Neuraminidase Protein Research Articles

Azithromycin, a 15-membered macrolide antibiotic, inhibits influenza A(H1N1)pdm09 virus infection by interfering with virus internalization process.

The pandemic influenza 2009 (A(H1N1)pdm09) virus currently causes seasonal and annual epidemic outbreaks. The widespread use of anti-influenza drugs such as neuraminidase and matrix protein 2 (M2) channel inhibitors has resulted in the emergence of drug-resistant influenza viruses. In this study, we aimed to determine the anti-influenza A(H1N1)pdm09 virus activity of azithromycin, a re-positioned macrolide antibiotic with potential as a new anti-influenza candidate, and to elucidate its underlying mechanisms of action. We performed in vitro and in vivo studies to address this. Our in vitro approaches indicated that progeny virus replication was remarkably inhibited by treating viruses with azithromycin before infection; however, azithromycin administration after infection did not affect this process. We next investigated the steps inhibited by azithromycin during virus invasion. Azithromycin did not affect attachment of viruses onto the cell surface, but blocked internalization into host cells during the early phase of infection. We further demonstrated that azithromycin targeted newly budded progeny virus from the host cells and inactivated their endocytic activity. This unique inhibitory mechanism has not been observed for other anti-influenza drugs, indicating the potential activity of azithromycin before and after influenza virus infection. Considering these in vitro observations, we administered azithromycin intranasally to mice infected with A(H1N1)pdm09 virus. Single intranasal azithromycin treatment successfully reduced viral load in the lungs and relieved hypothermia, which was induced by infection. Our findings indicate the possibility that azithromycin could be an effective macrolide for the treatment of human influenza.

Role of the Innate Cytokine Storm Induced by the Influenza A Virus.

Influenza A viruses (IAVs) can be classified into dozens of subtypes based on their hemagglutinin (HA) and neuraminidase (NA) proteins. To date, 18 HA subtypes and 11 NA subtypes of IAVs that spread in animals and humans have been found. Following infection, the IAV first induces the innate immune system, which can rapidly recruit innate immune cells and cytokines to the site of infection. Influenza-induced cytokine storms have been associated with uncontrolled proinflammatory responses, which may lead to significant immunopathy and severe disease. Cytokine storms are complicated by several types of cytokines and chemokines that have various activities. In addition to their direct effects, their crossregulation causes cytokine networks to form; these networks determine the outcome of viral infections. In this review, we focus on cytokine storms and their signaling pathways that are triggered by the different subtypes of IAV.

Curvature- and Phase-Induced Protein Sorting Quantified in Transfected Cell-Derived Giant Vesicles.

Eukaryotic cells possess a dynamic network of membranes that vary in lipid composition. To perform numerous biological functions, cells modulate their shape and the lateral organization of proteins associated with membranes. The modulation is generally facilitated by physical cues that recruit proteins to specific regions of the membrane. Analyzing these cues is difficult due to the complexity of the membrane conformations that exist in cells. Here, we examine how different types of membrane proteins respond to changes in curvature and to lipid phases found in the plasma membrane. By using giant plasma membrane vesicles derived from transfected cells, the proteins were positioned in the correct orientation and the analysis was performed in plasma membranes with a biological composition. Nanoscale membrane curvatures were generated by extracting nanotubes from these vesicles with an optical trap. The viral membrane protein neuraminidase was not sensitive to curvature, but it did exhibit strong partitioning (coefficient of K = 0.16) disordered membrane regions. In contrast, the membrane repair protein annexin 5 showed a preference for nanotubes with a density up to 10-15 times higher than that on the more flat vesicle membrane. The investigation of nanoscale effects in isolated plasma membranes provides a quantitative platform for studying peripheral and integral membrane proteins in their natural environment.

