variations were present in epitopes B and D, two sites in the RBS and two glycosylation sites in the Guangdong H3N2 HA1 gene. The majority of the Guangdong H3N2 isolates were sensitive to oseltamivir and zanamivir. Compared to the World Health Organization 2012 vaccine strains, Guangdong H3N2 strains varied genetically and antigenically to some degree.

Objective: To investigate the epidemiological features, genetic drift in the epitopes of hemagglutinin (HA) of the novel influenza A (H1N1) virus and oseltamivir-resistant variants characterized by H275Y and N295S mutations in children in Shanghai since the outbreak., Method: Between June 2009 and May 2012, a prospective surveillance study was carried out in Shanghainese children who attended the outpatient clinic of Children's Hospital of Fudan University for influenza-like illness. One-step real-time fluorescence quantitative RT-PCR was performed to detect seasonal influenza A and influenza B virus and the novel influenza A (H1N1) virus in the respiratory samples. Genetic drift from the vaccine strain in HA epitopes of the novel influenza H1N1 virus and the molecular markers associated with oseltamivir resistance in neuraminidase (NA) were analyzed., Result: Out of 3475 enrolled cases, the novel influenza A (H1N1) virus was confirmed virologically in 222 (6.4%) otherwise healthy children with 133 (59.9%) being boys and 89 (40.1%) girls. The median ages of children with the novel influenza A (H1N1) virus infection during the first wave from August 2009 to February 2010 and the second wave from December 2010 to February 2011 were 53.5 months and 32.0 months, respectively (Z = -4.601, P = 0.000); 119 (46.9%) had the close contact with persons suffering from fever or respiratory infection, of whom, 68 (57.1%) contacts were family members and 47 (39.5%) contacts were classmates. During the outbreak in 2009-2010 season, 66 (40.9%) were exposed to primary index cases, school students were the major exposure subjects, accounting for 50.0%. The nucleotide sequences of HA1 gene were highly homologous between the vaccine strain A/California/07/2009 and Shanghai circulating novel influenza A (H1N1) strains and only S83P mutation in epitope E of HA was detected inclusively in the circulating strains. The H275Y and N295S amino acid mutations associated with oseltamivir resistance were not found in the circulating novel influenza (H1N1) strains., Conclusion: Two major waves of the novel influenza A (H1N1) outbreaks occurred in Shanghainese children during 2009-2011. Institutional children were the major affected individuals during the 2009 pandemic wave. Households and schools were the main sites of transmission among children during influenza pandemic. Influenza vaccination should be enhanced in children and their close family contacts. The novel influenza A (H1N1) virus in Shanghai has not undergone significant genetic changes. Oseltamivir is effective for the treatment of the novel influenza A (H1N1) virus.

Background: Canine distemper virus (CDV) infects a variety of carnivores, including wild and domestic Canidae. In this study, we sequenced and phylogenetic analyses of the hemagglutinin (H) genes from eight canine distemper virus (CDV) isolates obtained from seven raccoon dogs (Nyctereutes procyonoides) and a giant panda (Ailuropoda melanoleuca) in China., Results: Phylogenetic analysis of the partial hemagglutinin gene sequences showed close clustering for geographic lineages, clearly distinct from vaccine strains and other wild-type foreign CDV strains, all the CDV strains were characterized as Asia-1 genotype and were highly similar to each other (91.5-99.8% nt and 94.4-99.8% aa). The giant panda and raccoon dogs all were 549Y on the HA protein in this study, irrespective of the host species., Conclusions: These findings enhance our knowledge of the genetic characteristics of Chinese CDV isolates, and may facilitate the development of effective strategies for monitoring and controlling CDV for wild canids and non-canids in China.

Measles virus (MV), an enveloped RNA virus belonging to the Paramyxoviridae family, enters the cell through membrane fusion mediated by two viral envelope proteins, an attachment protein hemagglutinin (H) and a fusion (F) protein. The crystal structure of the receptor-binding head domain of MV-H bound to its cellular receptor revealed that the MV-H head domain forms a tetrameric assembly (dimer of dimers), which occurs in two forms (forms I and II). In this study, we show that mutations in the putative dimer-dimer interface of the head domain in either form inhibit the ability of MV-H to support membrane fusion, without greatly affecting its cell surface expression, receptor binding, and interaction with the F protein. Notably, some anti-MV-H neutralizing monoclonal antibodies are directed to the region around the dimer-dimer interface in form I rather than receptor-binding sites. These observations suggest that the dimer-dimer interactions of the MV-H head domain, especially that in form I, contribute to triggering membrane fusion, and that conformational shift of head domain tetramers plays a role in the process. Furthermore, our results indicate that although the stalk and transmembrane regions may be mainly responsible for the tetramer formation of MV-H, the head domain alone can form tetramers, albeit at a low efficiency.

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.

