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Generation of Plasmid Vector Coding for Neuraminidase Gene NA1 of Highly Pathogenic Avian Influenza H5N1 Subtype
... A total of 10 highly pathogenic avian influenza virus H5N1 subtypes have been isolated in Egypt (Table 1) during the years 2010-2017, the isolates were previously identified by HA and HI, RT-PCR sequencing [5][6][7]. The isolates were propagated on embryonated chicken eggs via the allantoic route. ...
... Several previous studies showed that the DNA vaccine coding for both H5 and N1 genes elucidated protection rate up to 92% with nearly zero shedding [5][6][7]. To ensure the effectiveness of the DNA vaccine, one must investigate the circulating strain regarding to the cross reactivity with the vaccine which might me time consuming and work labor. ...
... Transformants were subcultured on LB broth-kanamycin (50ng/ml) at 37°C in a shaker incubator at a speed of 150 rpm/24 h. (Soliman, et al., 2016). ...
... The vaccine must then afford two main properties, first should give a high level of protection plus minimize shedding to zero to eliminate environmental load and prevent secondary epidemics (Capua and Alexander 2007). Inactivated vaccines widely used does not ensure prevention of viral shedding due to its minimal capability to induce a cell-mediated immune response, a property that is favored by the DNA vaccine (Hsu, et al., 2010, Eman, 2014and Maha, 2016. ...
Control of avian influenza infection depends mainly on biosafety measures and vaccination, DNA vaccination is a novel method to generate antigen-specific antibody and cell-mediated immunity. In the current study, a DNA vaccine for full-length N1 gene was developed in a trial to decrease the severity of avian influenza virus spread and shedding. Full-length N1 gene was cloned in entry clone pENTER SD/TOPO, followed by homologous recombination with the destination mammalian expression vector (PDEST 40). The PDEST 40-N1 was used in the immunization of SPF chickens. Potency was evaluated through the survival rate that reaches 65%, which was far less than the commercially available inactivated vaccine. Meanwhile, the shedding of the virus from dead birds was 0.46 Log 10 EID50. At the same time, the most surprising result was the shedding level of the vaccinated live birds that were zero shedding ;on the other hand, the inactivated vaccine could not reduce the shedding level which remains very high (3.2 Log 10 EID50 ). IFN-γ transcript level in the DNA vaccinated group was detected by the 3rd-day post-vaccination and remained upregulated till the 28th post-vaccination. After the challenge, the level of IFN-γ was much higher until 14 days post-challenge. The inactivated vaccine could not stimulate any detectable level post-vaccination. These data suggested the ability of DNA vaccine coding for N1 gene of avian influenza to combat the virus shedding from live birds and could be used in combination with DNA vaccine coding for H5 to produce maximum protection with zero shedding.
... Transformants were subcultured on LB broth-kanamycin (50 ng/mL) at 37°C in a shaker incubator at a speed of 150 rpm for 24 h. (Soliman et al., 2016) The transformants were analyzed by QPCR for the presence of the gene of interest. Briefly, the plasmid DNA ...
Control of avian influenza infection requires a good vaccine that could induce both humoral and cell-mediated immune response, specifically IFN-ɣ production, to maintain a high level of protection along with the minimal level of viral shedding after the infection to prevent secondary epidemics. In the current study, deoxyribonucleic Acid (DNA) vaccine coding for full-length H5 and N1 genes have been produced and evaluated in SPF-chicken. Humoral immune response estimated by haemagglutination inhibition (HI) assay revealed that the DNA vaccine gave a high titer of antibodies at the day 28-post vaccination and 14 days post-challenge. However, the shedding level was minimal with the DNA vaccine (0.1 Log 10 EID50). The IFN-ɣ transcript was upregulated at a higher level in the DNA vaccinated group. The results revealed that the DNA vaccine could induce a high level of humoral and IFN-ɣ level that maintains a high level of protection (92%) with the advantage of limiting the shedding level and thus, prevent secondary epidemics.