E-pharmacophore hypothesis strategy to discover potent inhibitor for influenza treatment

The surface protein of Influenza virus, Neuraminidase (NA), is believed to play a critical role in the release of new viral particle and thus spreads infection. It has been recognized as a valid drug target for anti-influenza therapy. Despite the number of available approved drugs for the influenza infection treatment, the emergence of resistant variants with novel mutations are the foremost challenges for the currently used NA inhibitors. Thus, the current investigation was carried out to ascertain potent inhibitors using computational strategies such as e-pharmacophore based virtual screening and docking approach. A three-dimensional e-pharmacophore hypothesis was generated based on the chemical features of complexes of the drugs and NA protein using PHASE module of Schrödinger suite. The generated hypothesis consisted of one hydrogen bond acceptor (A), two hydrogen bond donors (D), one negatively charged group (N) and one aromatic ring (R), ADDNR. The hypothesis was further evaluated for its integrity using enrichment analysis and used to filter out molecules with similar pharmacophoric features from approved, investigational and experimental subsets of DrugBank and ZINC database. In addition, ligand filtration was performed to curb down the molecules to an efficient collection of hit molecules by using Lipinski “rule of five and ADME analysis by using Qikprop module. Overall, the results from our analysis suggest that compound lisinopril and formoterol could serve as potent antiviral compounds for the treatment of influenza A virus infection. It is worth mentioning that the results correlate well with literature evidences.

B cell epitopes in the intrinsically disordered regions of neuraminidase and hemagglutinin proteins of H5N1 and H9N2 avian influenza viruses for peptide-based vaccine development.

Avian influenza viruses (AIV) are very active in several parts of the globe and are the cause of huge economic loss for the poultry industry and also human fatalities. Three dimensional modeling was carried out for neuraminidase (NA) and hemagglutinin (HA) proteins of AIV. The C-score, estimated TM-Score, and estimated root-mean-square deviation (RMSD) score for NA of H5N1 were -1.18, 0.57 ± 0.15, and 9.8 ± 7.6, respectively. The C-score, estimated TM-Score, and estimated RMSD score for NA of H9N2 were -1.43, 0.54 ± 0.15, and 10.5 ± 4.6, respectively. The C-score, estimated TM-Score, and estimated RMSD score for HA of H5N1 were -0.03, 0.71 ± 0.12, and 7.7 ± 4.3, respectively. The C-score, estimated TM-Score, and estimated RMSD score for HA of H9N2 were -0.57, 0.64 ± 0.13, and 8.9 ± 4.6, respectively. Intrinsically disordered regions were identified for the NA and HA proteins of H5N1 and H9N2 with the use of PONDR program. Linear B cell epitope was predicted using BepiPred 2 program for NA and HA of H5N1 and H9N2 avian influenza strains. Discontinuous epitopes were predicted by Discotope 2 program. The linear epitopes that were considered likely to be immunogenic and within the intrinsically disordered region for the NA of H5N1 was TKSTNSRSGFEMIWDPNGWTGTDSSFSVK, and for H9N2 it was VGDTPRNDDSSSSSNCRDPNNERGAP. In the case of HA of H5N1, it was QRLVPKIATRSKVNGQSG and ATGLRNSPQRERRRKK; for H9N2 it was INRTFKPLIGPRPLVNGLQG and SLKLAVGLRNVPARSSR. The discontinuous epitopes of NA of H5N1 and H9N2 were identified at various regions of the protein structure spanning from amino acid residue positions 90 to 449 and 107 to 469, respectively. Similarly, the discontinuous epitopes of HA of H5N1 and H9N2 were identified in the amino acid residue positions 27 to 517 and 136 to 521, respectively. This study has identified potential and highly immunogenic linear and conformational B-cell epitopes towards developing a vaccine against AIV both for human and poultry use.

Differential Effects of Influenza Virus NA, HA Head, and HA Stalk Antibodies on Peripheral Blood Leukocyte Gene Expression during Human Infection.

