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Orthomyxoviridae: the viruses and their replication, Fields BN, Knipe RM, Chanock MS, Fields Virology
... The disease-causing virus belongs to the Orthomyxoviridae family's genus Alphainfluenzavirus. This virus's genome is fragmented by 13.5 kb, includes single-stranded RNA, and is negative sense (Lamb, 2001;Maclachlan and Dubovi, 2010). Differences in the matrix (M1) protein and nucleoprotein (NP) lead to classification into four genera: A, B, C, and recently discovered D (Capua and Alexander, 2007). ...
... Differences in the matrix (M1) protein and nucleoprotein (NP) lead to classification into four genera: A, B, C, and recently discovered D (Capua and Alexander, 2007). Influenza A virus only infects birds (Lamb, 2001). ...
... These viruses were divided into four genera-A, B, C, and recently discovered D-based on variations in their matrix (M1) protein and nucleoprotein (NP) (Capua & Alexander, 2007). Only birds can contract a flu virus (Lamb, 2001). These viruses' neuraminidase (NA) and hemagglutinine (HA) spike structures enabled further subtyping into one of 16 antigenically distinct HA subtypes (H1 to H16) and one of nine NA subtypes (N1 to N9), while two subtypes found in bats (subtypes H17N10 and H18N11) represented the remaining 2 HA (17 to 18) and 2 NA subtypes (Tzarum et al., 2017). ...
This study was done at the college of Veterinary Medicine, University of Diyala, from September 2021 to April 2022 to assess the seroprevalence and molecular detection of avian influenza virus (AIV) H5N8 in layer chickens. All samples came from 8 commercial flocks of layer chickens. Clinical sings included, severe respiratory infection with enlargement of the head, neck, and prominent edema of the face, followed by excessive lacrimation, sinusitis, severe rales, and cyanosis in all parts of unfeather skin predominantly the wattles comb, and the legs. In this regard, ELISA was used as a serological test and RT-PCR as a molecular detection technique. Accordingly, (364) serum samples from 8 probable AIV (H5N8) infected layer farms were collected and subjected to ELISA test using commercial kit that specific for IgG antibodies to AIV H5N8. The results showed a significant increase of IgG antibodies in such serum samples at days 70 and 200 of age. According to the instruction manual of ELISA kit manufacturer and to mean titers of layers of present study, all farms are infected with AIVH5N8 strain. However, at the age of 200 days, 48 postmortem tissue samples including trachea, lung and liver of affected birds were collected from clinically and sub-clinically infected flocks from all farms. These samples were processed, RNA extracted and submitted to RT-PCR using specific primers for H5N8 strain. The results showed that 32 out of the 48 tissue samples (66.6%) tested positive for H5N8.The resulted Amplicon (320bp) was commercially sequenced and analyzed. The sequencing of the local AIV H5N8 revealed a 99% sequence identity with the reference sequences. The detected strain was registered in GenBank data (NCBI) under acc.number ON247929.1. Phylogenetic tree for locally detected virus in comparison to data from of NCBI was created and showed that the investigated S1(ON247929.1, AIV (A/laying hens/Iraq/(H5N8) segment 4 hemagglutinin (HA) gene, partial cds, local sample is closely related to reference isolates from the NCBI acc. no. of MW961428.1, MW961444.1, MW961476.1, MW961436.,1 and MW961484.1. These strains of the Influenza H5 virus have been deposited from Nigeria.
... The family Paramyxoviridae is a large group of enveloped, non-segmented negative stranded RNA viruses that include many important human pathogens, such as human parainfluenza viruses (hPIV) serotypes 1–4, mumps virus, measles virus, and human respiratory syncytial viruses (RSV). As a group the hPIVs and RSV are the leading cause of respiratory diseases in children [1][2][3]. The family is divided into two subfamilies, Paramyxovirinae and Pneumovirinae. ...
... Thus, immunity that develops through natural infection is not sufficient to provide complete protection. In infants and children, especially in the first six months of life, parainfluenza viruses are the most important causes of croup (hPIV1) and serious causes of bronchiolitis and pneumonia (hPIV3) [1,3,4]. Effective virus vaccines are available for only mumps and measles viruses. ...
... The Respirovirus and Rubulavirus genera have two surface glycoproteins, the hemaggltuinin-neuraminidase (HN) and the fusion protein (F). HN has three functions: (i) it recognizes sialic acid containing receptors on cell surfaces, (ii) it promotes the fusion activity of the F protein allowing the virus to penetrate the cell surface, and (iii) it acts as a neuraminidase (sialidase), removing sialic acids from progeny virus particles to prevent viral self-agglutination thereby aiding viral spread [3]. As paramyxovirus neuraminidases have an acid optimum (pH 4.8 to 5.5), it is likely that the neuraminidase acts in the trans Golgi network, removing sialic acid from glycosylated HN and F proteins destined for the cell membrane [3]. ...
Viruses of the Paramyxoviridae family are the leading cause of respiratory disease in children. The human parainfluenza viruses (hPIV) are members of the Paramyxovirinae subfamily, which also includes mumps virus, Newcastle disease virus (NDV), Sendai virus (SV) and simian type 5 virus (SV5). On the surface of these viruses is the glycoprotein hemagglutinin-neuraminidase (HN), which is responsible for cell attachment, promotion of fusion and release of progeny virions. This multifunctional nature of HN makes it an attractive target for the development of inhibitors as a treatment for childhood respiratory diseases. Here we report the crystal structure of NDV HN in complex with a derivative of 2-deoxy-2,3-dehydro-N-acetylneuraminic acid, Neu5Ac2en, that has a functional group designed to occupy a large conserved binding pocket around the active site. The purpose of this study was to examine the effect of a bulky hydrophobic group at the O4 position of Neu5Ac2en, given the hydrophobic nature of the binding pocket. This derivative, with a benzyl group added to the O4 position of Neu5Ac2en, has an IC(50) of approximately 10 microM in a neuraminidase assay against hPIV3 HN. The IC(50) value of the parent compound, Neu5Ac2en, in the same assay is approximately 25 microM. These results highlight the striking difference between the influenza neuraminidase and paramyxovirus HN active sites, and provide a platform for the development of improved HN inhibitors.
... Seal et al., 2000b) Paramyxovirion are pleomorphic, enveloped, and roughly spherical ranging between 150 to 400nm in size (Dubois-Dalcq et al., 1984). The envelope membrane contains 8-12nm glycoprotein spikes (Lamb & Kolakofsky, 2001). The NDV genome consists of a negative-sense; single-stranded RNA molecule of 5.2 to 5.7 x 10 6 Daltons molecular weight (Kolakofsky et al., 1974) and replicates entirely in the cytoplasm of host cells (Lamb & Kolakofsky, 2001). ...
... The envelope membrane contains 8-12nm glycoprotein spikes (Lamb & Kolakofsky, 2001). The NDV genome consists of a negative-sense; single-stranded RNA molecule of 5.2 to 5.7 x 10 6 Daltons molecular weight (Kolakofsky et al., 1974) and replicates entirely in the cytoplasm of host cells (Lamb & Kolakofsky, 2001). The genome has 15,186; 15,192 or 15,198 nucleotides (Phillips et al., 1998; Huang et al., 2004a; Czeglédi et al., 2006). ...