Highly pathogenic avian influenza (HPAI) H5N1 virus remains a public health threat and continues to cause outbreaks among poultry as well as human. Since its appearance, the virus has spread to numerous geographic areas and is now considered endemic in Egypt. Also widespread prevalence of H9N2 subtype in the Middle East region and its detection in Egypt in summer 2011 in poultry added another challenge to the Egyptian poultry industry as well as human health. Here, to evaluate the potential for avian to human transmission of avian influenza viruses H5 and H9, a serological study was conducted among 60 poultry workers and housewives and 143 participants randomly selected from Chest Hospital in Damanhour city in Behira governorate for determination of antibody titers against H5 and H9 avian influenza viruses by using hemagglutination inhibition assay. Further, participants were interviewed with a standardized questionnaire to assess the knowledge, attitudes, and practices regarding avian influenza infection. In addition, tissue samples from in contact poultry were assayed for virus detection by rapid test and positive samples were confirmed by real time RT-PCR. Our results showed some evidence of exposure to H5N1avian influenza virus with 3 (5%) poultry workers and 3 (2.1%) Chest hospital subjects having HI antibody titer of 1:160. All sera of poultry workers and housewives tested negative for H9. Only three poultry samples were positive for H5N1 virus. The level of community knowledge about AI disease was fair, but their practice and attitude were negative. Therefore, designing and implementing health educational programs about AI to improve the community practices should have the priority. Our findings support the low transmissibility of H5N1 to humans, but transmission to highly exposed persons cannot be excluded given that HI antibody titers of 1:80 and 1:40 were detected in some individuals. Prevalence of H9 antibodies was low, however, requires additional studies.
Historically, highly pathogenic avian influenza viruses (HPAIV) rarely resulted in infection or clinical disease in wild birds. However, since 2002, disease and mortality from natural HPAIV H5N1 infection have been observed in wild birds including gulls. We performed an experimental HPAIV H5N1 infection of black-headed gulls (Chroicocephalus ridibundus) to determine their susceptibility to infection and disease from this virus, pattern of viral shedding, clinical signs, pathological changes and viral tissue distribution. We inoculated sixteen black-headed gulls with 1 × 104 median tissue culture infectious dose HPAIV H5N1 (A/turkey/Turkey/1/2005) intratracheally and intraesophageally. Birds were monitored daily until 12 days post inoculation (dpi). Oropharyngeal and cloacal swabs were collected daily to detect viral shedding. Necropsies from birds were performed at 2, 4, 5, 6, 7, and 12 dpi. Sampling from selected tissues was done for histopathology, immunohistochemical detection of viral antigen, PCR, and viral isolation. Our study shows that all inoculated birds were productively infected, developed systemic disease, and had a high morbidity and mortality rate. Virus was detected mainly in the respiratory tract on the first days after inoculation, and then concentrated more in pancreas and central nervous system from 4 dpi onwards. Birds shed infectious virus until 7 dpi from the pharynx and 6 dpi from the cloaca. We conclude that black-headed gulls are highly susceptible to disease with a high mortality rate and are thus more likely to act as sentinel species for the presence of the virus than as long-distance carriers of the virus to new geographical areas.
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The online version of this article (doi:10.1186/s13567-014-0084-9) contains supplementary material, which is available to authorized users.
Antivirals play a critical role in the prevention and the management of influenza. One class of antivirals, neuraminidase inhibitors (NAIs), is effective against all human influenza viruses. Currently there are two NAI drugs which are licensed worldwide: oseltamivir (Tamiflu®) and zanamivir (Relenza®); and two drugs which have received recent approval in Japan: peramivir and laninamivir. Until recently, the prevalence of antiviral resistance has been relatively low. However, almost all seasonal H1N1 strains that circulated in 2008-09 were resistant to oseltamivir whereas about 1% of tested 2009 pandemic H1N1 viruses were found to be resistant to oseltamivir. To date, no studies have demonstrated widespread resistance to zanamivir. It seems likely that the literature on antiviral resistance associated with oseltamivir as well as zanamivir is now sufficiently comprehensive to warrant a systematic review.The primary objectives were to systematically review the literature to determine the incidence of resistance to oseltamivir, zanamivir, and peramivir in different population groups as well as assess the clinical consequences of antiviral resistance.
We searched MEDLINE and EMBASE without language restrictions in September 2010 to identify studies reporting incidence of resistance to oseltamivir, zanamivir, and peramivir. We used forest plots and meta-analysis of incidence of antiviral resistance associated with the three NAIs. Subgroup analyses were done across a number of population groups. Meta-analysis was also performed to evaluate associations between antiviral resistance and clinical complications and symptoms.
We identified 19 studies reporting incidence of antiviral resistance. Meta-analysis of 15 studies yielded a pooled incidence rate for oseltamivir resistance of 2.6% (95%CI 0.7% to 5.5%). The incidence rate for all zanamivir resistance studies was 0%. Only one study measured incidence of antiviral resistance among subjects given peramivir and was reported to be 0%. Subgroup analyses detected higher incidence rates among influenza A patients, especially for H1N1 subtype influenza. Considerable heterogeneity between studies precluded definite inferences about subgroup results for immunocompromised patients, in-patients, and children. A meta-analysis of 4 studies reporting association between oseltamivir-resistance and pneumonia yielded a statistically significant risk ratio of 4.2 (95% CI 1.3 to 13.1, p = 0.02). Oseltamivir-resistance was not statistically significantly associated with other clinical complications and symptoms.