In this study, we examined the relationships between anti-influenza virus serum antibody titers, clinical disease, and peripheral blood leukocyte (PBL) global gene expression during presymptomatic, acute, and convalescent illness in 83 participants infected with 2009 pandemic H1N1 virus in a human influenza challenge model. Using traditional statistical and logistic regression modeling approaches, profiles of differentially expressed genes that correlated with active viral shedding, predicted length of viral shedding, and predicted illness severity were identified. These analyses further demonstrated that challenge participants fell into three peripheral blood leukocyte gene expression phenotypes that significantly correlated with different clinical outcomes and prechallenge serum titers of antibodies specific for the viral neuraminidase, hemagglutinin head, and hemagglutinin stalk. Higher prechallenge serum antibody titers were inversely correlated with leukocyte responsiveness in participants with active disease and could mask expression of peripheral blood markers of clinical disease in some participants, including viral shedding and symptom severity. Consequently, preexisting anti-influenza antibodies may modulate PBL gene expression, and this must be taken into consideration in the development and interpretation of peripheral blood diagnostic and prognostic assays of influenza infection.IMPORTANCE Influenza A viruses are significant human pathogens that caused 83,000 deaths in the United States during 2017 to 2018, and there is need to understand the molecular correlates of illness and to identify prognostic markers of viral infection, symptom severity, and disease course. Preexisting antibodies against viral neuraminidase (NA) and hemagglutinin (HA) proteins play a critical role in lessening disease severity. We performed global gene expression profiling of peripheral blood leukocytes collected during acute and convalescent phases from a large cohort of people infected with A/H1N1pdm virus. Using statistical and machine-learning approaches, populations of genes were identified early in infection that correlated with active viral shedding, predicted length of shedding, or disease severity. Finally, these gene expression responses were differentially affected by increased levels of preexisting influenza antibodies, which could mask detection of these markers of contagiousness and disease severity in people with active clinical disease.

Influenza A virus surface proteins are organized to help penetrate host mucus.

Influenza A virus (IAV) enters cells by binding to sialic acid on the cell surface. To accomplish this while avoiding immobilization by sialic acid in host mucus, viruses rely on a balance between the receptor-binding protein hemagglutinin (HA) and the receptor-cleaving protein neuraminidase (NA). Although genetic aspects of this balance are well-characterized, little is known about how the spatial organization of these proteins in the viral envelope may contribute. Using site-specific fluorescent labeling and super-resolution microscopy, we show that HA and NA are asymmetrically distributed on the surface of filamentous viruses, creating a spatial organization of binding and cleaving activities that causes viruses to step consistently away from their NA-rich pole. This Brownian ratchet-like diffusion produces persistent directional mobility that resolves the virus's conflicting needs to both penetrate mucus and stably attach to the underlying cells, potentially contributing to the prevalence of the filamentous phenotype in clinical isolates of IAV.

Broad and Protective Influenza B Virus Neuraminidase Antibodies in Humans after Vaccination and their Clonal Persistence as Plasma Cells.

Although most seasonal inactivated influenza vaccines (IIV) contain neuraminidase (NA), the extent and mechanisms of action of protective human NA-specific humoral responses induced by vaccination are poorly resolved. Due to the propensity of influenza virus for antigenic drift and shift and its tendency to elicit predominantly strain-specific antibodies, humanity remains susceptible to waves of new strains of seasonal viruses and is at risk from viruses with pandemic potential for which limited or no immunity may exist. Here we demonstrate that the use of IIV results in increased levels of influenza B virus (IBV) NA-specific serum antibodies. Detailed analysis of the IBV NA B cell response indicates concurrent expansion of IBV NA-specific peripheral blood plasmablasts 7 days after IIV immunization which express monoclonal antibodies with broad and potent antiviral activity against both IBV Victoria and Yamagata lineages and prophylactic and therapeutic activity in mice. These IBV NA-specific B cell clonal lineages persisted in CD138+ long-lived bone marrow plasma cells. These results represent the first demonstration that IIV-induced NA human antibodies can protect and treat influenza virus infection in vivo and suggest that IIV can induce a subset of IBV NA-specific B cells with broad protective potential, a feature that warrants further study for universal influenza vaccine development.IMPORTANCE Influenza virus infections continue to cause substantial morbidity and mortality despite the availability of seasonal vaccines. The extensive genetic variability in seasonal and potentially pandemic influenza strains necessitates new vaccine strategies that can induce universal protection by focusing the immune response on generating protective antibodies against conserved targets such as regions within the influenza neuraminidase protein. We have demonstrated that seasonal immunization stimulates neuraminidase-specific antibodies in humans that are broad and potent in their protection from influenza B virus when tested in mice. These antibodies further persist in the bone marrow, where they are expressed by long-lived antibody-producing cells, referred to here as plasma cells. The significance in our research is the demonstration that seasonal influenza immunization can induce a subset of neuraminidase-specific B cells with broad protective potential, a process that if further studied and enhanced could aid in the development of a universal influenza vaccine.

