Dissertations / Theses on the topic 'Avian influenza virus M2e protein'

Within the avian influenza virus (AIV) history, H5N1 subtype is the most alarming in terms of its spread rate throughout the globe with its demonstrated unusual pattern of evolution. Persistency and constant circulation of this subtype in poultry population in a number of countries have resulted its establishment and declaration as enzootic. The affected countries are commonly characterised by high poultry populations and productions. They are also developing countries which have minimal funding allocated for precaution on disease incursion. Past observations showed that a single AIV epizootic is capable of causing significant economic burden throughout the world. Although epizootic, it still resulted sporadic cases of human infection and mortality. Therefore, H5N1 enzootic countries opt for vaccination strategy (usually with inactivated whole virus) to evade AIV incursions. However, this interferes with the AIV surveillance effort. This is due to the lack of diagnostic tool with the ability to differentiate AIV infected animal from vaccinated animal (DIVA). Following this realisation, several options are made available. Diagnostic tool development which is capable of DIVA requires a highly sensitive and specific target which at the same time is economic, and pose ease of application. In recent years, growing interest on the AIV matrix 2 extracellular domain (M2e) protein has propelled its exploration as the target for AIV serosurveillance diagnostic tool development. It has been demonstrated to be highly sensitive and specific in detection for AIV infection in an indirect enzyme-linked immunosorbent assay (ELISA) setting. The factor which made it highly interesting is its ability for DIVA application. M2e protein can only be found in low concentration on an AIV particle which is used in an inactivated vaccination strategy, while present in high concentration if cells are AIV infected. Therefore, this study has further explores the AIV M2e protein potential for AIV serosurveillance diagnostic tool development and successfully demonstrated an M2e-based test in a competitive ELISA format for DIVA. This particular ELISA format was of interest as it can be potentially used in multiple species application, as AIV is a multispecies pathogen. To ensure the universality of the competitor antibody, comparative mapping of anti-M2e antibodies from chicken, mouse and rabbit was done. Findings highlighted slight variations in the epitope identified for the M2e antigen by antibodies from different species. Mouse anti-M2e antibodies are more suitable to be used as the competitor antibodies against anti-M2e chicken sera in the M2e-based competitive ELISA test. Consequently, application of the mouse anti-M2e antibodies in the M2e-based competitive ELISA has demonstrated specific and sensitive indication of AIV infection in the H5N1 challenged chicken sera. Biotechnology developments has also introduced the single chain variable fragment (scFv) antibodies as specific and stable bait for antibodies detection against targeted pathogen’s protein (antigen). Taking advantage of this knowledge, this study has also successfully isolated reactive and specific anti-M2e scFv antibodies from avian sources. This is critical as an avian sourced antibodies to be used as bait for the targeted pathogen’s protein is highly relevant in the setting for AIV serosurveillance application in the poultry industry. These findings are significant in the effort to provide a highly sensitive and specific diagnostic tool, which are also cost effective, easy to apply with high throughput ability. Such ideal diagnostic tool for AIV serosurveillance is highly valuable, as this may hold the key to break the AIV continuous circulation.
Thesis (Ph.D.) -- University of Adelaide, School of Animal and Veterinary Sciences, 2017

碩士
國立嘉義大學
生物農業科技學系研究所
106
Avian influenza（Avian Influenza；AI）is commonly known as avian flu. It is one kind of animal infectious diseases caused by flu viruses. AI viruses can be grouped into highly pathogenic avian influenza (HPAI) and low pathogenic avian influenza (LPAI). The mortality of the poultry infected by HPAI can be higher than 80%. In this study, H1N1 viral M2 gene segment was constructed into the E. coli protein expression vector for expressing the M2 gene 's fusion protein with 8X-Histidine tag. The serum samples of vaccinated mice were assayed by dot blotting and ELISA. The specific antibody against the overexpressed M2 gene's protein produced in the vaccinated mice was determined by dot blotting assay. The M2 gene's fusion protein was also shown to have better immune response in association with the commercial adjuvant. These studies have laid a strong foundation for developing an effective AI subunit vaccine.

