Avian Influenza Virus Nucleoprotein Research Articles

Surveillance for avian influenza virus in captive wild birds and indigenous chickens in Nigeria.

Several reports of avian influenza virus (AIV) have been made on commercial chickens and wild birds in sub-Saharan Africa, but there is paucity of information of AIV among captive wild birds and indigenous chickens. Blood samples were obtained randomly from captive wild birds and chickens. AIV nucleoprotein antibody detection involved the use of enzyme immunoassay (ELISA) and subsequent subtyping with H5 and H7 AIV antigens (haemagglutination inhibition). Four hundred birds belonging to nine families and 14 species were sampled, and overall prevalence of 23% (92/400) was obtained (captive wild birds (10.4%, 5/48), indigenous birds (47.3%, 87/184) and exotic commercial birds (0.0%, 0/168)). Twelve ELISA-positive birds (13.04%) were positive to H7 antigen. Univariate analysis indicated statistical significance of AIV prevalence in captive wild birds (p < 0.0001) and exotic birds (p < 0.0001) using indigenous chickens as reference. This study gave an evidence of exposure of captive wild birds and indigenous chickens to AIV in Nigeria. Scavenging activities common among indigenously raised chickens, unrestricted movement of nonflying wild birds within the captive complex and free access by migrating wild birds to captive wild birds and local chickens were likely factors observed to promote AIV transmission. Continuous surveillance can further highlight the roles played by these birds in the epidemiology of AIV.

Monoclonal Antibody Preparation Against Nucleoprotein of Avian Influenza Virus Subtype H9N2

Background: In recent years, various poultry diseases have posed risks to this industry. Respiratory and infectious diseases are the most common diseases. According to previous findings, influenza disease is recognized as a significant life-threatening disease in the industry of poultry worldwide. Influenza virus type A, which belongs to the family Orthomyxoviridae, is responsible for a serious infectious disease. H9N2 AVI subtype circulates in the poultry worldwide, causing significant economic losses and infections in humans, also domestic and wild animals. Objectives: The purpose of the present experimental research was monoclonal antibodies (MAbs) production in contradiction of the NP of avian influenza virus (AIV) H9N2 subtype to detect AIV antigens and antibodies in the Department of Proteomics and Biochemistry, Razi Vaccine and Serum Research Institute, Agricultural Research Education and Extension Organization (AREEO), Karaj, Iran. in 2017. Methods: The conserved protein of NP from H9N2A/Chicken/Iran/259/2014 virus, with 60 kDa molecular weight, was isolated using the electroelution method. The purified protein was applied for Australian BALB/c mice immunization. After evaluating immunization by ELISA assay, the spleen of immunized mice was isolated and hybridized with SP2/0 myeloma cell. Next, the hybridized cell was cultured, and clone soups were collected after 15 days to examine antibodies via ELISA assay. The produced antibodies using Western blotting and antibody isotype kits were characterized. Results: Thirty antibody-producing clones were examined for reactivity against Nucleoprotein (NP), the antigen, at 1 µg/mL concentration. According to the ELISA assay of antibody titers, two (3/F10 and 2/D7) out of 30 antibodies were bound to the antigen with titer 0.863 and 1.641, respectively. Two hybrid clones, 2D7 and 3F10, which produced anti-NP antibodies, were isolated and cultured. Characterizing of the produced antibodies using Western blotting was performed using H9N2 virus; finally, two clone soups (3/F10 and 2/D7) reacted with the NP virus protein. According to the isotyping of antibodies produced by 3F10 and 2D7 clones, 3F10 clone produced IgG1 with a κ chain, and IgG1 concentration was 1.997, based on ELISA assay. Also, 2D7 clone produced IgG2a with a κ chain, and IgG2a concentration was obtained 1.951. Conclusions: According to the findings of our study, the produced antibody might be used in the diagnosis of influenza.