Community spread and late season increased incidence of oseltamivir-resistant influenza A(H1N1) viruses in Norway 2016.

BackgroundAntiviral resistance in Norwegian influenza viruses is rare. Only one A(H1N1)pdm09 virus from May 2015 had been found resistant to oseltamivir since the introduction of these viruses in 2009.ObjectivesSurveillance of antiviral resistance is part of the Norwegian surveillance system, to rapidly detect the development of antiviral‐resistant viruses and spread in the community. We describe the spread of oseltamivir‐resistant A(H1N1)pdm09 viruses in Norway in the 2016‐17 season, found as part of the routine surveillance.MethodsInfluenza H1N1 viruses were analysed for antiviral resistance by pyrosequencing, neuraminidase susceptibility assay and by Sanger sequencing of the HA and NA genes.ResultsDuring the 2015‐16 influenza season, 3% of all A(H1N1)pdm09 viruses screened for resistance in Norway were resistant to oseltamivir, possessing the H275Y substitution in the neuraminidase protein. In comparison, the overall frequency in Europe was 0.87%. Out of these, 37% (n = 10) were reported from Norway. Most cases in Norway were not related to antiviral treatment, and the cases were from several different locations of southern Norway. Genetic analysis revealed that resistant virus emerged independently on several occasions and that there was some spread of oseltamivir‐resistant influenza A(H1N1)6B.1 viruses in the community, characterised by a N370S substitution in the haemagglutinin and T48I in the neuraminidase.ConclusionsOur findings emphasise the importance of antiviral resistance surveillance in the community, not only in immunocompromised patients or other patients undergoing antiviral treatment.

Comprehensive profiling of translation initiation in influenza virus infected cells.

Translation can initiate at alternate, non-canonical start codons in response to stressful stimuli in mammalian cells. Recent studies suggest that viral infection and anti-viral responses alter sites of translation initiation, and in some cases, lead to production of novel immune epitopes. Here we systematically investigate the extent and impact of alternate translation initiation in cells infected with influenza virus. We perform evolutionary analyses that suggest selection against non-canonical initiation at CUG codons in influenza virus lineages that have adapted to mammalian hosts. We then use ribosome profiling with the initiation inhibitor lactimidomycin to experimentally delineate translation initiation sites in a human lung epithelial cell line infected with influenza virus. We identify several candidate sites of alternate initiation in influenza mRNAs, all of which occur at AUG codons that are downstream of canonical initiation codons. One of these candidate downstream start sites truncates 14 amino acids from the N-terminus of the N1 neuraminidase protein, resulting in loss of its cytoplasmic tail and a portion of the transmembrane domain. This truncated neuraminidase protein is expressed on the cell surface during influenza virus infection, is enzymatically active, and is conserved in most N1 viral lineages. We do not detect globally higher levels of alternate translation initiation on host transcripts upon influenza infection or during the anti-viral response, but the subset of host transcripts induced by the anti-viral response is enriched for alternate initiation sites. Together, our results systematically map the landscape of translation initiation during influenza virus infection, and shed light on the evolutionary forces shaping this landscape.

Molecular characteristic analysis of neuraminidase genes of avian influenza virus H9N2 in environments in Weining, Guizhou Province during 2015-2017