... (L) in the 3'-NP-P-M-F-HN-L-5' order (Lamb & Kolakofsky, 1996). Paramyxoviridae accessory genes V and W occur mostly as ORFs (open reading frames) that overlap within the P gene transcriptional unit (Lamb and Kolakofsky, 2001). The nucleocapsid protein (NP) is the major structural unit of the nucleocapsid (Samson, 1988). ...
... The CDSs with the highest dN/dS values were those for the G, M2-2, and SH CDSs. Interestingly, the fact that the SH, G, and M2-2 CDSs have the highest ratios of non-synonymous mutations within each subgroup correlates with the fact that these three proteins also have the lowest amino acid identity between RSV A and B [34]. In RSV B 59% of the variable sites are accounted for by differences between the published sequences from strains that date back more than 26 years and our sequences (2006–2010). ...
... The degree of variation in the intergenic sequences seen for the RSV Milwaukee strains group A and group B agreed with previous studies on RSV A clinical isolates from the USA and UK [36]. Unlike some Paramyxoviruses that present conserved or semi-conserved di-or trinucleotide IGS patterns [34,535455 RSV shows longer and more variable IGS regions. In RSV, transcription starts at a single promoter located at the 39 end of the genome. ...
Respiratory Syncytial Virus (RSV) is the leading cause of lower respiratory-tract infections in infants and young children worldwide. Despite this, only six complete genome sequences of original strains have been previously published, the most recent of which dates back 35 and 26 years for RSV group A and group B respectively.
We present a semi-automated sequencing method allowing for the sequencing of four RSV whole genomes simultaneously. We were able to sequence the complete coding sequences of 13 RSV A and 4 RSV B strains from Milwaukee collected from 1998-2010. Another 12 RSV A and 5 RSV B strains sequenced in this study cover the majority of the genome. All RSV A and RSV B sequences were analyzed by neighbor-joining, maximum parsimony and Bayesian phylogeny methods. Genetic diversity was high among RSV A viruses in Milwaukee including the circulation of multiple genotypes (GA1, GA2, GA5, GA7) with GA2 persisting throughout the 13 years of the study. However, RSV B genomes showed little variation with all belonging to the BA genotype. For RSV A, the same evolutionary patterns and clades were seen consistently across the whole genome including all intergenic, coding, and non-coding regions sequences.
The sequencing strategy presented in this work allows for RSV A and B genomes to be sequenced simultaneously in two working days and with a low cost. We have significantly increased the amount of genomic data that is available for both RSV A and B, providing the basic molecular characteristics of RSV strains circulating in Milwaukee over the last 13 years. This information can be used for comparative analysis with strains circulating in other communities around the world which should also help with the development of new strategies for control of RSV, specifically vaccine development and improvement of RSV diagnostics.
... Influenza as a member of the Orthomyxoviridae family is an enveloped virus, and contains a segmented RNA genome (8 segments encoding for at least 11 proteins) of negative polarity. The segmented nature of the genome allows for the generation of viral diversity [1]. Influenza can be subdivided into three types (A, B and C). ...
... The goal of the influenza vaccine is to induce antigen-specific memory T and B cells. The memory B cells secrete high-affinity neutralizing antibodies to HA and NA [1]. The memory T-cell compartment, consisting of CD4+ and CD8+ T cells, can quickly respond by CD8+ T cells acquiring effector functions to kill infected cells and secrete pro-inflammatory cytokines that inhibit virus replication and CD4+ T helper cells in aiding B-cells and CD8+ T cells in their functions [8]. ...
There is intense interest in the design and use of vaccine strategies against influenza to enhance protective immune responses in the elderly. To address the need for improved influenza vaccines for the aged, two inflammatory adjuvants, Imject(®) alum (a stimulator of the Nod-like receptor, Nalp3) and poly I:C (a toll-like receptor type 3 ligand), were used during vaccination with novel influenza virus-like particles (VLP). Adult (4 month old) or aged (24 month old) mice were vaccinated with VLPs alone or in combination with adjuvant. VLP-vaccinated adult mice were protected from a lethal influenza virus challenge without the use of either adjuvant. In contrast, only aged mice that were vaccinated with VLPs plus adjuvant survived challenge, whereas ∼33% of the mice vaccinated with VLP only survived challenge. Mice vaccinated with adjuvant only did not survive challenge despite similar levels of activation of CD11b(+)/CD11c(+) dendritic cells in the lungs. The protection was not associated with HAI titers or HA specific CD8(+) T cells, since both adjuvants boosted the VLP-induced serum HAI titers and CD8(+) responses in adult mice, but not aged mice. Influenza VLPs used in combination with two different inflammatory adjuvants during vaccination allow for the immune system to overcome the deficiency in the aged immune system to influenza virus infection.
... 4) with a growth rate (r Flu ) of 0.075 h − 1 . NS1 is highly abundant at early times post-infection, but appears relatively dynamic (Lamb et al., 2001), leading us to scale the NS1 growth rate (r NS1 ) at two-thirds the value of r Flu . We derived a carrying capacity (K) of 100 PFU for influenza virus within a cell based on the observation that 10 5 mouse embryo fibroblasts release 10 7 PFU/ml at 24 h post-infection (Goodman et al., 2010). ...
... Because NS1 inhibits PKR phosphorylation by 24 h p.i during an infection at 2 PFU/ml (Bergmann et al., 2000) but P58 IPK inhibits PKR phosphorylation by 8 h post-infection during an infection at 1 PFU/ml (Goodman et al., 2007), r PKR,21,P58 was greater than r PKR,21,NS1 . While half of the parameter values (r PKR,21,P58 , r PKR,21,NS1 , r Flu , r NS1 , and K) were derived from the literature (Borland and Mahy, 1968;Goodman et al., 2010;Lamb et al., 2001;Lee et al., 1994b;Lu et al., 1995), a weighted, non-linear least squares optimization algorithm was used to derive the remaining parameter values (r PKR,12,Flu , r P58,12 , r P58,21 , r eIF2α,12 , and r eIF2α,21 ). ...
Previously we showed that the cellular protein P58(IPK) contributes to viral protein synthesis by decreasing the activity of the anti-viral protein, PKR. Here, we constructed a mathematical model to examine the P58(IPK) pathway and investigated temporal behavior of this biological system. We find that influenza virus infection results in the rapid activation of P58(IPK) which delays and reduces maximal PKR and eIF2α phosphorylation, leading to increased viral protein levels. We confirmed that the model could accurately predict viral and host protein levels at extended time points by testing it against experimental data. Sensitivity analysis of relative reaction rates describing P58(IPK) activity and the downstream proteins through which it functions helped identify processes that may be the most beneficial targets to thwart virus replication. Together, our study demonstrates how computational modeling can guide experimental design to further understand a specific metabolic signaling pathway during viral infection in a mammalian system.
... When an Italian scientist, Edoardo Perroncito, reported what is believed to be the first documented evidence of "fowl plague" as a distinct disease which was reviewed in (Capua and Alexander, 2004). Avian influenza is caused by the infection with virus belong to Orthomyxoviridae and it was classified as influenza virus A genus (Lamb & Krug, 1996). Many bird species have been shown to be susceptible to infection with influenza A viruses. ...