Purification of Nucleoprotein of H9N2 Avian Influenza Virus Strain by Electroelution

Background: Avian influenza virus (AIV) belongs to the family of Orthomyxoviruses type A and causes avian influenza (AI) infectious disease. Currently, serological diagnostic techniques such as agar gel propagation (AGP), hemagglutination inhibition, and enzyme-linked immunosorbent assay (ELISA) are considered as important tools for the antibodies detection against viral antigens. Due to antigenic variation in the surface of AIV glycoproteins (hemagglutinin and neuraminidase), these proteins cannot be used in serological tests. Development of assays to detect AI surface glycoproteins is problematic because a great variety of combinations of these subtypes are found in nature. The internal antigen determinants on the nucleoprotein (NP) are highly conserved within influenza viruses, making this protein more appropriate for a serological test. Objectives: In the experimental present study, an effectual method was expanded to purify NP of H9N2 AIV based on Electroelution method. Methods: AIV strain A/flash chicken/Iran/772/1998 (H9N2) was acquired from the Department of Avian Influenza Reference Laboratory, Razi Vaccine and Serum Research Institute, Iran, about 2 cc, in 2017. Nucleoprotein of AIV (H9N2) was purified by an efficient and simple modified method directly from native polyacrylamide gel electrophoresis (PAGE) according to the Electroelution method. The purified protein concentration was defined by the Lowry method, and the purified NP protein (60 KDa) was examined by Tricine-SDS-PAGE. Results: The protein concentration of the virus solution was 4.62 mg/mL by the Lowry method. The purified Nucleoprotein concentration was 0.296 mg/mL by Lowry method and the Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis results showed only a 60-KDa protein band in the gel. Conclusions: The current technique was simple and rapid and made it possible to isolate NP from the H9N2 virus. The Nucleoprotein antigen is an appropriate candidate potential to detect antibodies against all subtypes of AIV and used as the main target antigen for the diagnosis of influenza virus due to its very high scale of sequence preserved among exist strains.

Establishment of xMAP for the simultaneous detection of antibodies to Newcastle disease virus and avian influenza virus

Using Luminex xMAP (x = analyte, MAP = multi-analyte profiling) technology, a serological method for the simultaneous detection of antibodies to Newcastle disease virus (NDV) and avian influenza virus (AIV) was established. Nano-magnetic beads coated with purified NDV protein and AIV nucleoprotein were incubated with serum samples. Using biotinylated rabbit anti-chicken IgY and streptavidin-R-phycoerythrin, the optical signals measured by a Luminex 200 detection system indicated the quantification of NDV or AIV antibodies in the serum. Specific pathogen-free (SPF) chicken serum was used as a negative control. The Luminex xMAP assay developed in this study demonstrated high specificity as there was no cross-reaction with antibodies to infectious laryngotracheitis virus, infectious bronchitis virus, infectious bursal disease virus, avian leukosis virus, and Marek's disease virus. The results from reproducibility experiments showed that intra-coefficients of variation were 3.36 and 9.23% and inter-coefficients of variation were 6.50 and 7.66% for NDV and AIV, respectively. The results also indicated that the Luminex xMAP assay was 16 times more sensitive for NDV antibody detection and 1,024 times more sensitive for AIV antibody detection compared to the enzyme-linked immunosorbent assay (ELISA). A total of 300 chicken serum samples were subjected to both Luminex xMAP assay and ELISA, showing the coincidence rates of 98.67 and 98% for NDV and AIV antibody detection, respectively. This study provides a new method for the simultaneous detection NDV and AIV antibodies in the serum with high specificity and sensitivity.

Production of monoclonal antibodies against recombinant nucleoprotein of avian influenza virus, serotype H9N2

Background: Avian influenza viruses (AIVs) including the subtype H9N2 cause considerable financial losses to poultry industries. Rapid and accurate diagnosis of avian influenza (AI) infection is important in control and eradication programs. OBJECTIVES: The aim of this study was to produce monoclonal antibodies (MAbs) specific for the nucleocapsid protein )NP (of AIV H9N2 subtype to improve diagnostic assays. METHODS: Recombinant NP protein was expressed in Escherichia coli and purified using amylose resin chromatography column and used as an antigen for mice immunization. Spleen cells of the immunized mice were fused with SP2/0 myeloma cells. Next, culture supernatants of primary hybridoma clones were screened by indirect ELISA. After three rounds of sub cloning, the reactivity of the MAbs with recombinant and natural antigens was assessed by Western blotting. RESULTS: Six MAbs showed specific binding to recombinant and natural NP from AIV H9N2 in Western blot analysis, enzyme-linked immunosorbent assay (ELISA) and immunofluorescence assay. Cross-reactivity with genetically non-related including Newcastle viruse (Paramyxoviridae family) was not detected. CONCLUSIONS: Based on the results, the MAbs generated in this study could be used for the development of rapid diagnostic assays for recognition of AIV.