Objective To understand the genetic variations of neuraminidase (NA) genes of avian influenza virus H9N2 in Weining, Guizhou Province, and to provide the scientific evidence for the prevention and control of avian influenza virus. Methods Ribonucleic acids (RNA) were extracted and NA genes were amplified and sequenced from 13 randomly selected H9N2 positive samples from the live poultry market (LPM) environments in north of Weining Yi and Hui and Miao autonomous county (Weining), Guizhou Province during 2015 to 2017. Then the homology, genetic evolution, and sites of stalk deletion areas, potential N-glycosylation, receptor binding regions and drug resistance of H9N2 subtype avian influenza viruses were analyzed by a series of bioinformation software. Results Homology analysis revealed that there were 93.0%-100.0% and 92.1%-100.0% similarity among 13 strains H9N2 avian influenza viruses in nucleotide and amino acid of the NA gene, respectively. All strains belonged to DK/HK/Y280/97 sub-lineage, but their genetic sources were complex and diverse. Thirteen strains had a stalk deletion of 3 amino acid residues TEI at positions 63-65 and 3 isolates had mutation QN to QK at positions 39-40. The potential N-glycosylation sites at amino acid residues 86, 146, 200, and 234 of the NA protein of all strains were highly conserved, while other N-glycosylation sites had quantity and site mutations. There were different mutation types at the three sialic acid binding site areas, especially at 399-404 area. All NA protease activity sites and key sites of the 13 strains had no mutations associated with resistance to the neuraminidase inhibitor drugs. Conclusions All 13 strains H9N2 viruses belongs to DK/HK/Y280/97 sub-lineage in Weining, Guizhou Province during 2015-2017, and their genetic sources are complex and diverse. The mutations on sites of stalk areas, potential N-glycosylation and sialic acid binding site areas are presented at different degrees. Hence, enhancing surveillance and controlling H9N2 avian influenza virus is necessary. Key words: Influenza A virus, H9N2 subtype; Neuraminidase gene; Molecular characteristic

Cellular immune response in infected mice to NSP protein encoded by the negative strand NS RNA of influenza A virus

ABSTRACT Influenza A virus belongs to a family of enveloped viruses with an RNA genome of negative polarity consisting of 8 RNA segments. The transcription of this RNA genome results in the synthesis of positive-sense mRNAs that translate up to 16 unique viral proteins with the help of splicing and translational shift mechanisms. The 8th NS segment encodes the NS1 protein (27 kDa), which is an active interferon antagonist, and the nuclear export protein NEP (14 kDa) through the standard negative polarity pathway. In addition, an alternative open reading frame for the synthesis of a third viral protein (NSP, negative-strand protein) by means of a direct translation of genome polarity RNA (the so-called positive polarity genome strategy) was identified in the NS segment. Since it is unknown as to whether the NSP protein can be synthesized in the infected organism post viral infection, the generation of spleen leucocytes specific to this protein was studied in mice after two sequential infections with influenza A viruses of H1N1 and H3N2 subtypes. It was found that leucocyte clones specifically recognizing a peptide domain in the central region of the NSP protein (amino acid positions 82-119) were generated in mice infected with influenza A viruses. In silico prediction has shown strong major histocompatibility complex-1 (MHC-I) and MHC-II specific epitopes in this central domain of the NSP. Comparative analysis of the influenza H3N2 viruses circulating in humans during 1968-2018 has shown high NSP variability, which was similar to that shown for the hemagglutinin (HA) and neuraminidase (NA) proteins. The highest variability was found to be in the N-and C-terminal parts of the NSP. These observations suggest that synthesis of the NSP protein occurs in infected animals and further support a bipolar (ambisense) strategy of the RNA genome of human influenza A virus.

Клеточный иммунный ответ у инфицированных животных против белка NSP, кодируемого негативной цепью NS RNA вируса гриппа А

Вирус гриппа А относится к оболочечным RNA-содержащим вирусам с негативно-полярным геномом, состоящим из 8 сегментов RNA, каждый из которых является матрицей для синтеза позитивно-полярных mRNA, которые, в свою очередь, служат матрицей для синтеза 16 вирусных белков с использованием для некоторых механизма сплайсинга или трансляционного шифта. Восьмой сегмент NS посредством классической негативной стратегии генома кодирует белок NS1, обладающий анти-интерфероновой активностью (27 кДа), и белок ядерного экспорта NEP (NS2) (nuclear export protein, 14 кДа). Кроме этого, в сегменте NS обнаружена дополнительная открытая рамка для синтеза третьего вирусного белка по альтер-нативному пути посредством прямой трансляции негативно полярной вирусной RNA. Эта стратегия генома получила название позитивно-полярной. Образование данного белка, названного NSP (negative strand protein), при инфекции вируса гриппа А в целостном организме пока не обнаружено. Анализ белка NSP in silico выявил наличие доменов MHC-I (major histocompatibility complex-1) и MHC-II. Сравнительный анализ вирусов гриппа подтипа H3N2, циркулировавших среди людей в период с 1968 по 2018 гг., показал высокую изменчивость гена белка NSP, которая сходна с изменчивостью поверхностных белков гемагглютинина (HA) и нейраминидазы (NA). Наибольшая изменчивость обнаруживалась в зонах, соответствующих N-и C-концевым участкам белка NSP. В настоящей работе изучена возможность образования иммунных лейкоцитов, специфичных к белку NSP, у мышей после инфекции вирусом гриппа А. Показано, что у мышей после заражения последовательно двумя вирусами гриппа А разных сероподтипов обнаруживаются иммунные лейкоциты, специфически распознающие вирусные домены в центральной зоне белка NSP (позиции аминокислот 82-119). Полученные данные с большой вероятностью позволяют утверждать, что при инфекции вирусом гриппа А в организме животных имеет место экспрессия гена NSP, что в свою очередь подтверждает концепцию о биполярной (амбисенс) стратегии генома вируса гриппа А.