... Figure 1 gives a general overview of the complete computational framework. The computer implementation of SFA has three main classes of parameters: i) values known from the standard immunology literature (Abbas et al., 2014); ii) parameters strictly correlated with the specific biological scenario we want to simulate, i.e. parameters that measure the influenza A virosome dynamics, matching its behavior and its interactions with those of the host immune system (Carrat et al., 2008;Doherty et al., 2006;Grayson and Holtzman, 2007;Baccam et al., 2006;Lamb and Krug, 2001); iii) parameters with unknown values which we set to plausible values after performing a series of tests. Table 1 details the values of the parameters retrieved from the literature, along with the specific parameters of the influenza A virosome. ...
Motivation:
Vaccines represent the most effective and cost-efficient weapons against a wide range of diseases. Nowadays new generation vaccines based on sub-unit antigens reduce adverse effects in high risk individuals. However, vaccine antigens are often poor immunogens when administered alone. Adjuvants represent a good strategy to overcome such hurdles, indeed they are able to: enhance the immune response; allow antigens sparing; accelerate the specific immune response; and increase vaccine efficacy in vulnerable groups such as newborns, elderly or immuno-compromised people. However, due to safety concerns and adverse reactions, there are only a few adjuvants approved for use in humans. Moreover, in practice current adjuvants sometimes fail to confer adequate stimulation. Hence, there is an imperative need to develop novel adjuvants that overcome the limitations of the currently available licensed adjuvants.
Results:
We developed a computational framework that provides a complete pipeline capable of predicting the best citrus-derived adjuvants for enhancing the immune system response using, as a target disease model, influenza A infection. In silico simulations suggested a good immune efficacy of specific citrus derived adjuvant (Beta Sitosterol) that was then confirmed in vivo.
Availability:
The model is available visiting the following URL: http://vaima.dmi.unict.it/AdjSim CONTACT: francesco.pappalardo@unict.it SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
... SeV has a single-stranded, negative-sense RNA genome $15–16 kb in length which codes for six structural proteins: two glycoproteins hemagglutinin-neuraminidase (HN) and fusion protein (F), a matrix protein (M), and three nucleocapsid proteins , Nucleoprotein (N), Phosphoprotein (P), and Large polymerase protein (L). The P gene which codes for the P protein also codes for an additional set of up to seven different nested polyproteins (P, V, W, C', C, Y1, and Y2) and may be referred to as 'P/V/C' (Lamb and Parks 2007). The HN glycoprotein plays critical role in mediating the virus entry into host cell and structural modeling was used to assess the impact mutations in this gene may have on protein function. ...
In vivo serial passage of non-pathogenic viruses has been shown to lead to increased viral virulence, and although the precise mechanism(s) are not clear, it is known that both host and viral factors are associated with increased pathogenicity. Under- or overnutrition leads to a decreased or dysregulated immune response and can increase viral mutant spectrum diversity and virulence. The objective of this study was to identify the role of viral mutant spectra dynamics and host immunocompetence in the development of pathogenicity during in vivo passage. Because the nutritional status of the host has been shown to affect the development of viral virulence, the diet of animal model reflected two extremes of diets which exist in the global population, malnutrition and obesity. Sendai virus was serially passaged in groups of mice with differing nutritional status followed by transmission of the passaged virus to a second host species, guinea pigs. Viral population dynamics were characterized using deep sequence analysis and computational modeling. Histopathology, viral titer and cytokine assays were used to characterize viral virulence. Viral virulence increased with passage and the virulent phenotype persisted upon passage to a second host species. Additionally, nutritional status of mice during passage influenced the phenotype. Sequencing revealed the presence of several non-synonymous changes in the consensus sequence associated with passage, a majority of which occurred in the hemagglutinin-neuraminidase and polymerase genes, as well as the presence of persistent high frequency variants in the viral population. In particular, an N1124D change in the consensus sequences of the polymerase gene was detected by passage 10 in a majority of the animals. In vivo comparison of an 1124D plaque isolate to a clone with 1124N genotype indicated that 1124D was associated with increased virulence.
... Influenza virus were enveloped ribonucleic acid viruses belonging to the family of orthomyxoviridae and were divided into 3 distinct types on the basis of antigenic differences of internal structural proteins. 15 Two influenza types, Type A and Type B, were responsible for yearly epidemic outbreaks of respiratory illness in humans and were further classified based on the structure of 2 major external glycoproteins, hemagglutinin (HA) and neuraminidase (NA). Type A strains infected a wide variety of avian and mammalian species. ...
To evaluate antibody persistence of Aleph inactivated split influenza vaccine, 3308 healthy Chinese people more than 3 years old were enrolled in a hemagglutination inhibition (HI) assay before vaccination, 641 were screened by HI assay negative, 437 of which received one dose of Aleph inactivated split influenza vaccine and 204 of which received one dose of control vaccine (recombinant hepatitis B). After vaccination, the receivers were collected blood at 1st month, 3rd month, 6th month and 12th month for Aleh influenza vaccine antibody persistence assess. The antibody test were determined by hemagglutination inhibition (HI) assay. There were significant difference in antibody geometric mean titer between experimental group and control at 1st month and 3rd month after vaccination. Influenza antibody could persist at least up to 3rd month. Because of the local spring influenza epidemic, we could not analyze the results of 6th and 12th month. Aleph influenza vaccines showed good immune persistence in healthy volunteers at least in the 3 months after vaccination. Influenza viruses are important human respiratory pathogens. Immunization is widely acknowledged to currently be the most effective method of minimizing the impact of pandemic influenza. Through we have checked many references about Influenza vaccine, the duration of protective antibody for influenza vaccines are still not available. Based on this situation and our previous work,1111.
Cheng K, Shen X, Yang S, Zhou Z, Xie J, Chen W, Weng Y, Yan Y. Research on growth and decline of antibody in H1N1 vaccine serum. Chinese Zoonosis 2012; 06:566-9View all references Influenza vaccine antibody duration analyze are necessary. This manuscript presents data on the persistence of Hemagglutination Inhibition (HI) immune response against the A/California/7/2009(H1N1), A/Peth/16/2009(H3N2) strain and B/Brisbane/60/2008. 641 were screened from 3302 volunteers by HI test of influenza A and confirmed enrollment based on the antibodies titer less than 1:10. After administered with one dose of Aleph influenza vaccine, blood samples were collected. 437 subjects (3–10y: 131; 11–17y: 110; 18–54y: 69; ≥55y: 127) were vaccinated influenza vaccine as test group. 204 subjects (3–10 y: 70; 11–17 y: 47; 18–54 y: 28; ≥55 y: 59) were vaccinated recombinant hepatitis B vaccine as control group. Immunogenicity end points were based on the European licensure criteria for pandemic influenza vaccines. The persistence of HI immune response against the vaccine strain was assessed through GMT. The immunogenicity of the Aleph influenza vaccine induced all reached the standards at 1st month and GMTs peak could persist at least up to 3rd month. (This study has been registered at clinicaltrials.gov under registration no. NCT01758185.) Because of the local spring influenza epidemic we could not analyze the results of 6th and 12th month. Aleph influenza vaccines showed good immune persistence in healthy volunteers at least in the 3 months after vaccination.