LOW SPECIFICITY OF A NESTED REVERSE TRANSCRIPTION POLYMERASE CHAIN REACTION TO DETECT AVIAN INFLUENZA VIRUS NUCLEOPROTEIN GENE

Reverse transcription polymerase chain reaction (RT-PCR) was used routinely to detect the avian influenza virus (AIV) nucleoprotein (NP) gene. The purpose of the present study was to compare the correctness of a nested RT-PCR (nRT-PCR), one conventional RT-PCR with its outer primer (oRT-PCR) and the other conventional RT-PCR with its inner primer (iRT-PCR) to detect AIV NP gene. A total of 365 AI-free fecal swabs (73 pools), 7 tracheal swabs and anllantoic fluid from 25 chicken embryos were used to determine the analytic specificities of those tests. Compared with the iRT-PCR, the nRT-PCR was more sensitive for AIV detection. However, the specificities of nRT-PCR, oRT-PCR and iRT-PCR were 48.6% (35/72), 100% (67/67) and 91.3% (84/92), respectively. The amplifying band was sequenced and confirmed to be the AIV NP gene as the positive control. The specificity of this nRT-PCR is too low to be used for the AIV screening test.

Production and application of a monoclonal antibody based peroxidase conjugate for detection of chicken IgG antibodies in ELISA

Viral infections are the cause of great economic losses in the poultry industry. Development of appropriate reagents and serological diagnostic kits will help to control these infections. The aim of this study is to prepare a peroxidase labeled anti-chicken IgG using a MAb (5B8) against chicken IgG, for detection of chicken antibodies in ELISA. Hybridoma cells producing the MAb 5B8 were cultured in RPMI 1640 medium and the MAb was purified from the cells supernatant using a sepharose matrix column, sensitized with chicken IgG. The purified MAb was labeled with Horseradish peroxidase (HRP) by periodate treatment. The peroxidase labeled MAb was compared with a commercial polyclonal product for detection of chicken antibodies against avian influenza virus nucleoprotein in ELISA. Therefore, type A recombinant nucleoprotein influenza virus was used as the antigen and chicken sera prepared from healthy and influenza virus infected chickens were used as primary antibodies. The results showed that there is a strong and direct correlation (r =0.972) between the optical densities of a commercial anti-chicken IgG and the prepared conjugate. In conclusion, the conjugated MAb is appropriate for the development of serological diagnostic tests for poultry infections.

Development of a duplex Fluorescent Microsphere Immunoassay (FMIA) for the detection of antibody responses to influenza A and newcastle disease viruses

Highly pathogenic avian influenza virus (HPAI) and virulent forms of avian paramyxovirus-1 (APMV-1) cause serious illnesses in domestic poultry, both of which are reportable to the World Organization of Animal Health (OIE). The clinical presentation of avian influenza (AI) and APMV-1 infections are difficult to differentiate, emphasizing the importance of rapid and sensitive serologic assays that are able to distinguish them. Currently, a variety of serological assays are used for the serologic diagnosis of both diseases, but these assays are not used in multiplex formats. In this study, development of a duplex fluorescent microsphere immunoassay (FMIA) based on Luminex xMAP Technology is described. The assay employs MagPlex magnetic microspheres that are covalently coated with recombinant avian influenza virus nucleoprotein and APMV-1 nucleocapsid antigens produced in a baculovirus insect cell expression system. The assay is able to detect AIV antibodies against all existing hemagglutinin (H1–H16) subtypes and simultaneously detect antibodies against APMV-1. In the process of this assay development different bead coupling conditions were compared. The assay has the capability of detecting serum antibodies from chickens and turkeys and optimization was accomplished by using 2462 chicken and 446 turkey field and experimental sera and had a comparable detection capability with currently used assays in the laboratory. Assay threshold values were calculated with Receiver Operating Characteristic Analysis (ROC) in non-parametric analysis due to a highly skewed data distribution; this analysis resulted in AIV nucleoprotein relative diagnostic sensitivity and specificity of 99.7%, and 97.3% respectively. The APMV-1 nucleocapsid relative diagnostic sensitivity and specificity were 95.4%, and 98.5% respectively.