Our results demonstrate that that a substantial number of patients may become oseltamivir-resistant as a result of oseltamivir use, and that oseltamivir resistance may be significantly associated with pneumonia. In contrast, zanamivir resistance has been rarely reported to date.
Efforts to develop a broadly protective vaccine against the highly pathogenic avian influenza A (HPAI) H5N1 virus have focused on highly conserved influenza gene products. The viral nucleoprotein (NP) and ion channel matrix protein (M2) are highly conserved among different strains and various influenza A subtypes. Here, we investigate the relative efficacy of NP and M2 compared to HA in protecting against HPAI H5N1 virus. In mice, previous studies have shown that vaccination with NP and M2 in recombinant DNA and/or adenovirus vectors or with adjuvants confers protection against lethal challenge in the absence of HA. However, we find that the protective efficacy of NP and M2 diminishes as the virulence and dose of the challenge virus are increased. To explore this question in a model relevant to human disease, ferrets were immunized with DNA/rAd5 vaccines encoding NP, M2, HA, NP+M2 or HA+NP+M2. Only HA or HA+NP+M2 vaccination conferred protection against a stringent virus challenge. Therefore, while gene-based vaccination with NP and M2 may provide moderate levels of protection against low challenge doses, it is insufficient to confer protective immunity against high challenge doses of H5N1 in ferrets. These immunogens may require combinatorial vaccination with HA, which confers protection even against very high doses of lethal viral challenge.
Proteolytic cleavage of hemagglutinin is required for cell entry by receptor-mediated endocytosis and plays a key role in pathogenicity of the influenza virus. Despite several studies describing relationships between bacterial proteases and influenza A viral activation in mammals, very little is known about the role of the normal bacterial flora of birds on hemagglutinin activation. We examined the indigenous intestinal microflora of 100 mixed-sex, 27-d-old Ross chickens from a commercial poultry facility for protease-secreting bacteria. Protease-secreting bacteria were isolated from 82 of 100 chickens with 50 birds exhibiting 2 or more protease-secreting bacterial species. A total of 20 protease-secreting bacterial species were identified: 17 gram-positive cocci, 2 gram-positive rods, and 1 gram-negative rod. Enterococcus faecalis, Enterococcus gallinarum, and Proteus mirabilis were the most frequently observed protease-secreting bacterial species. The presence of proteolytic bacteria in the intestinal tract of poultry in this study suggests the possibility of yet-to-be-described role(s) in cleavage of hemagglutinin that may alter the pathogenicity of avian influenza viruses.
The simple relation between the substrates and products of site-specific recombination raises questions about the control of directionality often observed in this class of DNA transactions. For bacteriophage lambda, viral integration and excision proceed by discrete pathways, and DNA substrates with the intrinsic property of recombining in only one direction can be constructed. These pathways display an asymmetric reliance on a complex array of protein binding sites, and they respond differently to changes in the concentrations of the relevant proteins. The Escherichia coli protein integration host factor (IHF) differentially affects integrative and excisive recombination, thereby influencing directionality. A four- to eightfold increase in intracellular IHF coincides with the transition from exponential to stationary phase; this provides a mechanism for growth phase-dependent regulation of recombination that makes the cellular physiology an intrinsic part of the recombination reaction.
Topoisomerase IB catalyzes recombinogenic DNA strand transfer reactions in vitro and in vivo. Here we characterize a new pathway
of topoisomerase-mediated DNA ligation in vitro (flap ligation) in which vaccinia virus topoisomerase bound to a blunt-end
DNA joins the covalently held strand to a 5′ resected end of a duplex DNA containing a 3′ tail. The joining reaction occurs
with high efficiency when the sequence of the 3′ tail is complementary to that of the scissile strand immediately 5′ of the
cleavage site. A 6-nucleotide segment of complementarity suffices for efficient flap ligation. Invasion of the flap into the
duplex apparently occurs while topoisomerase remains bound to DNA, thereby implying a conformational flexibility of the topoisomerase
clamp around the DNA target site. The 3′ flap acceptor DNA mimics a processed end in the double-strand-break-repair recombination
pathway. Our findings suggest that topoisomerase-induced breaks may be rectified by flap ligation, with ensuing genomic deletions
or translocations.