... CDV is a pleomorphic 150-250 nm diameter virion, with a single negative-stranded RNA that is enclosed in a helically symmetrical nucelocapsid (GREENE; APPEL, 2006; MACLACHLAN; DUBOVI, 2011). The virion proteins of CDV include three nucleocapsid proteins: an RNAbinding protein (N) previously referred to as nucleocapsid protein (NP), a phosphoprotein (P), and a polymerase protein (L); and three membrane proteins: a matrix protein (M); one fusion (F), and an attachment hemagglutinin protein (H) (LAMB; KOLAKOFSKY, 2001; MACLACHLAN; DUBOVI, 2011). The H protein is fundamental for infection, since it recognizes compatible ligands on the surface of the host cell (MACLACHLAN; DUBOVI, 2011), and activates the F protein by tissues-specific proteases resulting in infection (McCARTHY; SHAW; GOODMAN, 2007). ...
This review provides a critical review of current epidemiological trends of canine distemper virus (CDV) and syndromes related to canine distemper encephalitis (CDE) with specific reference to the situation in Brazil. Epidemiological data relative to susceptible animal populations, prevalence, seasonal occurrence, and age-related patterns associated with CDV are discussed. The participation of mongrel dogs in maintaining CDV within rural and semi-urban canine populations and their importance in the epidemiology of canine distemper is highlighted. The economic impact of treating the clinical manifestations associated with CDV-induced infections in Brazil is estimated. Additionally, neurological and neuropathological manifestations of CDV in Brazil are discussed, and a novel manifestation of CDE is proposed.
... As the first line of defense, APC and infected cells stimulate the innate immunity by secreting interferon α and β (IFN) molecules (Julkunen et al., 2000;Lamb R, 1996;Ronni et al., 1995;Sareneva et al., 1998), (Stark et al., 1998) which interact with healthy cells and convert them to an infection resistant state, thereby preventing the virus from spreading efficiently and allowing the adaptive immune response enough time to develop and eliminate the virus (Price et al., 2000). Another role of IFN is to stimulate symptoms such as fever which occurs in the early stages of infection. ...
... Nukleokapsida virusa oblikuje se u citoplazmi, a ovojnica virusa na površini stanice. Novi virion napušta stanicu domaćina pupanjem na staničnoj opni [22]. ...
... Mumps is a vaccine preventable childhood disease that tends to be mild; about 30 % of infections are asymptomatic. Transmission occurs through inhalation of respiratory droplets or by direct person-to-person contact, reinfection may occur either after natural infection or vaccination [13, 28]. The mumps virus (MuV) belongs to genus Rubulavirus, subfamily Paramyxovirinae, family Paramyxoviridae and order Mononegavirales. ...
Mumps is an acute and self-limiting disease characterized by parotitis, however in some cases it leads to aseptic meningitis, deafness, encephalitis and orchitis, which is a serious health concern. MMR vaccination was successful in eradicating the disease however, recent reports question the efficacy of MMR vaccine and countless outbreaks are observed in vaccinated populations throughout the world. Lack of specific treatment methods for mumps infection and inefficiency of MMR vaccine in vaccinated populations accentuates the need for the development of novel drugs to control mumps virus mediated serious infections. It was with this backdrop of information that the anti-mumps virus activity of Mimosa pudica was evaluated. Suspected mumps cases were collected to isolate a standard mumps virus by systematic laboratory testing which included IgM antibody assays, virus isolation, RT-PCR and phylogenetic analysis. The virus was quantified by TCID50 assay and anti-mumps virus property was evaluated by CPE reduction assay and cytotoxicity of the extract was measured by MTT assay and phytochemical analysis was done by gas chromatography-mass spectroscopy. The RT-PCR and phylogenetic tree analysis of the SH gene sequence of the clinical isolate showed it to be mumps virus genotype C. 150 μg/ml concentration of M. pudica completely inhibited mumps virus and the drug was found to be non-toxic up to 2 mg/ml. M. pudica was thus found to be a potent inhibitor of MuV.
... Transmission occurs through inhalation of respiratory droplets or by direct person-to-person contact, reinfection may occur either after natural infection or vaccination. [1,2] Although it is generally believed that mumps virus (MuV) is serologically monotypic, distinct genetic lineages of wild-type MuVs have been described and reported to be co-circulating globally. Recently World Health Organisation (WHO) had updated a proposal for standard nomenclature to describe genetic characteristics and emphasised the need for expanding virological surveillance of wild-type MuV, which was released in June 2012, having 12 genotypes A-N (namely A, B, C, D, F, G, H, I, J, K, L, N). ...
... Transmission occurs through inhalation of respiratory droplets or by direct person-to-person contact, reinfection may occur either after natural infection or vaccination. [1,2] Although it is generally believed that mumps virus (MuV) is serologically monotypic, distinct genetic lineages of wild-type MuVs have been described and reported to be co-circulating globally. Recently World Health Organisation (WHO) had updated a proposal for standard nomenclature to describe genetic characteristics and emphasised the need for expanding virological surveillance of wild-type MuV, which was released in June 2012, having 12 genotypes A-N (namely A, B, C, D, F, G, H, I, J, K, L, N). ...
... Transmission occurs through inhalation of respiratory droplets or by direct person-to-person contact, reinfection may occur either after natural infection or vaccination. [1,2] Although it is generally believed that mumps virus (MuV) is serologically monotypic, distinct genetic lineages of wild-type MuVs have been described and reported to be co-circulating globally. Recently World Health Organisation (WHO) had updated a proposal for standard nomenclature to describe genetic characteristics and emphasised the need for expanding virological surveillance of wild-type MuV, which was released in June 2012, having 12 genotypes A-N (namely A, B, C, D, F, G, H, I, J, K, L, N). ...
... In this paper, we discuss the property of symmetric items in transaction database, and then present an efficient algorithm to find all symmetric item sets using ZBDDs. We also show the experimental result for an actual biological database on the amino acid sequences of influenza viruses [8] [6]. We found a number of symmetric items from the database, some of which indicate an interesting relationship of amino acid mutation patterns. ...
(Abstract) In this paper, we present a method of finding symmetric items in a combinatorial item set database. The techniques for finding symmet- ric variables in Boolean functions have been studied for long time in the area of VLSI logic design, and the BDD (Binary Decision Diagram) -based meth- ods are presented to solve such a problem. Re- cently, we have developed an efficient method for handling databases using ZBDDs (Zero-suppressed
... Transmission occurs through inhalation of respiratory droplets or by direct person-to-person contact, reinfection may occur either after natural infection or vaccination. [1,2] Although it is generally believed that mumps virus (MuV) is serologically monotypic, distinct genetic lineages of wild-type MuVs have been described and reported to be co-circulating globally. Recently World Health Organisation (WHO) had updated a proposal for standard nomenclature to describe genetic characteristics and emphasised the need for expanding virological surveillance of wild-type MuV, which was released in June 2012, having 12 genotypes A-N (namely A, B, C, D, F, G, H, I, J, K, L, N). ...
Measles, mumps and rubella (MMR) vaccine failure had been reported globally and here, we report that it occurs in India now. MMR vaccinated people have developed acute mumps accompanied by anti-mumps immunoglobulin M. Genotypic characterisation revealed that the circulating mumps strain was genotype C, which is distinct from the vaccine strain of genotype N (L-Zagreb). This is the first report in India to suggest that genotype C is responsible for the present mumps infection. Thus, the present MMR vaccine must be revamped and optimised for its efficacy to prevent any future mumps epidemics.