Production and Characterization of Monoclonal Antibodies Against Nucleoprotein of Avian Influenza Virus

The present study was carried out with an aim to develop anti-nucleoprotein (anti-NP) monoclonal antibodies (MAbs) for use in immunodiagnostic testing for detection of avian influenza virus (AIV) antigen or antibodies. The NP gene of AIV, cloned in pET vector, was expressed in Escherichia coli BL 21 strain to produce a 6x-His tagged recombinant NP (rNP) antigen of ∼61 kDa molecular weight as soluble fraction. The rNP antigen was detected in soluble fraction of bacterial cell lysate with anti-His HRPO conjugate and reacted with the reference AIV antibody positive serum in immunoblotting. The rNP was used to immunize BALB/c mice to produce hybridoma secreting anti-NP MAbs. Out of 11 anti-NP MAbs produced, 8D2-H5, 8D2-H9, and 6D11-A7 were of IgM isotype and 5D10-C9 and 5D10-F11 were of IgG2b type, while 3F3-D2, 7D2-C9, 7D2-G7, and 7D2-G8 were of IgG1 isotype. The MAbs 3F3-D2 and 7D2-G8 showed high intensity positive reaction with rNP and a low intensity reaction with H5N1 virus in Western blot analysis. The anti-NP MAbs produced in the present work may be valuable in developing a competitive ELISA or immunochromatographic strip test-based assays for the rapid diagnosis of avian influenza.

Highly Pathogenic Avian Influenza Virus Nucleoprotein Interacts with TREX Complex Adaptor Protein Aly/REF

We constructed a novel chicken (Gallus gallus) lung cDNA library fused inside yeast acting domain vector (pGADT7). Using yeast two-hybrid screening with highly pathogenic avian influenza (HPAI) nucleoprotein (NP) from the strain (A/chicken/Malaysia/5858/2004(H5N1)) as bait, and the Gallus gallus lung cDNA library as prey, a novel interaction between the Gallus gallus cellular RNA export adaptor protein Aly/REF and the viral NP was identified. This interaction was confirmed and validated with mammalian two hybrid studies and co-immunoprecipitation assay. Cellular localization studies using confocal microscopy showed that NP and Aly/REF co-localize primarily in the nucleus. Further investigations by mammalian two hybrid studies into the binding of NP of other subtypes of influenza virus such as the swine A/New Jersey/1976/H1N1 and pandemic A/Malaysia/854/2009(H1N1) to human Aly/REF, also showed that the NP of these viruses interacts with human Aly/REF. Our findings are also supported by docking studies which showed tight and favorable binding between H5N1 NP and human Aly/REF, using crystal structures from Protein Data Bank. siRNA knockdown of Aly/REF had little effect on the export of HPAI NP and other viral RNA as it showed no significant reduction in virus titer. However, UAP56, another component of the TREX complex, which recruits Aly/REF to mRNA was found to interact even better with H5N1 NP through molecular docking studies. Both these proteins also co-localizes in the nucleus at early infection similar to Aly/REF. Intriguingly, knockdown of UAP56 in A549 infected cells shows significant reduction in viral titer (close to 10 fold reduction). Conclusively, our study have opened new avenues for research of other cellular RNA export adaptors crucial in aiding viral RNA export such as the SRSF3, 9G8 and ASF/SF2 that may play role in influenza virus RNA nucleocytoplasmic transport.