The 1918 influenza pandemic caused more than 20 million deaths worldwide. Thus, the potential impact of a re-emergent 1918 or 1918-like influenza virus, whether through natural means or as a result of bioterrorism, is of significant concern. The genetic determinants of the virulence of the 1918 virus have not been defined yet, nor have specific clinical prophylaxis and/or treatment interventions that would be effective against a re-emergent 1918 or 1918-like virus been identified. Based on the reported nucleotide sequences, we have reconstructed the hemagglutinin (HA), neuraminidase (NA), and matrix (M) genes of the 1918 virus. Under biosafety level 3 (agricultural) conditions, we have generated recombinant influenza viruses bearing the 1918 HA, NA, or M segments. Strikingly, recombinant viruses possessing both the 1918 HA and 1918 NA were virulent in mice. In contrast, a control virus with the HA and NA from a more recent human isolate was unable to kill mice at any dose tested. The recombinant viruses were also tested for their sensitivity to U.S. Food and Drug Administration-approved antiinfluenza virus drugs in vitro and in vivo. Recombinant viruses possessing the 1918 NA or both the 1918 HA and 1918 NA were inhibited effectively in both tissue culture and mice by the NA inhibitors, zanamivir and oseltamivir. A recombinant virus possessing the 1918 M segment was inhibited effectively both in tissue culture and in vivo by the M2 ion-channel inhibitors amantadine and rimantadine. These data suggest that current antiviral strategies would be effective in curbing the dangers of a re-emergent 1918 or 1918-like virus.
Antigenic profiles of post-2002 H5N1 viruses representing major genetic clades and various geographic sources were investigated
using a panel of 17 monoclonal antibodies raised from five H5N1 strains. Four antigenic groups from seven clades of H5N1 virus
were distinguished and characterized based on their cross-reactivity to the monoclonal antibodies in hemagglutination inhibition
and cell-based neutralization assays. Genetic polymorphisms associated with the variation of antigenicity of H5N1 strains
were identified and further verified in antigenic analysis with recombinant H5N1 viruses carrying specific mutations in the
hemagglutinin protein. Modification of some of these genetic variations produced marked improvement to the immunogenicity
and cross-reactivity of H5N1 strains in assays utilizing monoclonal antibodies and ferret antisera raised against clade 1
and 2 H5N1 viruses, suggesting that these sites represent antigenically significant amino acids. These results provide a comprehensive
antigenic profile for H5N1 virus strains circulating in recent years and will facilitate the recognition of emerging antigenic
variants of H5N1 virus and aid in the selection of vaccine strains.
Wild birds have been implicated in the expansion of highly pathogenic avian influenza virus (H5N1) outbreaks across Asia, the Middle East, Europe, and Africa (in addition to traditional transmission by infected poultry, contaminated equipment, and people). Such a role would require wild birds to excrete virus in the absence of debilitating disease. By experimentally infecting wild ducks, we found that tufted ducks, Eurasian pochards, and mallards excreted significantly more virus than common teals, Eurasian wigeons, and gadwalls; yet only tufted ducks and, to a lesser degree, pochards became ill or died. These findings suggest that some wild duck species, particularly mallards, can potentially be long-distance vectors of highly pathogenic avian influenza virus (H5N1) and that others, particularly tufted ducks, are more likely to act as sentinels.
We had examined the immunogenicity of a series of plasmid DNAs which include neuraminidase (NA) and nucleoprotein (NP) genes from avian influenza virus (AIV). The interleukin-15 (IL-15) and interleukin-18 (IL-18) as genetic adjuvants were used for immunization in combination with the N1 and NP AIV genes. In the first trial, 8 groups of chickens were established with 10 specific-pathogen-free (SPF) chickens per group while, in the second trial 7 SPF chickens per group were used. The overall N1 enzyme-linked immunosorbent assay (ELISA) titer in chickens immunized with the pDis/N1+pDis/IL-15 was higher compared to the chickens immunized with the pDis/N1 and this suggesting that chicken IL-15 could play a role in enhancing the humoral immune response. Besides that, the chickens that were immunized at 14-day-old (Trial 2) showed a higher N1 antibody titer compared to the chickens that were immunized at 1-day-old (Trial 1). Despite the delayed in NP antibody responses, the chickens co-administrated with IL-15 were able to induce earlier and higher antibody response compared to the pDis/NP and pDis/NP+pDis/IL-18 inoculated groups. The pDis/N1+pDis/IL-15 inoculated chickens also induced higher CD8+ T cells increase than the pDis/N1 group in both trials (P<0.05). The flow cytometry results from both trials demonstrated that the pDis/N1+pDis/IL-18 groups were able to induce CD4+ T cells higher than the pDis/N1 group (P<0.05). Meanwhile, pDis/N1+pDis/IL-18 group was able to induce CD8+ T cells higher than the pDis/N1 group (P<0.05) in Trial 2 only. In the present study, pDis/NP was not significant (P>0.05) in inducing CD4+ and CD8+ T cells when co-administered with the pDis/IL-18 in both trials in comparison to the pDis/NP. Our data suggest that the pDis/N1+pDis/IL-15 combination has the potential to be used as a DNA vaccine against AIV in chickens.