We investigate the causes of site-specific evolutionary-rate variation in influenza haemagglutinin (HA) between human and avian influenza, for subtypes H1, H3, and H5. By calculating the evolutionary-rate ratio, ω = dN/dS as a function of a residue's solvent accessibility in the three-dimensional protein structure, we show that solvent accessibility has a significant but relatively modest effect on site-specific rate variation. By comparing rates within HA subtypes among host species, we derive an upper limit to the amount of variation that can be explained by structural constraints of any kind. Protein structure explains only 20-40% of the variation in ω. Finally, by comparing ω at sites near the sialic-acid-binding region to ω at other sites, we show that ω near the sialic-acid-binding region is significantly elevated in both human and avian influenza, with the exception of avian H5. We conclude that protein structure, HA subtype, and host biology all impose distinct selection pressures on sites in influenza HA.

The most salient feature of influenza evolution in humans is its antigenic drift. This process is characterized by structural changes in the virus's B-cell epitopes and ultimately results in the ability of the virus to evade immune recognition and thereby reinfect previously infected hosts. Until recently, amino acid substitutions in epitope regions of the viral haemagglutinin were thought to be positively selected for their ability to reduce antibody binding and therefore were thought to be responsible for driving antigenic drift. However, a recent hypothesis put forward by Hensley and co-workers posits that cellular receptor binding avidity is the dominant phenotype under selection, with antigenic drift being a side effect of these binding avidity changes. Here, we present a mathematical formulation of this new antigenic drift model and use it to show how rates of antigenic drift depend on epidemiological parameters. We further use the model to evaluate how two different vaccination strategies can impact antigenic drift rates and ultimately disease incidence levels. Finally, we discuss the assumptions present in the model formulation, predictions of the model, and future work that needs to be done to determine the consistency of this hypothesis with known patterns of influenza's genetic and antigenic evolution.

Few questions on infectious disease are more important than understanding how and why avian influenza A viruses successfully emerge in mammalian populations, yet little is known about the rate and nature of the virus' genetic adaptation in new hosts. Here, we measure, for the first time, the genomic rate of adaptive evolution of swine influenza viruses (SwIV) that originated in birds. By using a curated dataset of more than 24 000 human and swine influenza gene sequences, including 41 newly characterized genomes, we reconstructed the adaptive dynamics of three major SwIV lineages (Eurasian, EA; classical swine, CS; triple reassortant, TR). We found that, following the transfer of the EA lineage from birds to swine in the late 1970s, EA virus genes have undergone substantially faster adaptive evolution than those of the CS lineage, which had circulated among swine for decades. Further, the adaptation rates of the EA lineage antigenic haemagglutinin and neuraminidase genes were unexpectedly high and similar to those observed in human influenza A. We show that the successful establishment of avian influenza viruses in swine is associated with raised adaptive evolution across the entire genome for many years after zoonosis, reflecting the contribution of multiple mutations to the coordinated optimization of viral fitness in a new environment. This dynamics is replicated independently in the polymerase genes of the TR lineage, which established in swine following separate transmission from non-swine hosts.

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.

Of the Orthomyxoviridae family of viruses, only influenza A viruses are thought to exist as multiple subtypes and has non-human maintenance hosts. In April 2011, nasal swabs were collected for virus isolation from pigs exhibiting influenza-like illness. Subsequent electron microscopic, biochemical, and genetic studies identified an orthomyxovirus with seven RNA segments exhibiting approximately 50% overall amino acid identity to human influenza C virus. Based on its genetic organizational similarities to influenza C viruses this virus has been provisionally designated C/Oklahoma/1334/2011 (C/OK). Phylogenetic analysis of the predicted viral proteins found that the divergence between C/OK and human influenza C viruses was similar to that observed between influenza A and B viruses. No cross reactivity was observed between C/OK and human influenza C viruses using hemagglutination inhibition (HI) assays. Additionally, screening of pig and human serum samples found that 9.5% and 1.3%, respectively, of individuals had measurable HI antibody titers to C/OK virus. C/OK virus was able to infect both ferrets and pigs and transmit to naive animals by direct contact. Cell culture studies showed that C/OK virus displayed a broader cellular tropism than a human influenza C virus. The observed difference in cellular tropism was further supported by structural analysis showing that hemagglutinin esterase (HE) proteins between two viruses have conserved enzymatic but divergent receptor-binding sites. These results suggest that C/OK virus represents a new subtype of influenza C viruses that currently circulates in pigs that has not been recognized previously. The presence of multiple subtypes of co-circulating influenza C viruses raises the possibility of reassortment and antigenic shift as mechanisms of influenza C virus evolution.

The association between a particular mutation in the HA1 subunit of the influenza virus haemagglutinin, D222G, and severe and fatal disease in cases of influenza A(H1N1)pdm09 in Norway during the 2009 pandemic was investigated using pyrosequencing. The prevalence of the variant among fatal cases was 8/26 and among severe non-fatal cases 5/52. No D222G mutations were found among the 381 mild cases. This difference could not be attributed to sampling differences, such as body location of sampling, or duration of illness. In cases with mutant virus where clinical specimens from different days of illness were available, transition from wild-type to mutant virus was commonly observed (4/5), indicating that the mutant virus emerged sporadically in individual patients. In patients with paired samples from both the upper and lower respiratory tract (n=8), the same viral genotypes were detected in both locations. In most of the D222G cases (11/13), the mutant virus was found as a quasispecies.