Using Rice Flour for Purification of Maltose Binding Fusion Proteins Expressed in Escherichia coli

Background: One disadvantage of expressing heterologous proteins in Escherichia coli is that the proteins are frequently expressed as insoluble inclusion bodies. To avoid this problem, heterologous proteins are typically expressed as a fusion protein. Maltose binding protein (MBP) is one of the widely used partners for production of recombinant fusion proteins in E. coli. MBP is among the most effective solubility enhancers. In addition, MBP can be used as an affinity tag for purification of recombinant proteins on a column of amylose resin. Objectives: The purpose of this study was to investigate the use of rice flour, a natural source of amylose, for purification of MBP fusion proteins. Materials and Methods: MBP and a fusion protein of MBP and avian influenza virus nucleoprotein (MBP-NP) were expressed in E. coli and subjected to purification by rice flour and a commercial amylose resin. The purified proteins were analyzed by SDS-PAGE. Results: The results indicated that MBP and MBP-NP, both were successfully purified by rice flour. Conclusions: Rice flour can be used for purification of MBP fusion proteins. Although the efficiency of purification by rice flour is less than amylose resin, however, the yield is sufficient to obtain a quantity of protein required for research purposes.

The pathogenesis of low pathogenic avian influenza in mallards.

Mallards are important natural hosts involved in the epidemiology of low pathogenic avian influenza viruses (LPAIVs). LPAIVs are mainly transmitted by a fecal-oral route and are excreted in high concentrations in the feces. We investigated the pathology, viral antigen distribution, and the expression of alpha2,3 sialic acid (SA) influenza virus receptors in mallards after intranasal inoculation with A/Mallard/MN/199106/99 (H3N8) or A/Mallard/MN/355779/00 (H5N2). Gross lesions were not observed. Avian influenza virus (AIV) nucleoprotein (NP) antigen was detected in rare epithelial cells of the larynx and trachea only at 1-day postinoculation (dpi) in the birds infected with H3N8 LPAIV, but infection with either virus was associated with lymphocytic tracheitis and laryngitis on 1 and 2 dpi. AIV NP antigen was detected in enterocytes of the lower intestine from 1 to 4 dpi and in epithelial cells of the bursa of Fabricius from 2 to 3 dpi in birds infected with either virus. Oropharyngeal and cloacal viral shedding was detected from 1 dpi, with higher cloacal viral shedding detected at 2 and 3 dpi with both viruses. Mallards abundantly expressed alpha2,3 sialic acid receptors in epithelial cells of the respiratory tract, lower intestine, and bursa of Fabricius. Some infected birds had decreased alpha2,3 sialic acid expression in epithelial cells of the bursa of Fabricius and in enterocytes of the ceca and colon. In conclusion, the main sites of LPAIV replication in mallards are the enterocytes of the lower intestinal tract and epithelial cells of the bursa of Fabricius in the first days after infection, when these birds are shedding AIV in high titers in the feces.

Prediction and Identification of T Cell Epitopes in the H5N1 Influenza Virus Nucleoprotein in Chicken

T cell epitopes can be used for the accurate monitoring of avian influenza virus (AIV) immune responses and the rational design of vaccines. No T cell epitopes have been previously identified in the H5N1 AIV virus nucleoprotein (NP) in chickens. For the first time, this study used homology modelling techniques to construct three-dimensional structures of the peptide-binding domains of chicken MHC class Ι molecules for four commonly encountered unique haplotypes, i.e., B4, B12, B15, and B19. H5N1 AIV NP was computationally parsed into octapeptides or nonapeptides according to the peptide-binding motifs of MHC class I molecules of the B4, B12, B15 and B19 haplotypes. Seventy-five peptide sequences were modelled and their MHC class I molecule-binding abilities were analysed by molecular docking. Twenty-five peptides (Ten for B4, six for B12, two for B15, and seven for B19) were predicted to be potential T cell epitopes in chicken. Nine of these peptides and one unrelated peptide were manually synthesized and their T cell responses were tested in vitro. Spleen lymphocytes were collected from SPF chickens that had been immunised with a NP-expression plasmid, pCAGGS-NP, and they were stimulated using the synthesized peptides. The secretion of chicken IFN-γ and the proliferation of CD8+ T cells were tested using an ELISA kit and flow cytometry, respectively. The significant secretion of chicken IFN-γ and proliferation of CD8+ T lymphocytes increased by 13.7% and 11.9% were monitored in cells stimulated with peptides NP89–97 and NP198–206, respectively. The results indicate that peptides NP89–97 (PKKTGGPIY) and NP198–206 (KRGINDRNF) are NP T cell epitopes in chicken of certain haplotypes. The method used in this investigation is applicable to predicting T cell epitopes for other antigens in chicken, while this study also extends our understanding of the mechanisms of the immune response to AIV in chicken.