... The disease ND, is caused by Newcastle disease virus (NDV), which is characterized by sudden appearance and rapid spread of the virus within the flock with high morbidity and mortality. Newcastle disease virus is synonymous with avian paramyxovirus type 1 (APMV-1) and has been classified in the order Mononegavirales, family Paramyxoviridae, subfamily Paramyxovirinae, genus Rubulavirus (Alexander, 1997; Alexander, 1998 and Lamb et al., 1996). The virus is an enveloped, negative-sense, single-stranded RNA genome that encodes for six proteins including RNA dependent RNA polymerase (L), fusion (F) protein, hemagglutinin-neuraminidase (HN) protein, matrix (M) protein, phosphoprotein (P), and nucleoprotein (NP) (De Leeuw et al., 1999). ...
A study was undertaken to determine the immune response of eight different imported live NDV vaccines in broiler chickens in the Department of Microbiology and Hygiene, Bangladesh Agricultural University, Mymensingh during the period from July to December 2008. A total of 55 broiler chickens (Ross breed) were divided into eleven groups such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 11 of which group 1, 3, 5, 7 and 9 were vaccinated primarily with Nobilis ® MA5+Clone30, Avipro ® ND LaSota vaccine respectively at day 21 of age by single eye instillation and 2, 4, 6, 8 and 10 were vaccinated with the same vaccines respectively by double eye instillation following the same schedule. Group 11 was kept as unvaccinated control. Sera samples were collected after 10 days of each vaccination and at day 5, 15, 20, 31 of age from non- vaccinated control and subjected to HI test for the determination of antibody titres. It was observed that after primary vaccination the geometric mean (GM) of HI titres of double eye vaccinated groups differed significantly (P
... The Paramyxovirus family includes numerous viruses that are of importance to animal and human health [1]. Within this family, human parainfluenza viruses (hPIV), belonging to the Rubulavirus genus, are responsible for upper and lower respiratory tract infections in young children, the elderly, and immunocompromised adults. ...
Human parainfluenza viruses (hPIV) are pathogens responsible for upper and lower respiratory tract infections. We previously described clinical variant strains of hPIV-2 that display unusual large syncytial cytopathic effects. Their molecular characterization revealed a recurrent conserved specific amino acid substitution: A96T in the F2 subunit of the fusion glycoprotein F. The objective of this study was to investigate the contribution of this A96T substitution to the specific hyperfusogenic properties of the hPIV-2 variant strains. Based on a transient expression strategy, quantification of cell-cell fusion assays, and flow cytometry, we have shown that the A96T mutation strongly alters the fusogenic properties of F hPIV-2, highlighting this key residue in the F2 subunit and its possible role in fusion regulation. This work highlights the benefits of monitoring genetic and phenotypic changes of circulating strains to complete our understanding of Paramyxovirus fusion and related pathogenesis.
... The genome encodes the envelope-associated protein M, two glycoproteins (hemagglutinin H and fusion protein F), two transcriptase-associated proteins (phosphoprotein P and large protein L), and the nucleocapsid protein N (van Regenmortel et al., 2000 ). The H protein is responsible for attachment to cell receptors in the first step of infection and promotes fusion activity of the F protein (Lamb and Parks, 2007). It has been reported that genetic variation in the H gene may favor the virus to avoid the immunological response generated by the " old strains " currently used in the vaccines (Bolt et al., 1997; Hirama et al., 2004; Iwatsuki et al., 1997 Iwatsuki et al., , 2000 ). ...
Canine distemper virus (CDV) is the etiological agent of a multisystemic infection that affects different
species of carnivores and is responsible for one of the main diseases suffered by dogs. Recent data have
shown a worldwide increase in the incidence of the disease, including in vaccinated dog populations,
which necessitates the analysis of circulating strains. The hemagglutinin (H) gene, which encodes the
major antigenic viral protein, has been widely used to determine the degree of genetic variability and to
associate CDVs in different worldwide circulating lineages. Here, we obtained the sequence of the first
full-length H gene of field South American CDV strains and compared it with sequences of worldwide
circulating field strains and vaccine viruses. In South America, we detect two co-circulating lineages with
different prevalences: the Europe 1 lineage and a new South America 2 lineage. The Europe 1 lineage
was the most prevalent in South America, and we suggest renaming it the Europe 1/South America 1
lineage. The South America 2 lineage was found only in Argentina and appears related to wild CDV
strains. All South American CDV strains showed high amino-acid divergence from vaccine strains. This
genetic variability may be a possible factor leading to the resurgence of distemper cases in vaccinated
dog populations.
... Together with the closely related Hendra virus (HeV) that appeared in Australia in 1994, NiV has been classified in the new genus called Henipavirus, in the Paramyxoviridae family. Placed in the order of the Mononegavirales, this family has nonsegmented single stranded negative-sense RNA genome (Lamb & Parks 2007). Henipavirus encodes 6 structural proteins: the nucleocapsid N, phosphoprotein P, the matrix protein M, fusion F, attachment G, and the large polymerase L. The P gene also codes for non-structural protein through two different strategies. ...
... Together with the closely related Hendra virus (HeV) that appeared in Australia in 1994, NiV has been classified in the new genus called Henipavirus, in the Paramyxoviridae family. Placed in the order of the Mononegavirales, this family has nonsegmented single stranded negative-sense RNA genome (Lamb & Parks 2007). Henipavirus encodes 6 structural proteins: the nucleocapsid N, phosphoprotein P, the matrix protein M, fusion F, attachment G, and the large polymerase L. The P gene also codes for non-structural protein through two different strategies. ...
H5N1 causes high mortality in domestic poultry. It is the causative agent of H5N1 flu and is the world's largest current pandemic threat. Haemagglutinin (HA) is a surface glycoprotein of the virus which facilitates viral attachment to target cell and its entry. HA frequently accumulates mutation to escape immune response of host. In this study, conserved amino acid sequences in HA2 subunit of H5N1 HA protein of both clade 2.2 and 2.3 were identified and the conserved sequence was further analyzed in silico for its antigenic index, hydrophilicity and surface probability. Six peptides showing potential antigenicity were selected and synthesized. Reactivity of the peptides were analysed by indirect ELISA using antisera against different subtypes of avian influenza. Five out of the six peptides showed reactivity. Thus, five epitopes in the conserved region of HA2 subunit of H5 could be identified which can detect positive serum against avian influenza.
Molecular diagnostic tests are commonly used to diagnose
avian influenza virus because they are sensitive, rapid and
cost effective. However, continuous shift and drift of avian
influenza viruses and H5N1 in particular, is a real challenge
for development of specific genetic based detection
techniques. Furthermore, most of current molecular diagnostic
commercial kits rely on the detection of H5 and/or
N1 genes without the presence of the M1 gene which represents the most conservative gene in the type A avian
influenza viruses (AIV). This enable spread of undetected
mutants.