Catalytic inactivation of influenza virus by iron oxide nanozyme.

Influenza poses a severe threat to human health in the world. However, developing a universal anti-viral strategy has remained challenging due to the presence of diverse subtypes as well as its high mutation rate, resulting in antigenic shift and drift. Here we developed an antiviral strategy using iron oxide nanozymes (IONzymes) to target the lipid envelope of the influenza virus.Methods: We evaluated the antiviral activities of our IONzymes using a hemagglutination assay, together with a 50% tissue culture infectious doses (TCID50) method. Lipid peroxidation of the viral envelope was analyzed using a maleic dialdehyde (MDA) assay and transmission electron microscopy (TEM). The neighboring viral proteins were detected by western blotting.Results: We show that IONzymes induce envelope lipid peroxidation and destroy the integrity of neighboring proteins, including hemagglutinin, neuraminidase, and matrix protein 1, causing the inactivation of influenza A viruses (IAVs). Furthermore, we show that our IONzymes possess a broad-spectrum antiviral activity on 12 subtypes of IAVs (H1~H12). Lastly, we demonstrate that applying IONzymes to a facemask improves the ability of virus protection against 3 important subtypes that pose a threat to human, including H1N1, H5N1, and H7N9 subtype.Conclusion: Together, our results clearly demonstrate that IONzymes can catalyze lipid peroxidation of the viral lipid envelope to inactivate enveloped viruses and provide protection from viral transmission and infection.

The selection pressure on the neuraminidase gene of influenza viruses isolated in Ukraine from 2009 to 2015

ABSTRACT A broad range of naturally occurring antigenic variants of the influenza virus is caused by its rapid evolutionary variability. The survival of viable influenza virus variants occurs through natural selection. The treatment of influenza infection with modern antiviral drugs – neuraminidase (NA) inhibitors – leads to the occurrence of mutations in the NA gene, which thereby result in the emergence of virus resistance to these drugs. The goal of this study was to determine the selection pressure on the NA protein of influenza viruses isolated in Ukraine from 2009 to 2015. The main method for assessing the selection pressure on proteins is to quantify the ratio of substitution rates at nonsynonymous (dN) and synonymous (dS) sites. With the help of this method, we showed that only a few codons in the NA gene were under positive selection resulting in mutations at the following sites: for influenza A viruses of the A(H1N1)pdm09 subtype – site 40, for viruses of the A(H3N2) subtype – sites 93 and 402, for Influenza B viruses of the B/Yamagata lineage – sites 74, 99, and 268, and for the viruses of the B/Victoria lineage – sites 358, 288, and 455. These sites are not associated with the NA active site, transmembrane domain, or the antigenic sites of this protein. We concluded that NA inhibitors are not a significant factor in the process of selection of the influenza viruses in Ukraine because the sites associated with the resistance of influenza viruses to NA inhibitors were not affected by positive selection. This finding could be explained by the limited use of NA inhibitors for the treatment of influenza infections in Ukraine.

An Infant Mouse Model of Influenza Virus Transmission Demonstrates the Role of Virus-Specific Shedding, Humoral Immunity, and Sialidase Expression by Colonizing Streptococcus pneumoniae.