Antisense oligonucleotides targeting the RNA binding region of the NP gene inhibit replication of highly pathogenic avian influenza virus H5N1

The H5N1 avian influenza virus (AIV) causes widespread infections in bird and human respiratory tracts, and vaccines and drug therapy are limited in their effectiveness. Recent studies of AIV structures have been published and provide new targets for designing antiviral drugs such as antisense oligonucleotides (AS ODNs), which effectively inhibit gene replication. In this study, we designed and synthesized three AS ODNs (NP267, NP628, NP749) that were specific for the RNA binding region of nucleoprotein (NP) based on AIV structure. Results showed that all three AS ODNs could inhibit viral replication in MDCK cells. The NP628 showed the best antiviral effect of all through viral titers, quantitative RT-PCR and indirect immunofluorescence (IFA) assays. In addition, the liposome mediated NP628 could partially protect the mice from a lethal H5N1 influenza virus challenge. Moreover, the NP628 group had a lower viral titer and lung index in the infected mice when compared with the viral control. Our results showed that AS ODN targeting of the AIV NP gene could potently inhibit AIV H5N1 reproduction, thus, formulating a candidate for an emergent therapeutic drug for the pathogenic H5N1 influenza virus infection.

Rapid detection of avian influenza virus in chicken fecal samples by immunomagnetic capture reverse transcriptase–polymerase chain reaction assay

Avian influenza virus (AIV) causes great economic losses for the poultry industry worldwide and threatens the human population with a pandemic. The conventional detection method for AIV involves sample preparation of viral RNA extraction and purification from raw sample such as bird droppings. In this study, magnetic beads were applied for immunoseparation and purification of AIV from spiked chicken fecal sample. The beads were conjugated with monoclonal antibodies against the AIV nucleoprotein, which is conserved in all the AIV. The bead-captured virus was detected by reverse transcriptase–polymerase chain reaction (RT-PCR) without RNA extraction because of effective removal of RT-PCR inhibitors. The developed bead-based assay showed a similar detection limit comparable to the RNA extraction and the classic virus isolation method. Using ready-to-use antibody-conjugated bead, the method requires less than 5 h. Furthermore, the method has potential to integrate into a Lab-on-a-chip system for rapid detection and identification of AIV.

Pathomorphological lesions and distribution of viral antigen in birds infected with the pathogenic strain of H5N1 avian influenza virus

The aim of this study was to determine pathomorphological lesions in the organs of birds infected with H5N1 avian influenza virus, presence and distribution of the viral antigen in tissue samples and the degree of their correlation. Examination was conducted on the carcasses of seven mute swans (Cygnus olor) and one domestic rooster (Gallus gallus domesticus). After necropsy, trachea, lungs, liver, spleen, pancreas, kidney and intestine tissues were sampled for histopathology and immunohistochemical examination. LSmethod and H5N1 avian influenza virus nucleoprotein polyclonal antibodies were used for detecting the viral antigen in the examined tissues. The most frequent gross lesions were hemorrhages and necroses of the pancreas. Major histological lesions were multifocal necroses in the pancreas, liver and spleen, non purulent encephalitis, with neuronal dystrophy and neuronophagia. Viral nucleoprotein was immunohistochemically proven in the pancreas, brain, liver, lungs, and in one swan in the kidney. A high correlation between pathomorphological lesions and distribution of the viral antigen was determined.

Reconstructing an annual cycle of interaction: natural infection and antibody dynamics to avian influenza along a migratory flyway