A replicon expression system based on the salmonid alphavirus (SAV) that encodes the infectious salmon anemia virus (ISAV) hemagglutinin-esterase (HE) was constructed and found to be an efficacious vaccine against infectious salmon anemia (ISA). Following a single intramuscular immunization, Atlantic salmon (Salmo salar) were effectively protected against subsequent ISAV challenge. Additional replicons coding for the ISAV fusion glycoprotein (F) or the ISAV matrix protein (M) were created and tested in combination with the replicon that encodes the HE. The ISAV HE was confirmed as a potent antigen, but neither the F nor the M proteins were found to be essential for immunization-induced protection. Innate immune response induced at the site of vaccination illustrated the immunogenicity of the SAV-based replicon and its ability to activate antiviral responses in Atlantic salmon. The successful testing of the SAV-based replicon as a vaccine model against ISA showed that the replicon approach may represent a novel immunization technology for the aquaculture industry. It offers potential benefits in terms of safety, efficacy, flexibility, and vaccine production complexity., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The measles virus (MV) is serologically monotypic. Life-long immunity is conferred by a single attack of measles or following vaccination with the MV vaccine. This is contrary to viruses such as influenza, which readily develop resistance to the immune system and recur. A better understanding of factors that restrain MV to one serotype may allow us to predict if MV will remain monotypic in the future and influence the design of novel MV vaccines and therapeutics. MV hemagglutinin (H) glycoprotein, binds to cellular receptors and subsequently triggers the fusion (F) glycoprotein to fuse the virus into the cell. H is also the major target for neutralizing antibodies. To explore if MV remains monotypic due to a lack of plasticity of the H glycoprotein, we used the technology of Immune Dampening to generate viruses with rationally designed N-linked glycosylation sites and mutations in different epitopes and screened for viruses that escaped monoclonal antibodies (mAbs). We then combined rationally designed mutations with naturally selected mutations to generate a virus resistant to a cocktail of neutralizing mAbs targeting four different epitopes simultaneously. Two epitopes were protected by engineered N-linked glycosylations and two epitopes acquired escape mutations via two consecutive rounds of artificial selection in the presence of mAbs. Three of these epitopes were targeted by mAbs known to interfere with receptor binding. Results demonstrate that, within the epitopes analyzed, H can tolerate mutations in different residues and additional N-linked glycosylations to escape mAbs. Understanding the degree of change that H can tolerate is important as we follow its evolution in a host whose immunity is vaccine induced by genotype A strains instead of multiple genetically distinct wild-type MVs.

Lentiviral vectors are vectors of choice for many gene therapy applications. Recently, efficient targeting of lentiviral vectors pseudotyped with the Measles virus (MV) glycoproteins has been reported. However, MV antibodies in patients might limit the clinical use of these vectors. We demonstrate here that lentiviral vectors can also be pseudotyped with the glycoproteins of Tupaia paramyxovirus (TPMV), the hemagglutinin (H) and fusion (F) protein. As this animal paramyxovirus has no known close relatives in humans, we do not expect TPMV antibodies in patients. Because TPMV normally does not infect human cells, 'detargeting' from natural receptors is unnecessary. Similar to the MV system, TPMV glycoproteins can mediate targeted cell entry by displaying different single-chain antibodies (scAb) directed against surface molecules on target cells on the viral hemagglutinin. We generated a panel of H and F proteins with truncated cytoplasmic tails and determined the variants that efficiently pseudotyped lentiviral vectors. The B-cell marker CD20 was used as a model antigen, and CD20-targeted TPMV vectors selectively transduced CD20-positive cells, including quiescent primary human B-cells. Lentiviral vectors pseudotyped with targeted TPMV envelope proteins might be a valuable vector choice when systemic application of targeted lentiviral vectors in humans is required.

Influenza A (H3N2) offers a well-studied, yet not fully understood, disease in terms of the interactions between pathogen population dynamics, epidemiology and genetics. A major open question is why the virus population is globally dominated by a single and very recently diverged (2-8 years) lineage. Classically, this has been modeled by limiting the generation of new successful antigenic variants, such that only a small subset of progeny acquire the necessary mutations to evade host immunity. An alternative approach was recently suggested by Recker et al. in which a limited number of antigenic variants are continuously generated, but most of these are suppressed by pre-existing host population immunity. Here we develop a framework spanning the regimes described above to explore the impact of rates of mutation and levels of competition on phylodynamic patterns. We find that the evolutionary dynamics of the subtype H3N2 influenza is most easily generated within this framework when it is mutation limited as well as being under strong immune selection at a number of epitope regions of limited diversity.

