H9N2 AIV Research Articles

H9N2 avian influenza infection altered expression pattern of Sphiogosine-1-phosphate Receptor 1 in BALB/c mice

BackgroundThe pathological damage inflicted by virulent AIV strains is often caused by inducing a positive feedback loop of cytokines in immune cells that cause excessive inflammation. Previous research has shown that a G protein-coupled receptor, sphingosine-1-phosphate receptor 1 (S1PR1), plays a crucial role in the development of excessive inflammation in influenza virus infection (Cell 146:861–862, 2011; Cell 146:980–991, 2011). BALB/c mice are common laboratory animals used in research of influenza virus; however the effects of influenza infections on expression patterns of S1PR1 in mice are unknown.MethodsWe investigated the expression patterns of S1PR1 in normal BALB/c mice and those infected by two distinct H9N2 AIV strains, one (A/chicken/Guangdong/V/2008,V) highly pathogenic, and the other (A/chicken/Guangdong/Ts/2004,Ts), non-pathogenic in mice, using quantitative PCR and immunohistochemistry (IHC) to detect S1PR1 mRNA and protein, respectively.ResultsS1PR1 mRNA was ubiquitously expressed in all the tissues examined, and significant differences were seen in mRNA expression between infected Ts, V and control mice in detected tissues, heart, liver, spleen, kidney and brain. S1PR1 protein was expressed in the cytoplasm and also demonstrated quantitative changes in expression in the various tissues between mice infected with the two strains of AIV.ConclusionsOur results provided the first look at differences in S1PR1 expression patterns in BALB/c mice infected by non-pathogenic and highly pathogenic H9N2 influenza viruses. This information will not only be helpful in designing experiments to better understand the role of S1PR1 in virus-host interactions but also in developing novel anti-influenza agents to minimize the mortality and morbidity associated with highly virulent strains in avian and human populations.

Survey on ELISA Based on anti Influenza A NS1 Antibodies to Differentiate the Infected and Vaccinated Poultries

Background: The Vaccination programs to control avian influenza (AI) infection in poultry, have limitations due to the difficulty in differentiating the vaccinated and naturally infected birds AI vaccination would bring greater global acceptance if a reliable test, which could clearly distinguish the naturally infected and vaccinated-only animals (DIVA), was available. Since the nonstructural protein (NS1) is expressed in influenza infected cells, and it is not presented as a virion, it could be a proper candidate for DIVA differential diagnostic test. Objectives: Vaccination programs for the control of avian influenza (AI) in poultry have limitations due to the problem of differentiating between vaccinated and virus-infected birds. The use of AI vaccination in poultry would have greater worldwide acceptance if a reliable test were available that clearly discriminated between naturally infected and vaccinated-only animals (DIVA). Because the nonstructural protein (NS1) is expressed in influenza virus–infected cells, and it is not packaged in the virion, it is an attractive candidate for a DIVA differential diagnostic test. Materials and Methods: A total of 300 day-old broiler chicks (Ross 308) divided into three equal groups (1 to 3) .The chicks in group 1 were immunized with killed AIV H9N2. The chicks in group 2 were infected with AI virus subtype H9N2. The chicks in group 3 were kept as controls and did not receive any vaccined or lived virus. Chicks sera were collected at day 42 usingrNS1-ELISA and Commercial ELISA kit. Results: Designed ELISA test for detection of antibody against influenza NS1 could experimentally distinguish the chicks infected with AIV and chicks immunized with killed influenza viruswith93.3% sensitivity and 100 ℅ specificity. 3 weeks after infection or vaccination, sera from all two treated groups were positively tested using commercial ELISA kit (IDEXX). In contrast, by NS1-ELISA, only infected groups sera were tested and the result was positive, and all sera samples from the vaccinated group were NS1-antibody titer were evaluated and the result were negative. Conclusions:: It was concluded that antibodies against AIV NS1 protein was only detected in the sera of chickens experimentally infected with AIV, not in the sera of chickens immunized with inactivated vaccine.

Astragalus polysaccharide enhances immunity and inhibits H9N2 avian influenza virus in vitro and in vivo.