Migratory animals may play an important role in connecting disparate ecosystems, including the introduction of various pathogens. The incidence of these pathogens may vary over time and space, such that events along the entire migratory flyway are likely to be important in the interaction between pathogens and their migratory hosts. On this premise, the annual cycle of a naturally occurring host–pathogen system was reconstructed by examining infection with and antibodies to avian influenza virus along the flyway of a long‐distance Arctic migrant, the Svalbard‐breeding pink‐footed goose Anser brachyrhynchus. A highly‐localized transmission period was identified in winter, in contrast to the north–south decline expected from dabbling ducks, indicating the dynamics of infection may differ among host species. In spring, 63% (95% CI: 57.1, 68.9) of adults had detectable antibodies to the nucleoprotein of avian influenza virus, compared to just 15% (95% CI: 8.7, 23.4) of juveniles, suggesting inter‐annual antibody maintenance. Nevertheless, adult seroprevalence declined by approximately 30% from spring to late summer, indicating significant seroreversion in the population. Integrating these findings in an epidemiological model, detectable antibodies to nucleoprotein were estimated to persist for just 343 days (95% CI: 221, 607); considerably shorter than for other wildlife diseases in long‐lived bird species. The investigation of wildlife diseases in migratory populations is an inherently complex task, yet, by integrating disease incidence and seroprevalence along a migratory flyway, our findings suggest that the ecological interactions and life history of the host, as well as the life‐history of the pathogen, can influence the dynamics of infection and host immune response.

A latex agglutination test using a recombinant nucleoprotein for detection of antibodies against avian influenza virus

A latex agglutination test (LAT) was developed for detecting antibodies against avian influenza virus. The recombinant avian influenza virus nucleoprotein expressed in Escherichia coli was purified, coupled with latex beads, and used as an antigen for the LAT. The LAT was capable of detecting anti-avian influenza virus antibodies irrespective of the avian-influenza subtype, and in most cases, the results correlated with the results of an agar gel precipitation test (AGPT). However, in comparison with the AGPT, the LAT could detect the anti-avian influenza virus antibodies for a longer period of time after the infection. The nonspecific agglutination observed in uninfected chicken sera was resolved by pretreating the sera with dried chicken-liver powder for 1 h. The LAT is easy to perform, and even after considering the time required for pretreatment of the serum, the total time required for obtaining the results is reduced in comparison to the time required in the case of the AGPT. This easy and rapid LAT is considered to be useful for monitoring avian influenza virus infection in the field.

MDV-1 VP22: a transporter that can selectively deliver proteins into cells

The tegument protein VP22 of Marek's disease virus (MDV) was previously shown to be able to travel between cells. To further characterize the transport property of VP22 and assess whether it can be used for protein delivery, we investigated the subcellular localization of VP22 fused to five heterologous proteins, including green fluorescent protein, nucleoprotein of avian influenza virus, bovine IFN-gamma, F protein of Newcastle disease virus and VP2 protein of infectious bursa disease virus. The transport of these fusion proteins in monolayer cells was assayed by immunofluorescence assay. The results showed that all except VP2 could be delivered by VP22 of MDV serotype 1 (MDV-1), at different efficiencies. After being transported to surrounding cells, VP22 fused to avian influenza nucleoprotein, bovine IFN-gamma, or to F was localized in the nucleus. Our data suggest that VP22 of MDV-1 can be used as transport tool in protein delivery and that the original localization of cargo proteins may be changed after transport by MDV-1 VP22. Finally, infectious bursa disease VP2 protein could not be transported by MDV-1 VP22.

Vaccination with Newcastle Disease Virus Vectored Vaccine Protects Chickens Against Highly Pathogenic H7 Avian Influenza Virus

A recombinant Newcastle disease virus (NDV) was engineered to express the hemagglutinin (HA) gene of avian influenza virus (AIV) subtype H7. The HA gene was inserted between the genes encoding NDV fusion and hemagglutinin-neuraminidase proteins. Within the H7 open reading frame, an NDV gene end-like sequence was eliminated by silent mutation. The expression of H7 protein was detected by western blot analysis and indirect immunofluorescence. The existence of H7 protein in the envelope of recombinant Newcastle disease virions was shown by immunoelectron microscopy. The protective efficacy of recombinant NDVH7m against virulent NDV, as well as against highly pathogenic avian influenza virus (HPAIV), was evaluated in specific-pathogen-free chickens. After a single immunization, all chickens developed NDV-specific, as well as AIV H7-specific, antibodies and were completely protected from clinical disease after infection with a lethal dose of virulent NDV or the homologous H7N1 HPAIV, while all control animals died within four days. Shedding of AIV challenge virus was strongly reduced compared to nonvaccinated control birds. Furthermore, the immunized birds developed antibodies against the AIV nucleoprotein after challenge infection. Thus, NDVH7m could be used as a marker vaccine against subtype H7 avian influenza.