In the present investigation we have optimized a primer
set for polymerase chain reaction (PCR)-based detection of
influenza A viruses H5, N1 and M1 genes that was validated
with a panel of influenza A virus reference strains representing
H5, H7, H9 and H13. Specificity test was carried
out by the use of eight type A AIV subtypes (H5N1, H5N2,
H5N9, H7N1, H7N3, H7N7, H9N2 and H13N6). Results
showed that this protocol is capable of produce two distinguished
bands represents the H5 and N1 genes in the
duplex format. While, it generated three distinguished
bands represents the H5, N1 and M1 genes (in the triplex
format). Moreover, the specificity test showed that the
used primers do not have cross-reactivity with the AIV subtypes
other than H5N1. In addition, Sensitivity test
revealed that the duplex format has a similar sensitivity
limit as the triplex one.
An avian paramyxovirus (APMV) isolated from goose feces (APMV/Shimane67) was biologically, serologically and genetically characterized. APMV/Shimane67 showed typical paramyxovirus morphology on electron microscopy. On hemagglutination inhibition test, antiserum against APMV/Shimane67 revealed low reactivity with other APMV serotypes and vice versa. The fusion (F) protein gene of APMV/Shimane67 contained 1,638 nucleotides in a single open reading frame encoding a protein of 545 amino acids. The cleavage site of F protein contained a pair of single basic amino acid (VRENR/L). The nucleotide and deduced amino acid sequences of the F gene of APMV/Shimane67 had relatively low identities (42.9-62.7% and 28.9-67.3%, respectively) with those of other APMVs. Phylogenetic analysis showed that APMV/Shimane67 was related to NDV, APMV-9 and APMV-12, but was distinct from those APMV serotypes. These results suggest that APMV/Shimane67 is a new APMV serotype, APMV-13.
Significance
Perturbation of transcription register by RNA polymerase, transcription slippage, is used to yield additional protein products. Known functionally important cases involve a small number of short sequences without secondary structure. The discovery reported here of the dependence of a newly identified motif on nascent RNA forming a stem loop structure within the RNA exit channel of the polymerase greatly extends the potential for a broad variety of putative slippage sequences, especially as the phenomenon has been observed with both bacterial and eukaryotic RNA polymerases. Characterization of the mechanism involved shows similarities with, and differences from, intrinsic transcription termination, which also depends on formation of RNA stem loop structures. Our findings reveal novel insights to RNA polymerase versatility and functioning.
Influenza virus is associated with upper respiratory tract infections. The fourth influenza pandemic was declared in 2009. The aim of this study was to determine the genetic variability of the 2009 H1N1 pandemic virus circulating in Paraguay. Nasal swabs were collected from 181 patients with flu symptoms managed at the Hospital of the Medical School in Asunción, Paraguay, between August and October 2009. Virus detection was carried out by real-time reverse transcription-polymerase chain reaction, followed by sequencing of the hemagglutinin and neuraminidase genes, and phylogenetic analysis. H1N1pdm09 was detected in 14.9% (27/181) of the suspected cases. Analysis of 13 samples showed that these viruses the clustered in a single genetic group. Neither the mutation related to exacerbation of disease (D239G in hemagglutinin) nor that related to antiviral resistance (H275Y in neuraminidase), both detected in neighboring countries, were found. This genetic analysis of H1N1pdm09 will help to understand the spread of the disease.
Significance
Respiratory syncytial virus (RSV) causes major disease in pediatric and elderly patients, urging the development of efficacious therapeutics. This study establishes a recombinant RSV reporter strain for drug discovery and identifies an entry inhibitor class targeting the viral fusion (F) protein. Biochemical, structural, and functional characterization of the inhibitor spotlights two microdomains governing the conformational stability of prefusion F. Mutations in these domains cause broad cross-resistance against the compound and RSV entry inhibitors in preclinical and clinical development, without mandatory loss of in vivo pathogenicity, challenging the possible clinical benefit of current RSV entry inhibitor classes. Anti-RSV campaigns should better target postentry steps or proactively circumvent resistance to entry inhibition. The resistant RSV reporter strain developed here establishes a strategy toward this goal.
Significance
Mycoviruses are viruses that infect fungi and replicate in fungi. Previously, no mycoviruses had been discovered with negative-stranded (−)ssRNA genomes. Here, we characterize a (−)ssRNA mycovirus that infects a fungal plant pathogen. Although its genome and organization are significantly different from those of current mononegaviruses, this virus is closely related to viruses in families Nyamiviridae and Bornaviridae that infect animals. This discovery may provide insights into the global ecology and evolution of (−)ssRNA viruses. Furthermore, since many (−)ssRNA viruses are serious human pathogens, this system is likely to provide a less hazardous way to study replication of a (−)ssRNA virus and could be useful in establishing a system to screen antiviral compounds against (−)ssRNA viruses.
Caffeic acid has been shown to inhibit the multiplication of influenza A virus in vitro, whereas caffeine, quinic acid and chlorogenic acid do not. Caffeic acid has also been shown to have antiviral activity against herpes simplex virus (DNA virus) and polio virus (RNA virus). In the present study, a comparison of the one-step growth curve of the influenza virus in the presence of caffeic acid with that in the absence of the reagent showed that an eclipse period of the virus multiplication in the infected cells was not affected by the reagent, while the progeny virus yield was markedly decreased in the presence of caffeic acid. In additional experiments, it was found that the addition of caffeic acid at an early time point post-infection (within 3 h post-infection) was mandatory for extensive antiviral activity, suggesting that a major target of the reagent exists in the early stages of infection. Simultaneously with the decrease in the progeny virus yield, both the virus-induced cytopathic effects and apoptotic nuclear fragmentation were markedly suppressed by the reagent, suggesting that caffeic acid suppresses, at least temporally, the degeneration of the virus-infected cells and that the observed antiviral activity is likely not the secondary result of the cytotoxic effects of the reagent. These results suggest the potential pharmacological use of caffeic acid or its derivatives as an antiviral drug against influenza A virus.
Influenza is a highly contagious, major respiratory tract
disease affecting millions of people each year. At present, two
classes of antivirals are available: the neuraminidase inhibitors and
the M2 proton channel blockers amantadine and rimantadine.
However, rapid emergence of M2 blockers resistance makes
imperative the development of new anti-influenza drugs. In the last
few years several groups have synthesized and evaluated several
analogs of amantadine. While several of them are active against
wild-type M2 channel only a few are able to inhibit the mutant ion
channels that lead to amantadine-resistance.
Significance
The activities of the fusion proteins that mediate virus–cell fusion are an absolute requirement for virus entry and infectivity of enveloped viruses such as HIV, influenza virus, measles virus, and respiratory syncytia virus, among others. Viral fusion proteins are translated initially in a metastable prefusion state and, upon triggering, undergo an extensive and irreversible refolding process. Membrane fusion is coupled to the energy released by the fusion proteins adopting a stable, low-energy postfusion state. Here we use oxidative footprinting of the parainfluenza virus 5 fusion protein to reveal new details of this critical event in the viral lifecycle. A greater understanding of the dynamic nature of these metastable proteins may reveal novel opportunities for the development of targeted therapeutics.