This study investigated the humoral immunization of Astragalus polysaccharide (APS) against H9N2 avian influenza virus (H9N2 AIV) infection in chickens.The effects of APS treatment on H9N2 infection was evaluated by an MTT [3(4, 5-dimethylthiazol-2-yl)-2, 3-diphenyl tetrazolium bromide] assay and analysis of MHC and cytokine mRNA expression. The effect on lymphocyte and serum antibody titers in vivo was also investigated. IL-4, IL-6, IL-10, LITAF, IL-12 and antibody titers to H9N2 AIV were enhanced in the first week after APS treatment. The results indicated that APS treatment reduces H9N2 AIV replication and promotes early humoral immune responses in young chickens.

Electrochemical Magnetoimmunosensing Approach for the Sensitive Detection of H9N2 Avian Influenza Virus Particles

A novel electrochemical magnetoimmunosensor for fast and ultrasensitive detection of H9N2 avian influenza virus particles (H9N2 AIV) was designed based on the combination of high-efficiency immunomagnetic separation, enzyme catalytic amplification, and the biotin-streptavidin system. The reusable, homemade magneto Au electrode (M-AuE) was designed and used for the direct sensing. Immunocomplex-coated magnetic beads (IMBs) were easily accumulated on the surface of the M-AuE to obtain the catalytically reduced electrochemical signal of H2 O2 after the immunoreaction. The transducer was regenerated through a simple washing procedure, which made it possible to detect all the samples on a single electrode with higher reproducibility. The magnetic-bead-based electrochemical immunosensor showed better analytical performance than the planar-electrode-based immunosensor with the same sandwich construction. Amounts as low as 10 pg mL(-1) H9N2 AIV could be detected even in samples of chicken dung. This electrochemical magnetoimmunosensor not only provides a simple platform for the detection of the virus with high sensitivity, selectivity, and reproducibility but also shows great potential in the early diagnosis of diseases.

Therapeutic Evaluation Of The Antiviral Activity Of Azadirachta indica In Comparison To Amantadine Hydrochloride Against Avian Influenza Virus Infection In Ducks

Twelve AI isolates were isolated in ECE from diseased backyard ducks showed signs of depression, anorexia, greenish or watery diarrhea, severe neurological signs, and high mortalities. All isolates were identified using HIT to be H5. RT-PCR was used for detection and subtyping of AIVs using specific pairs of primers to amplify the hemagglutinin gene of subtype H5 and neuraminidase gene of subtype N1. Analysis of RNA extracted yielded positive reactions of correct size as H5 subtype specific primers (545 bp) and N1 subtype specific primers (616 bp). The efficacy of Azadirachta indicaagainst AIV infection was evaluated in comparison toamantadinehydrochloride in 2-wk-old balady ducks inoculated with 0.1ml×106 EID50/ml of AIV H5N1. Aqueous Neem leaves extract provided ducks with 100% protection level, whereas neither clinical signs nor mortalities were recorded, while Adamine® presented 70% only.

Lack of evidence of avian-to-human transmission of avian influenza A (H5N1) virus among veterinarians, Guangdong, China, 2012

BackgroundCases of human infections with H5N1 avian influenza viruses have been reported all over the world with the reason of direct contact with sick or diseased poultry, which suggests the direct contact with poultry may be one of the major risk factors for human infection. ObjectivesIn this study, we estimated the seroprevalence of antibodies against avian influenza A (H5N1) virus in veterinarians with exposure to avians. Study designFrom May 21, 2011 through April 22, 2012, 406 veterinarians exposure to poultry in Guangdong province were interviewed a questionnaire. A serum specimen was collected from participants to test for H5N1 antibodies by HI and NT assay. ResultsNone of the 406 sera from occupationally exposed veterinarians was positive according to the HI test and the NT test with the H5N1 AIV. ConclusionOur seroepidemiologic survey suggests that the risk of avian-to-human transmission of the H5N1 AIV is very low based on the samples that we tested. However, prevention regarding the risk of H5N1 AIV transmission is essential and should be recommended as public health measures.