Alternative DNA vaccine constructs such as fully synthetic linear expressing cassettes (LECs) offer the advantage of accelerated manufacturing techniques as well as the lack of both antibiotic resistance genes and bacterial contaminants. The speed of manufacture makes LEC technology a possible future vaccination strategy for pandemic influenza outbreaks. Previously, we reported on a novel concept of DNA delivery to dermal tissue by a minimally invasive electroporation (EP) surface device powered using low voltage parameters. This device allows electroporation without penetration of electrodes into the skin. In addition to enhancing the delivery of traditional plasmid DNA vaccines, this device may also offer a safe, tolerable and efficient method to administer LECs. To assess immunogenicity and efficacy of EP-enhanced LEC delivery in mice, we designed and tested two influenza antigens in the form of LEC constructs delivered using the newly developed surface dermal EP device. Strong CTL and antibody responses were induced by the LEC versions of the DNA vaccine. When challenged with A/Canada/AB/RV1532/2009 viruses, mice immunized with LEC encoding the M2 and NP antigens recovered faster than naïve or mice immunized ID without EP. Mice immunized with equal-molar doses of LEC encoding the M2 and NP antigens demonstrated 100% survival following a lethal (100× LD50) challenge of the heterologuos and highly pathogenic H5N1 influenza virus (A/Vietnam/1203/04). These results suggest that influenza DNA vaccines based on LEC technology combined with the surface delivery platform are capable of fully protecting mice in a lethal challenge and the LEC based DNA constructs may serve as viable vaccine candidates.
Immunity to influenza infection in man is a multifactorial phenomenon, and the relative importance of virus virulence, innate immunity, specific serum IgG antibody, cell-mediated immunity and local antibody is difficult to establish. Observations in vitro or in well-controlled laboratory animal model infections, where each influencing factor can be isolated and examined, form an important background to the interpretation of events in man; from the latter studies alone, only limited observations and correlations can be made. The genetic basis of influenza virus virulence is not established to date, but this probably has a marked effect on the expression of immunity. The over-all immunological response to a virus with 7 large proteins each with multiple antigenic determinants is likely to be extremely complex. Nevertheless, studies in man show a clear correlation between levels of serum IgG antibody to virus H or N antigens and immunity to infection; rather less clear is the role of locally produced IgA to these virus proteins; and least clear is the role of cell-mediated immunity.
The neuraminidase inhibitor 2-deoxy-2,3-dehydro-N-trifluoroacetylneuraminic acid (FANA) inhibits the mutlicycle replication of influenza viruses in tissue culture. Influenza virus grown in the presence of FANA contains neuraminic acid on its envelope which then serves as receptor for other virus particles causing extensive aggregation. Thus, FANA inhibits influenza virus replication by preventing the enzymatic removal of neuraminic acid from the virus envelope.
In Escherichia coli, the miniF plasmid CcdB protein is responsible for cell death when its action is not prevented by polypeptide CcdA. We report the isolation, localization, sequencing and properties of a bacterial mutant resistant to the cytotoxic activity of the CcdB protein. This mutation is located in the gene encoding the A subunit of topoisomerase II and produces an Arg462----Cys substitution in the amino acid sequence of the GyrA polypeptide. Hence, the mutation was called gyrA462. We show that in the wild-type strain, the CcdB protein promotes plasmid linearization; in the gyrA462 strain, this double-stranded DNA cleavage is suppressed. This indicates that the CcdB protein is responsible for gyrase-mediated double-stranded DNA breakage. CcdB, in the absence of CcdA, induces the SOS pathway. SOS induction is a biological response to DNA-damaging agents. We show that the gyrA462 mutation suppresses this SOS activation, indicating that SOS induction is a consequence of DNA damages promoted by the CcdB protein on gyrase-DNA complexes. In addition, we observe that the CcdBS sensitive phenotype dominates over the resistant phenotype. This is better explained by the conversion, in gyrA+/gyrA462 merodiploid strains, of the wild-type gyrase into a DNA-damaging agent. These results strongly suggest that the CcdB protein, like quinolone antibiotics and a variety of antitumoral drugs, is a DNA topoisomerase II poison. This is the first proteinic poison-antipoison mechanism that has been found to act via the DNA topoisomerase II.
Crystallographic studies of neuraminidase-sialic acid complexes indicate that sialic acid is distorted on binding the enzyme. Three arginine residues on the enzyme interact with the carboxylate group of the sugar which is observed to be equatorial to the saccharide ring as a consequence of its distorted geometry. The glycosidic oxygen is positioned within hydrogen-bonding distance of Asp-151, implicating this residue in catalysis.