Although avian influenza virus (AIV) infections in domestic poultry are uncommon, transmission of avian influenza from wild waterfowl reservoirs does occur. Depopulation of the infected flock is the typical response to AIV outbreaks in domestic chicken production, causing a loss in profits and accumulation of unexpected expenses. Because it is impossible to know which of many virus subtypes will cause an outbreak, it is not feasible for the U.S. to stockpile vaccines against all possible avian influenza threats. Currently, the U.S. does not routinely vaccinate chickens against influenza due to the inability to differentiate infected from vaccinated animals (DIVA), which would place limitations on its trade markets. A Sindbis virus vector expressing the PR8 influenza strain's M2e peptide was developed as a potential universal DIVA vaccine. M2e is a conserved peptide amongst influenza A viruses; M2e-specific antibodies induce antibody-dependent cytotoxicity or phagocytosis of infected cells, reducing production and shedding of AIV during infection. In this study, chickens were vaccinated at one-month-of-age with parental (E2S1) or recombinant Sindbis viruses expressing the PR8 M2e peptide (E2S1-M2e) by subcutaneous or intranasal routes at high (106 pfu) or low (103 pfu) dosages. Chickens were boosted at 2-weeks post-initial vaccination using the same virus, route, and dosage, then challenged with low pathogenic H5N3 AIV at 0.2 mL of 106/mL EID50 2-weeks post-boost. Serum samples were collected at 1-week and 2-weeks post-vaccination, 2-weeks post-boost, and 2-weeks post-challenge and screened for PR8 M2e-specific IgY antibody production by ELISA. Both high and low dose subcutaneously, as well as high dose intranasally vaccinated E2S1-M2e groups produced significantly higher levels of PR8 M2e-specific IgY antibodies as early as 1-week post-vaccination, while the uninoculated control and E2S1 groups remained negative for all pre-challenge time points. M2e-specific IgY antibodies capable of binding the challenge H5N3 M2e peptide were detected in groups with existing vaccine-induced M2e-specific antibodies pre-challenge, suggesting antibody M2e cross-reactivity. After challenge, all groups developed M2e-specific IgY antibodies and high HI titers, verifying successful AIV infection during challenge and production of hemagglutinin-specific antibodies. Viral shedding titers 4-days post-challenge were used to measure vaccine efficacy and were similar amongst all groups. Microneutralization assay results confirmed that post-boost serum samples, containing only M2e-specific antibodies, were unable to neutralize AIV in vitro. Although the E2S1-M2e vaccine was capable of producing high levels of M2e-specific IgY antibodies when inoculated subcutaneously, these antibodies were not able to reduce viral shedding and therefore did not protect chickens from AIV.

碩士
國立臺灣大學
獸醫學研究所
94
From 1986 to 2003, the avian influenza viruses (AIV) isolated in Taiwan were all low pathogenic. Some low pathogenic H5N2 AIVs were isolated in Taiwan in 2004, resulting in stamping 380 thousands chickens out from 22 poultry flocks. To test if the virus titers increased during passaging, H5N2 and H6N1 isolates were passaged in specific-pathogen-free embryonated eggs. After 45 passages, the virus titer of H5N2 increased 100 times, and two amino acid residues in HA1 of H5N2 virus were changed. The partial fragments of HA1 relating to neutralizing epitopes of the H5N2 and the H6N1 isolates were cloned in pGEX-5X-1 vector (prokaryotic expression) and pcDNA3.1 vector (eukaryotic expression) for expression. The purified recombinant proteins were intramuscualarly injected in 4-week-old SPF chickens for two times. Two weeks after the second immunization, the serum from H6-immunized chickens showed hemagglutination inhibition (HI) titers of 24 to 26, but no antibody was detected in H5-immunized chickens. To test the increase efficiency of liposome compounds on vaccine, Newcastle disease vaccines coated with liposome and with liposome/chitosan were used to immunize chickens. The results showed that Newcastle disease vaccine coated with liposome/chitosan increased HI titers in vaccinated chickens but not vaccine coated with lipsome alone.