The pandemic potential of influenza A viruses (IAV) depends on the infectivity of the host, transmissibility of the virus, and susceptibility of the recipient. While virus traits supporting IAV transmission have been studied in detail using ferret and guinea pig models, there is limited understanding of host traits determining transmissibility and susceptibility because current animal models of transmission are not sufficiently tractable. Although mice remain the primary model to study IAV immunity and pathogenesis, the efficiency of IAV transmission in adult mice has been inconsistent. Here we describe an infant mouse model that supports efficient transmission of IAV. We demonstrate that transmission in this model requires young age, close contact, shedding of virus particles from the upper respiratory tract (URT) of infected pups, the use of a transmissible virus strain, and a susceptible recipient. We characterize shedding as a marker of infectiousness that predicts the efficiency of transmission among different influenza virus strains. We also demonstrate that transmissibility and susceptibility to IAV can be inhibited by humoral immunity via maternal-infant transfer of IAV-specific immunoglobulins and modifications to the URT milieu, via sialidase activity of colonizing Streptococcus pneumoniae Due to its simplicity and efficiency, this model can be used to dissect the host's contribution to IAV transmission and explore new methods to limit contagion.IMPORTANCE This study provides insight into the role of the virus strain, age, immunity, and URT flora on IAV shedding and transmission efficiency. Using the infant mouse model, we found that (i) differences in viral shedding of various IAV strains are dependent on specific hemagglutinin (HA) and/or neuraminidase (NA) proteins, (ii) host age plays a key role in the efficiency of IAV transmission, (iii) levels of IAV-specific immunoglobulins are necessary to limit infectiousness, transmission, and susceptibility to IAV, and (iv) expression of sialidases by colonizing S. pneumoniae antagonizes transmission by limiting the acquisition of IAV in recipient hosts. Our findings highlight the need for strategies that limit IAV shedding and the importance of understanding the function of the URT bacterial composition in IAV transmission. This work reinforces the significance of a tractable animal model to study both viral and host traits affecting IAV contagion and its potential for optimizing vaccines and therapeutics that target disease spread.

Recombinant Newcastle disease virus (NDV) La Sota expressing the haemagglutinin–neuraminidase protein of genotype VII NDV shows improved protection efficacy against NDV challenge

ABSTRACTIntensive vaccination strategies against Newcastle disease (ND) have been implemented in many countries for a long time, but ND outbreaks still occur frequently, with most isolates belonging to genotype VII of Newcastle disease virus (NDV). Many researchers have revealed that vaccines closely matched to epidemic viruses provide better protection. Therefore, using a previously established reverse genetics system, we generated a recombinant NDV vaccine strain (rLa Sota-HN) based on the La Sota vaccine strain expressing the haemagglutinin–neuraminidase (HN) protein of genotype VII NDV. The pathogenicity of the recombinant virus was confirmed by the mean death time in 9-day-old specific-pathogen-free embryonated chicken eggs and the intracerebral pathogenicity index in 1-day-old specific-pathogen-free chickens. Subsequently, 1-day-old chickens were immunized with commercial vaccine La Sota and recombinant virus rLa Sota-HN and then challenged with virulent genotype VII NDV strain. The results indicated that recombinant virus rLa Sota-HN provided increased protection of vaccinated chickens from morbidity and mortality, and inhibited the shedding of virulent virus after challenging with genotype VII virus, compared with the conventional vaccine La Sota. Our findings indicated that rLa Sota-HN is a promising vaccine candidate to improve the protection efficiency against ND in chickens, thereby preventing frequent outbreaks of this disease.

Immune Correlates of Protection Induced by Virus-Like Particles Containing 2009 H1N1 Pandemic Influenza HA, NA or M1 Proteins