It is the enzyme neuraminidase, projecting from the surface of influenza virus particles, which allows the virus to leave infected cells and spread in the body. Antibodies which inhibit the enzyme limit the infection, but antigenic variation of the neuraminidase renders it ineffective in a vaccine. This article describes the crystal structure of influenza virus neuraminidase, information about the active site which may lead to development of specific and effective inhibitors of the enzyme, and the structure of epitopes (antigenic determinants) on the neuraminidase. The 3-dimensional structure of the epitopes was obtained by X-ray diffraction methods using crystals of neuraminidase complexed with monoclonal antibody Fab fragments. Escape mutants, selected by growing virus in the presence of monoclonal antibodies to the neuraminidase, possess single amino acid sequence changes. The crystal structure of two mutants showed that the change in structure was restricted to that particular sidechain, but the change in the epitope was sufficient to abolish antibody binding even though it is known in one case that 21 other amino acids on the neuraminidase are in contact with the antibody.
Three different influenza virus neuraminidase (NA) genes have been subcloned into the vector pSC11 and expressed from the recombinant vaccinia viruses. These genes are from influenza viruses A/Tokyo/3/67 (N2); A/tern/Australia/G70c/75 (N9); and B/Hong Kong (HG)(NA of B/Lee/40). Cells infected with recombinants containing the NA gene express enzymatically active NA on the cell surface. The expressed protein results in the infected cells beings stripped of sialic acid, the receptor for influenza virus. This is not due to cleavage by NA from detached cells since at low multiplicity of infection only cells present at plaques are devoid of sialic acid. Thus NA is able to cleave sialic acid from neighboring glycoconjugates on the same membrane.
Two temperature sensitive mutants, ts3 and ts11, of the WSN (HON1) strain of influenza virus, belonging to the same recombination group, have a 1000- to 10,000-fold lower infectivity titer and lack hemagglutinating and neuraminidase activity when grown at nonpermissive temperature (39.5°), compared with virus grown at permissive temperature (33°). The patterns of viral polypeptides synthesized in cells infected with these mutants are similar to those found with wild type virus. Neuraminidase activity of the mutant viruses is more temperature labile than the enzyme of the wild type. Despite the low yield, morphologically intact virus particles are found at 39.5°, but in contrast to virus grown at 33° large aggregates of virus accumulate near the cell surface. These aggregated virus particles contain neuraminic acid as demonstrated by a colloidal iron stain. Thus, it is likely that a neuraminic acid containing protein serves as receptor for the hemagglutinin of other virus particles, resulting in the extensive aggregation. Hemagglutinating activity of virus grown at 39.5° is restored by treatment of the aggregates with neuraminidase from Vibrio cholerae. These results suggest that the lack of hemagglutinating activity of mutant virus grown at 39.5° is a consequence of the formation of aggregates of virus particles carrying neuraminic acid on their surface, and that the ts defect is in the neuraminidase but not the hemagglutinin molecule. It is postulated that neuraminidase is essential for the replication of influenza viruses and is required to remove neuraminic acid from the viral envelope to avoid aggregation of the progeny virus.
In October of 1993, there was decreased egg production and increased mortality among Mexican chickens, in association with serologic evidence of an H5N2 influenza virus. First isolated from chickens in May of 1994, after spreading widely in the country, the virus caused only a mild respiratory syndrome in specific pathogen-free chickens. Because eradication of the virus by destruction of infected birds posed major obstacles to the poultry industry in Mexico, we were able to conduct a "field experiment" to determine the fate of an avirulent virus after repeated cycles of replication in millions of chickens. By the end of 1994, the virus had mutated to contain a highly cleavable hemagglutinin (HA), but remained only mildly pathogenic in chickens. Within months, however, it had become lethal in poultry. Nucleotide sequence analysis of the HA cleavage site of the original avirulent strain revealed R-E-T-R, typical of avirulent viruses and unlike the K-K-K-R sequence characterizing viruses responsible for the 1983 outbreak in poultry in the United States. Both mildly and highly pathogenic isolates contained insertions and a substitution of basic residues in the HA connecting peptide, R-K-R-K-T-R, which made the HA highly cleavable in trypsin-free chicken embryo fibroblasts. Phylogenetic analysis of the HA of H5 avian influenza viruses, including the Mexican isolates, indicated that the epidemic virus had originated from the introduction of a single virus of the North American lineage into Mexican chickens. This sequence of events demonstrates, apparently for the first time, the stepwise acquisition of virulence by an avian influenza virus in nature.