碩士
國立屏東科技大學
生物科技研究所
94
Avian influenza virus（AIV）belongs to orthomyxoviridae family. Highly pathogenetic avian influenza virus causes flowl plague in chickens and turkeys. The HA protein of AIV 1209 strain was expressed by Pichia pastoris yeast expression system. The results indicated that the tagged fusion protein was present both in medium and pellet. The HA protein could be cleaved into HA1（47 kDa） and HA2（22 kDa）. The AIV CE9 could induce apoptosis in MDCK cell. To further study the molecular mechanism of AIV-induced apoptosis, the NA protein of AIV H5N2 CE9 strain was cloned into pcDNA3.1(-), and the recombinant DNA transfected into MDCK cell lines. The results indicated that at the same focus, caspase 3 were not detected in the MDCK cell expressing the GFP-NA fusion protein. Phylogenesis analysis of HA and NA gene of strains Taiwanese 1209 and CE9 was done to sequence with other H5N2 viruses in the world using MEGA 3.1 software. The result indicate the 1209 and CE9 strain belong to the same group as the Italy strain.

Although wild birds are natural hosts of avian influenza viruses (AIVs), these viruses can be highly contagious to poultry and a zoonotic threat to humans. The propensity of AIV for genetic variation through genetic shift and drift allows virus to evade vaccine mediated humoral immunity. An alternative approach to current vaccine development is induction of CD8+ T cells which responds to more conserved epitopes than humoral immunity and targets a broader spectrum of viruses. Since the memory CD8+ T lymphocyte responses in chickens to individual AIV proteins have not been defined, the modulation of responses of the memory CD8+ T lymphocytes to H5N9 AIV hemagglutinin (HA) and nucleocapsid (NP) proteins over a time course were evaluated. CD8+ T lymphocyte responses induced by intramuscular inoculation of chickens with AIV HA and NP expressing cDNA plasmids or a non-replicating human adenovirus vector were identified through ex vivo stimulation with virus infected, major histocompatibility complex (MHC) matched antigen presenting cells (APCs). The IFN? production by activated lymphocytes was evaluated by macrophage production of nitric oxide and ELISA. MHC-I restricted memory T lymphocyte responses were determined at 10 days and 3, 5, 7 and 9 weeks post-inoculation (p.i). The use of non-professional APCs and APC driven proliferation of cells with CD8+ phenotype correlated with the activation of CD8+ T lymphocytes. The responses specific to nucleocapsid protein (NP) were consistently greater than those to the hemagglutinin (HA) at 5 weeks when the CD8+ T cell responses were maximum. By 8 to 9 weeks p.i., responses to either protein were undetectable. The T lymphocytes also responded to stimulation with a heterologous H7N2 AIV infected APCs. Administration of booster dose induced secondary effector cell mediated immune responses which had greater magnitudes than primary effector responses at 10 days p.i. Flow cytometric analysis (FACS) of the T lymphocytes demonstrated that memory CD8+ T lymphocytes of chickens can be distinguished from naive lymphocytes by their higher expression of CD44 and CD45 surface antigens. CD45 expression of memory lymphocytes further increases upon ex vivo stimulation with APCs expressing AIV. This is the first characterization of avian memory responses following both primary and secondary expression of any individual viral protein.