Background: During the vaccination campaigns, puppies younger than 3 months old are not targeted and remain unvaccinated for at least the first year of their lives. Almost half of the reported rabid dogs are 6 months or younger. Hence, we should recommend the vaccination against rabies of young puppies. Unfortunately, owing to the exposure of puppies to infections with either canine parvovirus (CPV) or distemper virus (CDV) after the intervention of the vaccinators, owners are reluctant to vaccinate puppies against rabies. Therefore, it is necessary to include the CPV and CDV valences in the vaccine against rabies. Multivalent DNA-based vaccination in dogs, including rabies and distemper valences, could help in raising vaccine coverage., Methods: We have designed monovalent and multivalent DNA-based vaccine candidates for in vitro and in vivo assays. These plasmids encode to the rabies virus glycoprotein and/or the canine distemper virus hemagglutinin. The first strategy of multivalent DNA-based vaccination is by mixing plasmids encoding to a single antigen each. The second is by simply fusing the genes of the antigens together. The third is by adding the foot and mouth disease virus (FMDV) 2A oligopeptide gene into the antigen genes. The last strategy is by the design and use of a bicistronic plasmid with an "Internal Ribosome Entry Site" (IRES) domain., Results: The monovalent construct against canine distemper was efficiently validated by inducing higher humoral immune responses compared to cell-culture-derived vaccine both in mice and dogs. All multivalent plasmids efficiently expressed both valences after in vitro transfection of BHK-21 cells. In BALB/c mice, the bicistronic IRES-dependant construct was the most efficient inducer of virus-neutralizing antibodies against both valences. It was able to induce better humoral immune responses compared to the administration of either cell-culture-derived vaccines or monovalent plasmids. The FMDV 2A was also efficient in the design of multivalent plasmids., Conclusions: In a single shot, the design of efficient multivalent plasmids will be very beneficial for DNA-based vaccination against numerous diseases.

The highly conserved first 23 residues of the influenza hemagglutinin HA2 subunit constitute the fusion domain, which plays a pivotal role in fusing viral and host-cell membranes. At neutral pH, this peptide adopts a tight helical hairpin wedge structure, stabilized by aliphatic hydrogen bonding and charge-dipole interactions. We demonstrate that at low pH, where the fusion process is triggered, the native peptide transiently visits activated states that are very similar to those sampled by a G8A mutant. This mutant retains a small fraction of helical hairpin conformation, in rapid equilibrium with at least two open structures. The exchange rate between the closed and open conformations of the wild-type fusion peptide is ~40 kHz, with a total open-state population of ~20%. Transitions to these activated states are likely to play a crucial role in formation of the fusion pore, an essential structure required in the final stage of membrane fusion.

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.

Background: The influenza A virus has evolved into 16 hemagglutinin (HA) subtypes with different antigenic properties. Thus far typing has been primarily assay based, but the many sequences available from the US National Center for Biotechnology Information (NCBI) offer alternative ways of characterizing the HA gene., Methods: All available HA sequences from the NCBI were analyzed. The software package HMMER was used to score how a training sequence fitted a profile hidden Markov model (profile HMM) constructed from the consensus sequence of one particular HA subtype, Hx, where x=1 to 16. Scores from sequences of the same subtype and from other subtypes were then compared to see if they were separable. This approach was implemented in a stepwise manner, utilizing a sliding window of 100 amino acids with 10-amino-acid increments to build many subtype-specific models, and then assessing which 100-amino acid segments yielded the desired differentiability., Results: Segment-based analysis revealed domains that correlate to HA sequence heterogeneity from one subtype to the others. For example, we showed that H1 segments covering only the second half of HA are not statistically separable from H2, H5 and H6 within the same region, suggesting evolutionary relatedness for these subtypes. The HA1 domain was found to be mostly differentiable between subtypes, which is in line with wet-lab findings that the domain is antigenicity-rich. We also reported a couple of regions that can be conveniently used to characterize all HA subtypes., Conclusion: We established an analysis framework for assessing sequence-subtype association to provide insights into HA subtypes with close evolutionary relationships., (Copyright © 2011. Published by Elsevier B.V.)

The first UK epizootic of highly pathogenic (HP) H5N1 influenza in wild birds occurred in 2008, in a population of mute swans that had been the subject of ornithological study for decades. Here we use an innovative combination of ornithological, phylogenetic and immunological approaches to investigate the ecology and age structure of HP H5N1 in nature. We screened samples from swans and waterbirds using PCR and sequenced HP H5N1-positive samples. The outbreak's origin was investigated by linking bird count data with a molecular clock analysis of sampled virus sequences. We used ringing records to reconstruct the age-structure of outbreak mortality, and we estimated the age distribution of prior exposure to avian influenza. Outbreak mortality was low and all HP H5N1-positive mute swans in the affected population were <3 years old. Only the youngest age classes contained an appreciable number of individuals with no detectable antibody responses to viral nucleoprotein. Phylogenetic analysis indicated that the outbreak strain circulated locally for ~1 month before detection and arrived when the immigration rate of migrant waterbirds was highest. Our data are consistent with the hypothesis that HP H5N1 epizootics in wild swans exhibit limited mortality due to immune protection arising from previous exposure. Our study population may represent a valuable resource for investigating the natural ecology and epidemiology of avian influenza.

In 2011, vaccinia virus caused an outbreak of bovine vaccinia, affecting dairy cattle and dairy workers in Brazil. Genetic and phenotypic analyses identified this isolate as distinct from others recently identified, thereby reinforcing the hypothesis that different vaccinia virus strains co-circulate in Brazil.