vian influenza (AI) has emerged as a disease with significant potential to disrupt commercial poultry production often resulting in extensive losses (1). Influenza is caused by a zoonotic virus that occurs in lower animals and birds as well as in humans. Influenza viruses belong to the Orthomyxoviridae family of RNA viruses and are divided into five genera: Influenza A, B, C virus, Thogtovirus and Isavirus (2, 3). A viruses can be divided into subtypes on the basis of the possession of one of 16 antigenically distinct Haemagglutinin (HA) antigens and one of the 9 Neuraminidase (NA) antigens (4). Virtually all HA and NA combinations have been isolated from birds (3). Live Bird Markets (LBM) have been recognized as a productive source and important man-made reservoir of AI virus (AIV) linked to outbreaks of influenza in commercial poultry farms and humans. LBMs provide an ideal tool for genetic mixing and spreading of the influenza virus. Because they bring together numerous hosts (e.g. Chickens, Ducks, turkeys and quail) in close contact and high density. So viral reassortment and inter species transmission have accrued. Long term replication of AIV in even unnatural host species can lead to accelerated mutation rates for AIV. LBMs are therefore hypothesized to be a missing link in the epidemiology of AIV and it is important that the LBMs be routinely monitored for AIV. Low & High Pathogenic AIV has been isolated repeatedly from LBMs. Presence of this virus in the LBMs poses a significant risk to the commercial poultry in any region (3, 5). Investigations conducted in Hong Kong following the first H5N1 outbreak in humans in 1997 determined that exposure to poultry in LBM was a key risk factor for human disease (6, 7) Since 1998, H9N2 AI outbreaks have been one of the major problems in Iranian poultry industry. In 2006, H5N1 report in swans in northern of Iran, but to this time we don't have official report from commercial flocks in Iran
Herpesvirus entry functions of the conserved glycoproteins gB and gH-gL have been delineated, but their role in regulating cell-cell fusion is poorly understood. Varicella-zoster virus (VZV) infection provides a valuable model for investigating cell-cell fusion because of the importance of this process for pathogenesis in human skin and sensory ganglia. The present study identifies a canonical immunoreceptor tyrosine-based inhibition motif (ITIM) in the gB cytoplasmic domain (gBcyt) and demonstrates that the gBcyt is a tyrosine kinase substrate. Orbitrap mass spectrometry confirmed that Y881, central to the ITIM, is phosphorylated. To determine whether the gBcyt ITIM regulates gB/gH-gL-induced cell-cell fusion in vitro, tyrosine residues Y881 and Y920 in the gBcyt were substituted with phenylalanine separately or together. Recombinant viruses with these substitutions were generated to establish their effects on syncytia formation in replication in vitro and in the human skin xenograft model of VZV pathogenesis. The Y881F substitution caused significantly increased cell-cell fusion despite reduced cell-surface gB. Importantly, the Y881F or Y881/920F substitutions in VZV caused aggressive syncytia formation, reducing cell-cell spread. These in vitro effects of aggressive syncytia formation translated to severely impaired skin infection in vivo. In contrast, the Y920F substitution did not affect virus replication in vitro or in vivo. These observations suggest that gB modulates cell-cell fusion via an ITIM-mediated Y881 phosphorylation-dependent mechanism, supporting a unique concept that intracellular signaling through this gBcyt motif regulates VZV syncytia formation and is essential for skin pathogenesis.
The paramyxovirus parainfluenza virus 5 (PIV5) enters cells by fusion of the viral envelope with the plasma membrane through the concerted action of the fusion (F) protein and the receptor binding protein hemagglutinin-neuraminidase. The F protein folds initially to form a trimeric metastable prefusion form that is triggered to undergo large-scale irreversible conformational changes to form the trimeric postfusion conformation. It is thought that F refolding couples the energy released with membrane fusion. The F protein is synthesized as a precursor (F0) that must be cleaved by a host protease to form a biologically active molecule, F1,F2. Cleavage of F protein is a prerequisite for fusion and virus infectivity. Cleavage creates a new N terminus on F1 that contains a hydrophobic region, known as the FP, which intercalates target membranes during F protein refolding. The crystal structure of the soluble ectodomain of the uncleaved form of PIV5 F is known; here we report the crystal structure of the cleavage-activated prefusion form of PIV5 F. The structure shows minimal movement of the residues adjacent to the protease cleavage site. Most of the hydrophobic FP residues are buried in the uncleaved F protein, and only F103 at the newly created N terminus becomes more solvent-accessible after cleavage. The conformational freedom of the charged arginine residues that compose the protease recognition site increases on cleavage of F protein.
Many pleomorphic, lipid-enveloped viruses encode matrix proteins that direct their assembly and budding, but the mechanism of this process is unclear. We have combined X-ray crystallography and cryoelectron tomography to show that the matrix protein of Newcastle disease virus, a paramyxovirus and relative of measles virus, forms dimers that assemble into pseudotetrameric arrays that generate the membrane curvature necessary for virus budding. We show that the glycoproteins are anchored in the gaps between the matrix proteins and that the helical nucleocapsids are associated in register with the matrix arrays. About 90% of virions lack matrix arrays, suggesting that, in agreement with previous biological observations, the matrix protein needs to dissociate from the viral membrane during maturation, as is required for fusion and release of the nucleocapsid into the host's cytoplasm. Structure and sequence conservation imply that other paramyxovirus matrix proteins function similarly.
Influenza virus is a continuous and severe global threat to mankind. The continuously re-emerging disease gives rise to thousands of deaths and enormous economic losses each year, which emphasizes the urgency and necessity to develop high-quality influenza vaccines in a safer, more efficient and economic way. The influenza subunit and VLP vaccines, taking the advantage of recombinant DNA technologies and expression system platforms, can be produced in such an ideal way. This review summarized the recent advancements in the research and development of influenza subunit and VLP vaccines based on the recombinant expression of hemagglutinin antigen (HA), neuraminidase antigen (NA), Matrix 2 protein (M2) and nucleocapsid protein (NP). It would help to get insight into the current stage of influenza vaccines, and suggest the future design and development of novel influenza vaccines.
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.Gene Therapy advance online publication, 5 January 2012; doi:10.1038/gt.2011.209.
The paramyxovirus hemagglutinin-neuraminidase (HN) protein plays multiple roles in viral entry and egress, including binding to sialic acid receptors, activating the fusion (F) protein to activate membrane fusion and viral entry, and cleaving sialic acid from carbohydrate chains. HN is an oligomeric integral membrane protein consisting of an N-terminal transmembrane domain, a stalk region, and an enzymatically active neuraminidase (NA) domain. Structures of the HN NA domains have been solved previously; however, the structure of the stalk region has remained elusive. The stalk region contains specificity determinants for F interactions and activation, underlying the requirement for homotypic F and HN interactions in viral entry. Mutations of the Newcastle disease virus HN stalk region have been shown to affect both F activation and NA activities, but a structural basis for understanding these dual affects on HN functions has been lacking. Here, we report the structure of the Newcastle disease virus HN ectodomain, revealing dimers of NA domain dimers flanking the N-terminal stalk domain. The stalk forms a parallel tetrameric coiled-coil bundle (4HB) that allows classification of extensive mutational data, providing insight into the functional roles of the stalk region. Mutations that affect both F activation and NA activities map predominantly to the 4HB hydrophobic core, whereas mutations that affect only F-protein activation map primarily to the 4HB surface. Two of four NA domains interact with the 4HB stalk, and residues at this interface in both the stalk and NA domain have been implicated in HN function.