ABSTRACTInfluenza virus-like particle (VLPs) vaccines are a promising alternative to conventional egg-based vaccines. Evaluation of vaccine efficacy induced by HA-M1 VLPs, NA-M1 VLPs or M1 VLPs against virus challenge infection would provide important insight into vaccine design strategy. In this study, we generated VLPs containing hemagglutinin (HA), neuraminidase (NA) or M1 proteins derived from the A/California/04/09. Mice were immunized intramuscularly with HA-M1, NA-M1 or M1 VLPs and protective immunity was evaluated by assessing lung virus loads against low (5LD50) or high (100LD50) lethal dose of homologous virus challenges. High levels of virus-specific serum IgG antibody responses were induced in mice after HA-M1 VLPs immunization, whereas low or no IgG antibody responses were detected from immunization with NA-M1 VLPs or M1 VLPs, independently. Mice that were immunized with HA-M1 VLPs showed below the limit of detection on lung virus loads against low dose (5LD50) of challenge and significant reduction against high dose (100LD50) of challenge infection. Mice that were immunized with NA-M1 or M1 VLPs also displayed reduced lung viral loads compared to naïve control. In vitro cultures of cells from mouse spleen and bone marrow revealed that HA-M1 VLPs and NA-M1 VLPs induced higher levels of antibody-secreting cell (ASC) responses compared to naïve control, whereas M1 VLPs showed no ASC responses. HA-M1, NA-M1 or M1 VLPs immunization demonstrated varying degree of protection with respect to body weight changes and survival rates, which are consistent with the levels of antibody responses in sera and ASC responses from spleen and bone marrow.

Genetic characteristics of hemagglutinin and neuraminidase of avian influenza A (H7N9) virus in Guizhou province, 2014-2017

Objective: To understand the molecular characteristics of hemagglutinin (HA) and neuraminidase (NA) as well as the disease risk of influenza virus A H7N9 in Guizhou province. Methods: RNAs were extracted and sequenced from HA and NA genes of H7N9 virus strains obtained from 18 cases of human infection with H7N9 virus and 6 environmental swabs in Guizhou province during 2014-2017. Then the variation and the genetic evolution of the virus were analyzed by using a series of bioinformatics software package. Results: Homology analysis of HA and NA genes revealed that 2 strains detected during 2014-2015 shared 98.8%-99.2% and 99.2% similarities with vaccine strains A/Shanghai/2/2013 and A/Anhui/1/2013 recommended by WHO, respectively. Two strains detected in 2016 and 14 strains detected in 2017 shared 98.2%-99.3% and 97.6%-98.8% similarities with vaccine strain A/Hunan/02650/2016, respectively. Other 6 stains detected in 2017 shared 99.1%-99.4% and 98.9%-99.3% similarities with strain A/Guangdong/17SF003/2016, respectively. Phylogenetic analysis showed that all the strains were directly evolved in the Yangtze River Delta evolution branch, but they were derived from different small branch. PEVPKRKRTAR↓GLF was found in 6 of 24 strains cleavage site sequences of HA protein, indicating the characteristic of highly pathogenic avian influenza virus. Mutations A134V, G186V and Q226L at the receptor binding sites were found in the HA. All the strains had a stalk deletion of 5 amino acid residue "QISNT" in NA protein, and drug resistance mutation R294K occurred in strain A/Guizhou-Danzhai/18980/2017. In addition, potential glycosylation motifs mutations NCS42NCT were found in the NA of 9 of 24 strains. Conclusions: HA and NA genes of avian influenza A (H7N9) virus showed genetic divergence in Guizhou province during 2014-2017. The mutations of key sites might enhance the virulence of the virus, human beings are more susceptible to it. Hence, the risk of infection is increasing.

Full sequence analysis of hemagglutinin and neuraminidase genes and proteins of highly pathogenic avian influenza H5N1 virus detected in Iran, 2015.

Over the last two decades, the highly pathogenic avian influenza H5N1 virus has gained a lot of attention due to its zoonotic and mutative nature. Iran is among the countries significantly affected by the virus as it hosts migratory birds during seasonal migration. In this study, the molecular characterizations of hemagglutinin (HA) and neuraminidase (NA) genes and proteins of H5N1 strain A/chicken/Iran/8/2015 detected in backyard poultry, Mazandaran province, were investigated. Phylogenetic analysis classified this virus as a member of subclade 2.3.2.1c, with the cleavage site motif of "PQRERRRK-R/GLF". HA carried a few mutations altering affinity to mammalian cells; however, the virus was categorized as avian. NA protein had the 20-amino acid deletion at aa position 49-69 similar to those isolated since 2000. Mutations of H253Y and H274Y contributing to antiviral resistance were present in NA. From this analysis, it can be concluded that the wild migratory birds flying from Western Asia to Eastern Africa are probably the main carriers of seasonal H5N1 in the country.