Continual and rapid mutation of seasonal influenza viruses by antigenic drift necessitates the almost annual reformulation of flu vaccines, which may offer little protection if the match to the dominant circulating strain is poor. S139/1 is a cross-reactive antibody that neutralizes multiple HA strains and subtypes, including those from H1N1 and H3N2 viruses that currently infect humans. The crystal structure of the S139/1 Fab in complex with the HA from the A/Victoria/3/1975 (H3N2) virus reveals that the antibody targets highly conserved residues in the receptor binding site and contacts antigenic sites A, B, and D. Binding and plaque reduction assays show that the monovalent Fab alone can protect against H3 strains, but the enhanced avidity from binding of bivalent IgG increases the breadth of neutralization to additional strains from the H1, H2, H13, and H16 subtypes. Thus, antibodies making relatively low affinity Fab interactions with the receptor binding site can have significant antiviral activity when enhanced by avidity through bivalent interactions of the IgG, thereby extending the breadth of binding and neutralization to highly divergent influenza virus strains and subtypes.

Immunity induced by DNA vaccines containing the hemagglutinin (H) and nucleoprotein (N) genes of wild-type and attenuated canine distemper virus (CDV) was investigated in mink (Mustela vison), a highly susceptible natural host of CDV. All DNA-immunized mink seroconverted, and significant levels of virus-neutralizing (VN) antibodies were present on the day of challenge with wild-type CDV. The DNA vaccines also primed the cell-mediated memory responses, as indicated by an early increase in the number of interferon-gamma (IFN-γ)-producing lymphocytes after challenge. Importantly, the wild-type and attenuated CDV DNA vaccines had a long-term protective effect against wild-type CDV challenge. The vaccine-induced immunity induced by the H and N genes from wild-type CDV and those from attenuated CDV was comparable. Because these two DNA vaccines were shown to protect equally well against wild-type virus challenge, it is suggested that the genetic/antigenic heterogeneity between vaccine strains and contemporary wild-type strains are unlikely to cause vaccine failure.

Endocytosis has been proposed as one of the primary mechanisms for cellular entry of cell-penetrating peptides (CPPs) and their cargoes. However, a major limitation of endocytic pathway is entrapment of the CPP-cargo in intracellular vesicles from which the cargo must escape into the cytoplasm to exert its biological activity. Here we demonstrate that a CPP tagged with an endosomolytic fusion peptide derived from the influenza virus hemagglutinin-2 (HA2) remarkably enhances the cytosolic delivery of proteins in human A549 cells. To determine the endosome-disruptive effects, recombinant DNA plasmids containing coding sequences of HA2, CPPs and red fluorescent proteins (RFPs) were constructed. The fusion proteins were purified from plasmid-transformed Escherichia coli, and their effects on protein transduction were examined using live cell imaging and flow cytometry. Our data indicate that endocytosis is the major route for cellular internalization of CPP-HA2-tagged RFP. Mechanistic studies revealed that the fusogenic HA2 peptide dramatically facilitates CPP-mediated protein entry through the release of endocytosed RFPs from endosomes into the cytoplasm. Furthermore, incorporating the HA2 fusion peptide of the CPP-HA2 fusion protein improved cytosolic uptake without causing cytotoxicity. These findings strongly suggest that the CPP-HA2 tag could be an efficient and safe carrier that overcomes endosomal entrapment of delivered therapeutic drugs., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Recently, the D222G substitution was observed in the HA of pandemic (H1N1) 2009 viruses isolated from fatal cases in several countries. We made a similar observation in one fatal case in Tunisia showing a D222G substitution in a virus isolate. The man was 47 years old and had no other subjacent pathologies or any known risk factors. He died after three days, suffering from severe respiratory symptoms of flu. The causal link of the D222G substitution in Tunisia with virulence must be verified. Further study is warranted to elucidate the intriguing relationship between the D222G substitution and severe disease. Constant molecular surveillance is important to monitor the pathogenicity of circulating strains and evaluate vaccine efficacy.

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.