After the first report of induced pluripotent stem cells (iPSCs), considerable efforts have been made to develop more efficient methods for generating iPSCs without foreign gene insertions. Here we show that Sendai virus vector, an RNA virus vector that carries no risk of integrating into the host genome, is a practical solution for the efficient generation of safer iPSCs. We improved the Sendai virus vectors by introducing temperature-sensitive mutations so that the vectors could be easily removed at nonpermissive temperatures. Using these vectors enabled the efficient production of viral/factor-free iPSCs from both human fibroblasts and CD34(+) cord blood cells. Temperature-shift treatment was more effective in eliminating remaining viral vector-related genes. The resulting iPSCs expressed human embryonic stem cell markers and exhibited pluripotency. We suggest that generation of transgene-free iPSCs from cord blood cells should be an important step in providing allogeneic iPSC-derived therapy in the future.
Virus-like particles are a new type of vaccine platform that presents an attractive alternative to more traditional live-attenuated or inactivated/subunit vaccines. Virus-like particles (VLP) are composed of viral structural proteins that assemble spontaneously in cells, mimicking the live virus without the possibility of replication. They are readily recognized by the immune system, inducing both humoral and cellular immune responses. Here we review the development of VLP as vaccine delivery systems at mucosal surfaces. We first summarize the current status of VLP vaccines in general, and then discuss their use in mucosal vaccination approaches for several viruses that enter the host via the urogenital, respiratory or gastrointestinal tract.
Nipah virus (NiV) and Hendra virus are the type species of the highly pathogenic paramyxovirus genus Henipavirus, which can cause severe respiratory disease and fatal encephalitis infections in humans, with case fatality rates approaching 75%. NiV contains two envelope glycoproteins, the receptor-binding G glycoprotein (NiV-G) that facilitates attachment to host cells and the fusion (F) glycoprotein that mediates membrane merger. The henipavirus G glycoproteins lack both hemagglutinating and neuraminidase activities and, instead, engage the highly conserved ephrin-B2 and ephrin-B3 cell surface proteins as their entry receptors. Here, we report the crystal structures of the NiV-G both in its receptor-unbound state and in complex with ephrin-B3, providing, to our knowledge, the first view of a paramyxovirus attachment complex in which a cellular protein is used as the virus receptor. Complex formation generates an extensive protein–protein interface around a protruding ephrin loop, which is inserted in the central cavity of the NiV-G β-propeller. Analysis of the structural data reveals the molecular basis for the highly specific interactions of the henipavirus G glycoproteins with only two members (ephrin-B2 and ephrin-B3) of the very large ephrin family and suggests how they mediate in a unique fashion both cell attachment and the initiation of membrane fusion during the virus infection processes. The structures further suggest that the NiV-G/ephrin interactions can be effectively targeted to disrupt viral entry and provide the foundation for structure-based antiviral drug design.
• crystallography
• viral attachment
M2 is an integral protein of influenza A virus that functions as an ion channel. The ratio of M2 to HA in influenza A virions differs from that found on the cell surface, suggesting selective incorporation of M2 and HA into influenza virions. To examine the sequences that are important for M2 incorporation into virions, we used an incorporation assay that involves expressing M2 from a plasmid, transfecting the plasmid into recipient cells, and then infecting those cells with influenza virus. To test the importance of the different regions of the protein (extracellular, transmembrane, and cytoplasmic) in determining M2 incorporation, we created chimeric mutants of M2 and Sendai virus F proteins, exchanging corresponding extracellular, transmembrane, and cytoplasmic domains. Of the six possible chimeric mutants, only three were expressed on the cell surface. Of these three chimeric proteins, only one mutant (with the extracellular domain from M2 and the rest from F) was incorporated into influenza virions. These results suggest that the extracellular domain of M2 is important for its incorporation into virions.
Fundamentally new approaches are required for the development of vaccines to pre-empt and protect against emerging and pandemic influenzas. Current strategies involve post-emergent homotypic vaccines that are modelled upon select circulating 'seasonal' influenzas, but cannot induce cross-strain protection against newly evolved or zoonotically introduced highly pathogenic influenza (HPI). Avian H5N1 and the less-lethal 2009 H1N1 and their reassortants loom as candidates to seed a future HPI pandemic. Therefore, more universal 'seasoned' vaccine approaches are urgently needed for heterotypic protection ahead of time. Pivotal to this is the need to understand mechanisms that can deliver broad strain protection. Heterotypic and heterosubtypic humoral immunities have largely been overlooked for influenza cross-protection, with most 'seasoned' vaccine efforts for humans focussed on heterotypic cellular immunity. However, 5 years ago we began to identify direct and indirect indicators of humoral-herd immunity to protein sites preserved among H1N1, H3N2 and H5N1 influenzas. Since then the evidence for cross-protective antibodies in humans has been accumulating. Now proposed is a rationale to stimulate and enhance pre-existing heterotypic humoral responses that, together with cell-mediated initiatives, will deliver pre-emptive and universal human protection against emerging epidemic and pandemic influenzas.
Influenza A virus can infect a wide variety of animal species including humans, pigs, birds and other species. Viral ribonucleoprotein (vRNP) was involved in genome replication, transcription and host adaptation. Currently, firefly luciferase (Fluc) reporter system was used in vRNP functional assay. However, its limitation for the testing by virus infection resulted in an increased need for rapid, sensitive, and biosafe techniques. Here, an influenza A virus UTR-driven gene reporter for vRNP assay based on secreted Gaussia luciferase (Gluc) activity was evaluated.
By measuring Gluc levels in supernatants, reporter gene activity could be detected and quantitated after either reconstitution of influenza A virus polymerase complex or viral infection of 293T and A549 cells, respectively. As compared with Fluc reporter, Gluc-based reporter was heat-tolerant (65°C for 30 min) and produced 50-fold higher bioluminescent activity at 24 h posttransfection. Signals generated by Gluc reporter gene could be detected as early as 6 h post-infection and accumulated with time. Testing by viral infection, stronger signals were detected by Gluc reporter at a MOI of 0.001 than that of 1 and the effects of PB2-627K/E or amantadine on influenza vRNP activity were elucidated more effectively by the Gluc reporter system.
This approach provided a rapid, sensitive, and biosafe assay of influenza vRNP function, particularly for the highly pathogenic avian influenza viruses.
IFNs play a critical role in innate immunity against viral infections. Melanoma differentiation-associated protein 5 (MDA5), an RNA helicase, is a key component in activating the expression of type I IFNs in response to certain types of viral infection. MDA5 senses noncellular RNA and triggers the signaling cascade that leads to IFN production. Synthetic double-stranded RNAs are known activators of MDA5. Natural single-stranded RNAs have not been reported to activate MDA5, however. We have serendipitously identified a viral mRNA from parainfluenza virus 5 (PIV5) that activates IFN expression through MDA5. We provide evidence that the signaling pathway includes the antiviral enzyme RNase L. The L mRNA of PIV5 activated expression of IFN-β. We have mapped the RNA to a region of 430 nucleotides within the L mRNA of PIV5. Our results indicate that a viral mRNA, with 5'-cap and 3'-poly (A), can activate IFN expression through an RNase L-MDA5 pathway.
Eight Newcastle disease virus isolates from Pakistan were sequenced and characterized. A PCR matrix gene assay, designed to
detect all avian paramyxovirus 1, did not detect four of the isolates. A new matrix gene test that detected all isolates was
developed. Phylogenetic analysis and pathotyping confirmed that virulent viruses of different genotypes are circulating in
Pakistan.
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