Proteolytic cleavage of the influenza virus hemagglutinin glycoprotein (HA) by cellular proteases is a prerequisite for virus infectivity, spread of the virus in the infected organism, tissue tropism, and viral pathogenicity. Production of infectious virus depends upon the structure at the HA cleavage site as well as the substrate specificity and the distribution of appropriate enzymes. Differences exist in the specificities of the endoproteases that recognize the different sequence motifs at the cleavage site. With avian influenza viruses that cause lethal systemic infections, the cleavage site consists of multibasic amino acids. Furin, which activates this type of HA, is a member of the subtilisin family and represents the prototype of ubiquitously occurring membrane-bound proteases. On the other hand, serine proteases secreted from a restricted number of cell types and some bacterial enzymes recognize a monobasic cleavage signal at HA of the mammalian and the apathogenic avian influenza viruses. The limited occurrence of these proteases results in only localized infection. Implementation of these defined conditions for virus activation may represent a novel type of disease control.
In this study, we investigated the role of the conserved neuraminidase (NA) cytoplasmic tail residues in influenza virus replication. Mutants of influenza A virus (A/WSN/33 [H1N1]) with deletions of the NA cytoplasmic tail region were generated by reverse genetics. The resulting viruses, designated NOTAIL, contain only the initiating methionine of the conserved six amino-terminal residues. The mutant viruses grew much less readily and produced smaller plaques than did the wild-type virus. Despite similar levels of NA cell surface expression by the NOTAIL mutants and wild-type virus, incorporation of mutant NA molecules into virions was decreased by 86%. This reduction resulted in less NA activity per virion, leading to the formation of large aggregates of progeny mutant virions on the surface of infected cells. A NOTAIL virus containing an additional mutation (Ser-12 to Pro) in the transmembrane domain incorporated three times more NA molecules into virions than did the NOTAIL parent but approximately half of the amount incorporated by the wild-type virus. However, aggregation of the progeny virions still occurred at the cell surface. All NOTAIL viruses were attenuated in mice. We conclude that the cytoplasmic tail of NA is not absolutely essential for virus replication but exerts important effects on the incorporation of NA into virions and thus on the aggregation and virulence of progeny virus. In addition, the relative abundance of long filamentous particles formed by the NOTAIL mutants, compared with the largely spherical wild-type particles, indicates a role for the NA cytoplasmic tail in virion morphogenesis.
Analysis of the structure of the avian influenza (AI) virus hemagglutinin (HA) gene and protein has yielded a wealth of information on the virulence mechanisms of influenza viruses. The AI hemagglutinin appears to be unique in its capacity to accept basic amino acids at its proteolytic cleavage site (PCS). The association of multiple basic (MB) amino acids, HA cleavage, tissue spread and virulence by AI strains first proposed in the late 1970s and early 1980s [Klenk, H.D., Rott, R., Orlich, M., 1977. J. Gen. Virol. 36, 151-161; Bosch, F.X., Garten, W., Klenk, H.D., Rott, R., 1981. Virology 113, 725-735] has held fast for two decades now. While other structural characteristics and other genes can certainly influence virulence, the presence of MB amino acids at the PCS has provided a hallmark structural feature which justifies continuing sequence analysis of emerging field isolates of AI strains. In addition to this structural feature, the distal tip of the HA is prone to appearance and disappearance of glycosylation sites, some of which have been associated with virulence. The recent outbreaks of highly pathogenic AI in Mexico, Australia, Pakistan, Hong Kong and in the ongoing outbreak of moderately pathogenic H7 avian influenza in the northeast US have all provided new and useful information regarding the role of HA RNA and protein structure in both virulence and host adaptation. We have previously noted that stable RNA secondary structure near the PCS is related to the acquisition of virulence and have proposed that the secondary structure may promote the insertion of basic amino acids. In this report we evaluate the phylogenetic relationships for three recent isolates of highly pathogenic avian influenza viruses and the possible virulence factors associated with their primary and secondary structure.
As a result of numerous genome sequencing projects, large numbers of candidate open reading frames are being identified, many of which have no known function. Analysis of these genes typically involves the transfer of DNA segments into a variety of vector backgrounds for protein expression and functional analysis. We describe a method called recombinational cloning that uses in vitro site-specific recombination to accomplish the directional cloning of PCR products and the subsequent automatic subcloning of the DNA segment into new vector backbones at high efficiency. Numerous DNA segments can be transferred in parallel into many different vector backgrounds, providing an approach to high-throughput, in-depth functional analysis of genes and rapid optimization of protein expression. The resulting subclones maintain orientation and reading frame register, allowing amino- and carboxy-terminal translation fusions to be generated. In this paper, we outline the concepts of this approach and provide several examples that highlight some of its potential.
The rapid evolution of influenza A and B viruses contributes to annual influenza epidemics in humans. In addition, pandemics of influenza are also caused by influenza A viruses, whereas influenza B does not have the potential to cause pandemics because there is no animal reservoir of the virus. Study of the genetic differences between influenza A and influenza B viruses, which are restricted to humans, may be informative in understanding the factors that govern mammalian adaptation of influenza A viruses.