As part of our ongoing efforts on animal influenza surveillance in Argentina, an H9N2 virus was isolated from a wild aquatic bird (Netta peposaca), A/rosy-billed pochard/Argentina/CIP051-559/2007 (H9N2) - herein referred to as 559/H9N2. Due to the important role that H9N2 viruses play in the ecology of influenza in nature, the 559/H9N2 isolate was characterized molecularly and biologically. Phylogenetic analysis of the HA gene revealed that the 559/H9N2 virus maintained an independent evolutionary pathway and shared a sister-group relationship with North American viruses, suggesting a common ancestor. The rest of the genome segments clustered with viruses from South America. Experimental inoculation of the 559/H9N2 in chickens and quail revealed efficient replication and transmission only in quail. Our results add to the notion of the unique evolutionary trend of avian influenza viruses in South America. Our study increases our understanding of H9N2 viruses in nature and emphasizes the importance of expanding animal influenza surveillance efforts to better define the ecology of influenza viruses at a global scale., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Raccoon dogs (Nyctereutes procyonoides) are widespread and common in South Korea. In 2011, we obtained serum samples from 102 wild raccoon dogs to survey their exposure to canine distemper virus (CDV). Forty-five of the 102 animals (44.1%) were seropositive. Field cases of canine distemper in wild raccoon dogs from 2010 to 2011 were investigated. Fourteen cases of CDV infection were identified by a commercially available CDV antigen detection kit. These cases were used for virus isolation and molecular analysis. Sequence analysis of hemagglutinin genes indicated that all viruses isolated belonged to the Asia-2 genotype. H protein residues which are related to the receptor and host specificity (residues 530 and 549) were analyzed. A glutamic acid (E) residue is present at 530 in all isolates. At 549, a histidine (H) residue was found in five isolates and tyrosine (Y) residue was found in 6 isolates. Our study demonstrated that CDV infection was widespread in wild raccoon dogs in South Korea., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The newly emerging canine influenza virus (CIV) causes considerable concerns for both veterinary and public health. During 2009-2010, six strains of H3N2 influenza virus were isolated from dogs in Jiangsu Province, China. Sequence and phylogenetic analysis of eight gene segments revealed that the six viruses were most similar to a recent canine-derived subtype H3N2 influenza virus isolated in cats from South Korea, which originated from avian strain. By comparing the deduced amino acid sequences of the hemagglutinin 1 (HA1) and neuraminidase (NA) genes of the six Jiangsu isolates against the most similar avian strains, we found that all isolates had several common mutations at the receptor-binding sites, potential glycosylation sites and cleavage site in HA1, and antigenic sites in both the HA1 and NA segments. Significantly, a unique two amino acid insertion in the NA stalk was found. Experimental infection of BALB/c mice revealed that viral RNA could be detected in the major rodent organs, such as brain, heart, spleen, kidney, liver and intestine, as well as the lung. All the sampled organs from infected mice showed significant lesions and viral antigen staining. This study highlights the potential of domesticated animals to become a reservoir for influenza virus and the need for surveillance programs to detect cross-species transmission., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The H1N1 2009 influenza pandemic took the health care workers by surprise in spite of warning about influenza pandemic. Influenza A virus has the ability to overcome immunity from previous infections through the acquisition of genetic changes by shift or drift. Thus, understanding the evolution of the viruses in human is important for the surveillance and the selection of vaccine strains. A total of 23 pandemic A/H1N1 2009 viral HA gene sequences were downloaded from NCBI submitted during March and May 2010 by NIV and were analysed. Along with that the vaccine strain A/California/07/2009 was also downloaded from NCBI. All the sequences were used to analyse the evolution of the haemagglutinin (HA) by phylogenetic analysis. The HA gene could be divided into four groups with shift from 1 to lV revealing that the HA genes of the influenza A viruses evolved in a sequential way, in comparison to vaccine strain A/California/07/2009. Amino acid sequence analysis of the HA genes of the A/H1N1 2009 isolates, revealed mutations at positions 100, 220 and additional mutations in different positions 114, 171, 179, 190, 208, 219, 222, 239, 240, 247, 251, 260 and 285 .The mutations identified showed the adaptation of the new virus to the host that could lead to genetic changes inherent to the virus resulting in a reassortant which could be catastrophic, hence continuous monitoring of strains is mandatory.

Protein array technology is an emerging tool that enables high-throughput screening of protein-protein or protein-lipid interactions and identification of immunodominant antigens during the course of a bacterial or viral infection. In this work, we developed an Influenza virus protein array using the sortase-mediated transpeptidation reaction known as "Sortagging". LPETG-tagged Influenza virus proteins from bacterial and eukaryotic cellular extracts were immobilized at their carboxyl-termini onto a preactivated amine-glass slide coated with a Gly3 linker. Immobilized proteins were revealed by specific antibodies, and the newly generated Sortag-protein chip can be used as a device for antigen and/or antibody screening. The specificity of the Sortase A (SrtA) reaction avoids purification steps in array building and allows immobilization of proteins in an oriented fashion. Previously, this versatile technology has been successfully employed for protein labeling and protein conjugation. Here, the tool is implemented to covalently link proteins of a viral genome onto a solid support. The system could readily be scaled up to proteins of larger genomes in order to develop protein arrays for high-throughput screening.

Influenza A virus possesses a segmented genome of eight negative-sense, single-stranded RNAs. The eight segments have been shown to be represented in approximately equal molar ratios in a virus population; however, the exact copy number of each viral RNA segment per individual virus particles has not been determined. We have established an experimental approach based on multicolor single-molecule fluorescent in situ hybridization (FISH) to study the composition of viral RNAs at single-virus particle resolution. Colocalization analysis showed that a high percentage of virus particles package all eight different segments of viral RNAs. To determine the copy number of each RNA segment within individual virus particles, we measured the photobleaching steps of individual virus particles hybridized with fluorescent probes targeting a specific viral RNA. By comparing the photobleaching profiles of probes against the HA RNA segment for the wild-type influenza A/Puerto Rico/8/34 (PR8) and a recombinant PR8 virus carrying two copies of the HA segment, we concluded that only one copy of HA segment is packaged into a wild type virus particle. Our results showed similar photobleaching behaviors for other RNA segments, suggesting that for the majority of the virus particles, only one copy of each RNA segment is packaged into one virus particle. Together, our results support that the packaging of influenza viral genome is a selective process.