碩士
國立臺灣大學
獸醫學研究所
102
NS1 is an indictor Ag of influenza virus (IV) infection and it’s only produced during IV replication in very early stage of infection. It can be used in determining whether the chicken was infected or not, which also can be applied on early diagnosis of IV infection. Therefore, my study aimed to characterize the anti-NS1 MAb (αNS1 MAb) by indirect ELISA (iELISA), indirect immunofluorescence assay (IFA), western blotting (WB), immunohistochemistry stain (IHC) and mapping the antibody binding site by eukaryotic expression system (EES). Subsequently, to develop a rapid, sensitive and specific diagnostic MAbs-based NS1 Ag sandwich ELISA that might be incorporated with NP and M Ag ELISA kit for detecting the IV infection. The parental hybridoma have been prepared by immunizing Balb/c mice with E. coli expressed recombinant nonstructural protein 1 (rNS1) oringinated from IV isolates of A/chicken/Taiwan/2838V/00 (H6N1/2838). 16 αNS1 MAbs have been characterized by WB, IFA and iELISA with NS1 Ag from several IV isolates such as A/chicken/Miaoli/2904/00 (H6N1/2904), A/chicken/Taiwan/1209/03 (H5N2/1209). Meanwhile, the epitope mapping was studied in EES. Expression of full length rNS1 (residues 1-230) were all positive and 4 αNS1 MAb (4M4, 4M6, 4N5, 4R3) were negative in C-terminal deletion (residues 1-207). Accroding to epitope mapping analysis results, which can divide 16 αNS1 MAb into four groups (A, B, C &; D). The predictive epitopes of 16 αNS1 MAb mainly recognize the effector domain (residues 74-230) and group B recognize the C-terminal tail (residues 202-230) of NS1 protein. MAb-based NS1 Ag sandwich ELISA was designed as sixteen MAb individually conjugated with HRP as the tracer Ab paired with each non-conjugated MAb acted as docking Ab respectively to compare the binding ability of those combinations with E. coli expressed rNS1. Current results indicated that MP-5 (4R11 – 4M2/HRP), MP-9 (4R11 – 4J12/HRP) and MP-10 (4M2 – 4M2/HRP) can detect NS1 Ag from 15 subtypes of IV (H1~H15). It is believed the three MP (MP-5, MP-9 &; MP-10) we studied have potential to be applied on detecting the IV NS1 Ag.

A proper vaccination program can play a critical role in prevention and control of avian influenza (AI) in commercial poultry. Low pathogenic avian influenza viruses (LPAIV) of H5 and H7 AI subtypes cause serious economic losses to the poultry industry and have the potential to mutate to highly pathogenic AI (HPAI) strains. Due to trade implications, differentiation of infected from vaccinated animals (DIVA) is an important issue in the control of AI. Therefore, the development and characterization of vaccine candidates with DIVA properties is critical in improving vaccination programs. Keeping these aspects in mind, we investigated the role of an NS1 mutant virus as a potential live attenuated DIVA vaccine. The NS1 protein of influenza virus plays a major role in blocking the host's antiviral response. Using an eight-plasmid reverse genetics system, we recovered the low pathogenic parental (H5N3) and NS1 mutant (H5N3/NS1/144) viruses. H5N3/NS1/144 expresses only the first 144 amino acids of the NS1 protein compared to the 230 of the parental H5N3. The growth properties of H5N3 and H5N3/NS1/144 were compared in cell culture and in different age embryonated chicken eggs. Our results confirmed that NS1 is involved in down regulation of interferon as shown by IFN-beta mRNA expression analysis and by the inability of H5N3/NS1-144 to efficiently grow in older age, interferon competent, chicken embryos. However with regards to safety the virus reverted to virulence within five back passages in chickens and was therefore not a safe vaccine candidate. However the killed form of H5N3/NS1-144 was a safer alternative and it also induced antibody titers and protection not significantly different from the parental H5N3 as vaccine. To further understand the reversion of H5N3/NS1/144 to virulence, we carried out 3 independent serial passages of H5N3/NS1/144 in increasing age of embryonated chicken eggs and examined the NS1 gene for presence of mutations. RT-PCR and sequence analysis of the NS gene in all three lineages showed the presence of a 54 amino acid deletion resulting in the generation of a 87 amino acids long NS1 ORF with a point mutation (L80V) at the site of deletion. In addition, the NS1 ORF in lineages L2 and L3 presented two additional point mutations in the RNA binding domain (Q40R and T73M). To determine if these mutations played a role in increased virulence, recombinant viruses expressing these mutant NS1 proteins in the background of parental virus were generated by reverse genetics and their replication properties and pathogenicity was examined in vitro, in ovo and in vivo systems. Our results showed that the 87 amino acid long NS1 protein clearly increased virus replication and virulence specifically in interferon competent systems. In addition, the two point mutations in the RNA binding domain of NS1 ORF expressing 87 a protein slightly increased the virus virulence. Overall this study reinforces the role of NS1 in influenza virus pathogenicity and supports the use of killed inactivated NS1 mutant virus vaccines as potential DIVA vaccines.