Aquatic birds provide the natural reservoir for influenza A viruses, but in general, avian influenza is asymptomatic in feral birds. Occasionally, however, highly pathogenic strains of influenza cause serious systemic infections in domestic poultry. The pathogenicity of these strains is related to the presence of a polybasic cleavage sequence in the precursor of the surface glycoprotein haemagglutinin, which makes the glycoprotein susceptible to activation by ubiquitous proteases such as furin and PC6.
However, the mechanism of pathogenicity may differ in highly pathogenic strains of human influenza, such as the H1N1 pandemic strain of 1918 and the H5N1 strain involved in the outbreak in Hong Kong in 1997. Binding of host proteases by the viral neuraminidase to assist activation of the haemagglutinin, shortening of the neuraminidase and substitutions in the polymerase gene, PB2, have all been suggested as alternative molecular correlates of pathogenicity of human influenza viruses. Additionally, systemic spread in humans of pathogenic subtypes has not been demonstrated and host factors such as interferons may be crucial in preventing the spread of the virus outside the respiratory tract. Copyright
The hemagglutination (HA) assay is a tool used to screen cell culture or amnioallantoic fluid harvested from embryonating chicken eggs for hemagglutinating agents, such as type A influenza. The HA assay is not an identification assay, as other agents also have hemagglutinating properties. Live and inactivated viruses are detected by the HA test. Amplification by virus isolation in embryonating chicken eggs or cell culture is typically required before HA activity can be detected from a clinical sample. The test is, to some extent, quantitative [1 hemagglutinating unit (HAU) is equal to approximately 5-6 logs of virus]. It is inexpensive and relatively simple to conduct. Several factors (quality of chicken erythrocytes, laboratory temperature, laboratory equipment, technical expertise of the user) may contribute to slight differences in the interpretation of the test each time it is run. This chapter will describe the methods validated and used by the National Veterinary Services Laboratories (NVSL) for screening and identification of hemagglutinating viruses.
Control of directionality in site-specific recombinationThe paper requires good English revision Update: Isolation of avian influenza A (H5N1) viruses from human – Hong Kong
- W Bushman
- J F Thompson
- L Vargas
- A Landy
Bushman,W.; Thompson,J.F.; Vargas,L. and Landy.A.
1985. Control of directionality in site-specific
recombination.Science 230,906-911.The paper requires
good English revision.
Centers for Disease Control (1998). Update: Isolation of
avian influenza A (H5N1) viruses from human – Hong
Kong, 1997 – 1998,Morb.Mortal.Wkly Rep. 46: 1245–
1247.
Incidence of Avian Influenza Among Commercial and Native Breeds In West Delta Region
- M Ashraf
- Y Sahar
- A Abdelsatar
- Arafa
Ashraf,M. Awad; Sahar, Y.; Abdelsatar, A.
Arafa,(2013).Incidence of Avian Influenza Among
Commercial and Native Breeds In West Delta
Region.Alexandria Journal of Veterinary Sciences 2013,
39: 31-39 ISSN 110-2047
HA) cleavage site sequence of H5 and H7 avian in¯uenza viruses: amino acid sequence at the HA cleavage site as a marker of pathogenicity potential characterization of an avian influenza A (H5N1) virus isolated from a child with a fatal respiratory illness
Survey of the hemagglutinin (HA) cleavage site sequence
of H5 and H7 avian in¯uenza viruses: amino acid
sequence at the HA cleavage site as a marker of
pathogenicity potential. Avian Dis. 40, 425±437.
Subbarao, K., A. Klimov, and
J.Katz, (1998).
characterization of an avian influenza A (H5N1) virus
isolated from a child with a fatal respiratory illness.
Science 279: 393–396.
Molecular genotyping of highly pathogenic avian influenza H5N1 in Egypt
- A N Maha
- Gamal
- Y A Soliman
- S A Khalil
Maha, A.N. Gamal ; Soliman, Y.A. and Khalil, S. A
(2012).Molecular genotyping of highly pathogenic avian
influenza H5N1 in Egypt.Researcher, 4(4) : 69-76.
Update: Isolation of avian influenza A (H5N1) viruses from human -Hong Kong
Centers for Disease Control (1998). Update: Isolation of
avian influenza A (H5N1) viruses from human -Hong
Kong, 1997 -1998,Morb.Mortal.Wkly Rep. 46: 1245-1247.
Immunologic and proteolytic analysis of HIV-1 reverse transcriptase structure
- S H Hughes
Hughes, SH(1990). "Immunologic and
proteolytic analysis of HIV-1 reverse transcriptase
structure."(PDF).
Virology.
175
(2):
456-64.
doi:10.1016/0042-6822(90)90430-y. PMID 1691562.