Oncolytic vesicular stomatitis virus (VSV) has potent antitumor activity, but infects a broad range of cell types. Here, we used the measles virus (MV) hemagglutinin (H) and fusion (F) envelope glycoproteins to redirect VSV entry and infection specifically to tumor-associated receptors. Replication-defective VSV, deleted of its glycoprotein gene (VSVΔG), was pseudotyped with MV-F and MV-H displaying single-chain antibodies (scFv) specific for epidermal growth factor receptor (EGFR), folate receptor (FR), or prostate membrane-specific antigen (PSMA). Viral titers were ∼10(5) PFU/ml, but could be concentrated to 10(7) PFU/ml. Immunoblotting confirmed incorporation of the MV-H-scFv and MV-F into functional VSV virions. Although VSV-G was able to infect all tumor cell lines tested, the retargeted VSV infected only cells that expressed the targeted receptor. In vivo specificities of the EGFR-, FR-, and PSMA-retargeted VSV were assessed by intratumoral injection into human tumor xenografts. Analysis of green fluorescent protein reporter gene expression indicated that VSV infection was restricted to receptor-positive tumors. In summary, we have demonstrated for the first time that VSV can be efficiently retargeted to different cellular receptors using the measles display technology, yielding retargeted VSV vectors that are highly specific for tumors that express the relevant receptor.

Despite the availability of live attenuated measles virus (MV) vaccines, a large number of measles-associated deaths occur among infants in developing countries. The development of a measles subunit vaccine may circumvent the limitations associated with the current live attenuated vaccines and eventually contribute to global measles eradication. Therefore, the goal of this study was to test the feasibility of producing the recombinant globular head domain of the MV hemagglutinin (H) protein by stably transfected human cells and to examine the ability of this recombinant protein to elicit MV-specific immune responses. The recombinant protein was purified from the culture supernatant of stably transfected HEK293T cells secreting a tagged version of the protein. Two subcutaneous immunizations with the purified recombinant protein alone resulted in the production of MV-specific serum IgG and neutralizing antibodies in mice. Formulation of the protein with adjuvants (polyphosphazene or alum) further enhanced the humoral immune response and in addition resulted in the induction of cell-mediated immunity as measured by the production of MV H-specific interferon gamma (IFN-γ) and interleukin 5 (IL-5) by in vitro re-stimulated splenocytes. Furthermore, the inclusion of polyphosphazene into the vaccine formulation induced a mixed Th1/Th2-type immune response. In addition, the purified recombinant protein retained its immunogenicity even after storage at 37°C for 2 weeks., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Using the complete haemagglutinin (HA) gene and partial phosphoprotein (P) gene we investigated the genotype of canine distemper virus (CDV) strains recovered from two wildlife species in Mecklenburg-Vorpommern, Germany. Phylogenetic analyses demonstrated significant differences between the strains from raccoons Procyon lotor (family Procyonidae) obtained in 2007 and strains from red foxes Vulpes vulpes (family Canidae) obtained in 2008. The raccoon strains belonged to the CDV European wildlife lineage whereas the red fox strains belonged to the CDV Europe lineage. We combined our genetic sequence data with published data from 138 CDV stains worldwide to investigate the proposed importance of amino acid substitutions in the SLAM binding region of the CDV HA protein at position 530 (G/E to R/D/N) and 549 (Y to H) to the spread of domestic dog-adapted CDV strains to other carnivores. We found no evidence that amino acid 530 was strongly affected by host species. Rather, site 530 was conserved within CDV lineages, regardless of host species. Contrary to expectation, strains from non-dog hosts did not exhibit a bias towards the predicted substitution Y549H. Wild canid hosts were more frequently infected by strains with 549Y, a pattern similar to domestic dogs. Non-canid strains showed no significant bias towards either H or Y at site 549, although there was a trend towards 549H. Significant differences between the prevalence of 549Y and 549H in wild canid strains and non-canid strains suggests a degree of virus adaptation to these categories of host., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The eradication of rinderpest virus (RPV) from the globe was possible through the availability of a safe and effective live attenuated vaccine and a suitable companion diagnostic test. However, the inability to serologically 'Differentiate between naturally Infected and Vaccinated Animals' (DIVA) meant that both the time taken to complete the eradication programme and the economic burden on countries involved was significantly greater than if a vaccine and companion diagnostic test that fulfilled the DIVA concept had been available. During the RPV eradication campaign serosurveillance for RPV was primarily based on a competitive ELISA using a RPV specific (C1) monoclonal antibody (mAb) directed against the viral haemagglutinin (H) protein but this test was not able to meet DIVA requirements. To provide proof of concept for the generation of novel morbillivirus DIVA vaccines we have identified, by phage display, and mutated residues critical for C1 mAb binding and assessed the functionality of mutants in an in vitro fusion assay. Finally we have incorporated mutated epitopes into a full length clone and rescued recombinant RPV using reverse genetics techniques. Here we describe a novel mechanism of marking morbillivirus vaccines, using RPV as a proof of concept, and discuss the applicability of this method to the development of marked vaccines for peste des petits ruminants virus (PPRV)., (Crown Copyright © 2012. Published by Elsevier Ltd. All rights reserved.)