碩士
中興大學
獸醫學系暨研究所
95
Abstract Avian influenza (AI) is a serious infectious disease caused by avian influenza virus (AIV) belonging to type A Orthomyxovirus. Vaccination programs for controlling avian influenza (AI) in poultry have limitations because of differentiating between vaccinated and virus-infected birds. Nonstructural (NS) proteins can be used as markers because viral infection can induce antibodies against both structural and nonstructural antigens. While immunized with inactivated viral vaccines, animals elicit antibodies from only structural proteins. Two nonstructural proteins (NS1 and NS2) are present in cells when infected with influenza virus. The NS1 protein of AIV is the major nonstructural protein and is highly conserved among AIV subtypes. The antigenic analyses of the purified NSl protein from several subtypes have indicated cross-reactivity among all influenza A virus strains. The presence of an anti-NSl response provides a useful measure in influenza viral infection, and highly circulating anti-HA antibodies. In order to use the immune response of the NSl protein in a routine diagnostic setting, it is necessary to develop an enzyme-linked immunosorbent assay (ELISA) with recombinant NS1. We have used NS1, the conserved nonstructural protein of influenza A virus, as a differential diagnostic marker for influenza virus infection. Experimentally infected poultry were evaluated for the ability to induce antibodies reactive to NS1 recombinant protein produced in Escherichia coli, which was cloned from H6N1 AIV A / Chicken / Taiwan / NCHU-0507 / 99 strain. Immune sera were obtained from SPF chickens inoculated with live AI virus, inactivated semi-purified AI virus, or inactivated AI virus. Animals that received live AI virus, inactivated purified AI virus, or inactivated AI virus were found to possess antibodies against AI virus, as measured by the standard agar gel precipitin (AGP) test. Seroconversion to positive for antibodies to the NS1 protein was achieved in birds experimentally infected with live AI virus, as determined by enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. In contrast, animals inoculated with inactivated AI virus had faint seroconversion to positive for antibodies to the NS1 protein, and animals vaccinated with inactivated semi-purified AI virus had low, but detectable, levels of NS1 antibodies. These results demonstrate the potential benefit of a simple, specific ELISA for anti-NS1 antibodies that may have diagnostic value for the poultry industry, this ELISA is useful for serological diagnosis to distinguish poultry infected with influenza viruses from those immunized with inactivated vaccine.

碩士
國立中興大學
獸醫微生物學研究所
94
Avian influenza viruses are members of the orthomyxoviridae family and are grouped into type A influenza virus according to antigenic characteristics of their core proteins. These viruses can be further classified by their surface proteins hemagglutinin (H1-H16) and neuraminidase (N1-N9). During 1999 to 2000, outbroke of low or high pathogenic H7N1 avian influenza occured in Italy and led to a sobering economic loss. In consequence, the government of Italy developed an effective DIVA methodology as a tool for the eradication of AI. The goal of this study is to establish a DIVA methodology for rapid subtyping of NA-antibodies. The method is to use Bac-to-Bac® baculovirus express system to construct recombinant baculovirus containing N1 and N2 genes. Sf9 and High FiveTM insect cells were then used to produce recombinant NA (rNA) protein by infection with recombinant baculovirus. Possible glycosylations of the full length N1 and N2 recombinant proteins were observed based on the molecular weight of SDS-PAGE. However, the amount of recombinant N1 and N2 proteins expressed in insect cells were low. These two rNA proteins were probed with antibodies of 9 NA subtypes by multi-screen channel Western blot analysis, and the result showed that both N1 and N2 recombinant protein could discriminate the N1 and N2 sera, respectively. Moreover, the two rNA proteins were used as antigens to detect antibodies of 9 NA subtypes by indirect immunofluorescence assay (iIFA) analysis, and the result showed that recombinant N1 can discriminate each subtype sera except for the N2. The recombinant N1 and N2 proteins produced in this study could be used to establish a DIVA method that could help the surveillance and prevention of AIV in Taiwan.