Analyses and visualizations by the ISSCOR method of the influenza virus hemagglutinin genes of three different A-subtypes revealed some rather striking temporal (for A/H3N3), and spatial relationships (for A/H5N1) between groups of individual gene subsets. The application to the A/H1N1 set revealed also relationships between the seasonal H1, and the swine-like novel 2009 H1v variants in a quick and unambiguous manner. Based on these examples we consider the application of the ISSCOR method for analysis of large sets of homologous genes as a worthwhile addition to a toolbox of genomics-it allows a rapid diagnostics of trends, and possibly can even aid an early warning of newly emerging epidemiological threats., (Copyright © 2012 Académie des sciences. Published by Elsevier SAS. All rights reserved.)

The hemagglutination-inhibition (HI) titers of a panel of 25 mouse monoclonal antibodies (MAbs) against 44 isolates of highly pathogenic avian influenza virus H5N1 were determined. A two-dimensional antigenic dendrogram was constructed by hierarchical clustering of HI titers. Viruses with similar reactivity patterns were clustered horizontally, whereas MAbs were clustered vertically. In this 2-D dendrogram, with 40% similarity as a cut-off, four virus clusters and four MAbs clusters were identified. A phylogenetic tree based on the deduced amino acid sequence of the hemagglutinin gene of tested viruses was constructed and its topology was compared to the antigenic dendrogram. Interestingly, viruses with high genetic homology in the phylogenetic tree also had high similarity in their reactivity patterns, as indicated by their relatedness in the tree and close clustering in the dendrogram, respectively. However, the reverse and the converse were also true. Of the five pairs of viruses in the dendrogram with bootstrap values higher than 75, four pairs were in concordance with their genetic relatedness. However, one pair contained viruses belong to two distinct genetic clades. These results were discussed in the context of antigenic variation, genetic polymorphism, and the potential application of MAbs in antigenic analysis., (© 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

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.

The outbreak of the 2009 influenza pandemic underscored the important role of swine in influenza virus evolution and the emergence of novel viruses with pandemic potential. Vaccination is the most common practice to control swine influenza in swine industry. Influenza virus-like particle (VLP) vaccines are an alternative approach and have been demonstrated to be immunogenic and confer protection against influenza virus challenge in chickens, mice and ferrets. In this study, we generated VLPs consisting of HA, NA and M1 proteins derived from pandemic virus A/California/04/2009 in insect cells. The immunogenicity and efficacy following vaccination of VLPs were evaluated in swine. Our data showed that vaccination using VLPs elicited robust levels of serum IgG, mucosal IgA, and viral neutralizing antibodies against A/Sw/Manitoba/MAFRI32/2009 H1N1. Following challenge with pandemic H1N1 2009, vaccinated pigs were protected, displaying reduced lung lesions, virus shedding and inhibition of virus replication in the lungs compared to non-vaccinated control pigs. Thus, VLPs can serve as a promising vaccination strategy to control influenza in swine., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Background: Highly pathogenic avian influenza A(H5N1) viruses are an important health problem in many Asian and African countries. The current increase in human cases demonstrates that influenza A(H5N1) is still a significant global pandemic threat. Many health organizations have recognized the need for new strategies to improve influenza global surveillance. Specifically, the World Health Organization through the global technical consultation for influenza surveillance have called for a detailed picture of the current limitations, especially at the nation level, to evaluate, standardize and strength reporting systems. The main goal of our study is to demonstrate the value of genetic surveillance as part of a strategic surveillance plan. As a proof of concept, we evaluated the current situation of influenza A(H5N1) in Asian and Africa., Results: Our analysis revealed a power-law distribution in the number of sequences of A(H5N1) viruses analyzed and/or reported to influenza surveillance networks. The majority of the Asian and African countries at great risk of A(H5N1) infections have very few (approximately three orders of magnitude) sequenced A(H5N1) viruses (e.g. hemagglutinin genes). This suggests that countries under pandemic alert for avian influenza A(H5N1) have very limited participation (e.g. data generation, genetic analysis and data share) in avian influenza A(H5N1) surveillance. More important, this study demonstrates the usefulness of influenza genetic surveillance to detect emerging pandemic threat viruses., Conclusions: Our study reveals that some countries suffering from human cases of avian influenza have limited participation (e.g. genetic surveillance or data share) with global surveillance networks. Also, we demonstrate that the implementation of genetic surveillance programs could increase and strengthen worldwide epidemic and pandemic preparedness. We hope that this work promotes new discussions between policy makers and health surveillance organizations to improve current methodologies and regulations.

Influenza impressively reflects the paradigm of a viral disease in which continued evolution of the virus is of paramount importance for annual epidemics and occasional pandemics in humans. Because of the continuous threat of novel influenza outbreaks, it is essential to gather further knowledge about viral pathogenicity determinants. Here, we explored the adaptive potential of the influenza A virus subtype H1N1 variant isolate A/Hamburg/04/09 (HH/04) by sequential passaging in mice lungs. Three passages in mice lungs were sufficient to dramatically enhance pathogenicity of HH/04. Sequence analysis identified 4 nonsynonymous mutations in the third passage virus. Using reverse genetics, 3 synergistically acting mutations were defined as pathogenicity determinants, comprising 2 mutations in the hemagglutinin (HA[D222G] and HA[K163E]), whereby the HA(D222G) mutation was shown to determine receptor binding specificity and the polymerase acidic (PA) protein F35L mutation increasing polymerase activity. In conclusion, synergistic action of all 3 mutations results in a mice lethal pandemic H1N1 virus.