碩士
國立中興大學
獸醫學系
93
Avian influenza virus is a type A influenza virus. According to virus surface glycoprotein, fifteen HA subtypes（H1-H15）and nine NA subtypes（N1-N9）have been identified. During 1999 and 2000, chickens in Italy were infected by H7N1 viruses of low or high pathogenicity, which resulted in a drastic economic loss. Because of this impact, the government of Italy developed an effective DIVA methodology as a tool for the eradication of AI in Italy. In this study, recombinant baculovirus containing the N1, N3 and N7 genes were constructed by Bac-N-BlueTM or Bac-to-Bac® expression system. High Five insect cells were used to produce recombinant NA（rNA） protein. Possible glycosylations of the full length N3 and N7 recombinant proteins were observed by SDS-PAGE. These two rNA proteins were used to detect antibodies of 9 NA subtypes by multi-screen channel Western blot analysis, and the result showed that N3 recombinant protein could discriminate the N3-specific sera from sera of other subtypes. Moreover, N7 recombinant protein could discriminate the N7-specific sera from sera of other subtypes, except for the N6- and N8-specific sera. The two rNA proteins were used as antigens to detect antibodies of 9 NA subtypes by indirect immunofluorescence assay（iIFA）analysis, and the result showed that sera of the same subtype had strong positive fluorescence against the rNA proteins. Therefore, this iIFA can discriminate sera of N3 and N7 subtypes from sera of other subtypes, especially from N1, N2, N5 and N9 subtypes. The recombinant N3 and N7 proteins produced in this study could be used to establish a DIVA method that could help the surveillance and prevention of AIV.

Influenza A viruses (IAV) are prominent human pathogens and understanding the molecular biology of their replication is of utmost importance for the development novel anti-viral treatments and surveillance practices. IAV genome replication proceeds in the nucleus of infecting cells; where the virus takes advantage of host cell transcription machinery, particularly mRNA processing enzymes. The role of nuclear localization in the viral replication cycle is not clear and the viral and host cell determinants that are required for efficient nuclear import and export are an active area of study in our laboratory and others. Specifically, we are studying the role of IAV Nuclear Export Protein (NEP) in nuclear export in virus replication, biogenesis, and pathogenesis. A previously identified host nuclear export protein, CRM-1, has been identified as a critical mediator of the nuclear egress of newly replicated viral ribonucleoproteins (vRNPs). To define the virus-host interactions required for pathogenesis in humans and ferrets, the preferred animal model for IAV infection, we have cloned and expressed human and ferret CRM-1 and fused them to GFP and RFP to allow in vitro analysis of NEP/CRM-1 interactions in the context of IAV replication and biogenesis. We have developed a mammalian two-hybrid system for co-transfection of human or ferret CRM-1 and NEP from a panel of human and highly pathogenic avian influenza virus strains. This mammalian two-hybrid system has allowed for the identification of critical amino acid residues required for efficient nuclear export and virus biogenesis in human or ferret cells with a focus on the role of these later steps in replication in the dissemination of virus in the infected host or in the transmission to other susceptible hosts. In addition, using microRNA (miRNA) targeting sequences of CRM-1 and/or NEP inhibiting their expression, we examined the interaction of these host and viral proteins in cells and the role of this interaction in replication efficiency. Our results are consistent with a critical role for these interactions in virus biogenesis and pathogenesis for influenza virus infection in both humans and ferrets providing novel anti-viral therapeutic targets for antiviral development
acase@tulane.edu