Potential Role of HPA Axis and Sympathetic Nervous Responses in Depletion of B Cells Induced by H9N2 Avian Influenza Virus Infection

Except severe pulmonary disease caused by influenza virus infection, an impaired immune system is also a clinic characteristic. However, the mechanism(s) of influenza virus infection-induced depletion of B cells was unknown. Here, we compared the effect of two variant virulence H9N2 virus infections on mouse B cells. Our study found that the infection with highly pathogenic virus (V) of led to depletion of spleen B cells and bone marrow (BM) early B cells, compared to lowly pathogenic virus (Ts). Moreover, high apoptosis and cell cycle arrest in spleen and BM were detected, suggesting important factors for the reduction of B cells in both organs. Further, this effect was not caused by virus replication in spleen and BM. Compared to Ts virus infection, V virus resulted in higher glucocorticoids (GCs) and lower leptin level in plasma. Intraperitoneal GCs receptor antagonist RU486 injection was sufficient to prevent the loss of spleen B cell and BM pro- and immature B cells, but similar result was not observed in leptin-treated mice. Depletion of spleen B cells and BM pro-B cells was also reversed by chemical sympathectomy mediated by the norepinephrine (NE) analog 6-hydroxydopamine (6-OHDA), but the treatment didn't affect the GCs level. This study demonstrated that depletion of B cells induced by H9N2 AIV was dependent on HPA axis and sympathetic response.

Pinon shell polysaccharide enhances immunity against H9N2 avian influenza virus in chickens

This study investigated the potential of pinon shell polysaccharide (PSP) to enhance immunity against H9N2 subtype avian influenza virus (H9N2 AIV) in chickens. The effect of PSP treatment and H9N2 virus infection after PSP pretreatment were investigated by [3-(4, 5-dimethylthiazol-2-yl)-2, 3-diphenyl tetrazolium bromide] assay and analysis of major histocompatibility complex and cytokine mRNA expression. The in vivo effects on lymphocytes and serum antibody titers were also investigated. The IL-6 expression and antibody titers were enhanced in the first week after PSP treatment. These data indicate that prophylactic PSP administration reduces H9N2 AIV replication and promotes early humoral immune responses in young birds.

Complete Genome Sequence of a Novel H9N2 Subtype Influenza Virus FJG9 Strain in China Reveals a Natural Reassortant Event

A/chicken/FJ/G9/09 (FJ/G9) is an H9N2 subtype avian influenza virus (H9N2 AIV) strain causing high morbidity that was isolated from broilers in Fujian Province of China in 2009. FJ/G9 has been used as the vaccine strain against H9N2 AIV infection in Fujian Province of China. Here, we report the complete genome sequence of FJ/G9 with natural six-way reassortment, which is the most complex genotype strain in China and even in the world so far. The present findings will aid in understanding the complexity and diversity of H9N2 subtype avian influenza virus.

Enhancement of Th1-biased protective immunity against avian influenza H9N2 virus via oral co-administration of attenuated Salmonella enterica serovar Typhimurium expressing chicken interferon-α and interleukin-18 along with an inactivated vaccine

BackgroundControl of currently circulating re-assorted low-pathogenicity avian influenza (LPAI) H9N2 is a major concern for both animal and human health. Thus, an improved LPAI H9N2 vaccination strategy is needed to induce complete immunity in chickens against LPAI H9N2 virus strains. Cytokines play a crucial role in mounting both the type and extent of an immune response generated following infection with a pathogen or after vaccination. To improve the efficacy of inactivated LPAI H9N2 vaccine, attenuated Salmonella enterica serovar Typhimurium was used for oral co-administration of chicken interferon-α (chIFN-α) and chicken interleukin-18 (chIL-18) as natural immunomodulators.ResultsOral co-administration of S. enterica serovar Typhimurium expressing chIFN-α and chIL-18, prior to vaccination with inactivated AI H9N2 vaccine, modulated the immune response of chickens against the vaccine antigen through enhanced humoral and Th1-biased cell-mediated immunity, compared to chickens that received single administration of S. enterica serovar Typhimurium expressing either chIFN-α or chIL-18. To further test the protective efficacy of this improved vaccination regimen, immunized chickens were intra-tracheally challenged with a high dose of LPAI H9N2 virus. Combined administration of S. enterica serovar Typhimurium expressing chIFN-α and chIL-18 showed markedly enhanced protection compared to single administration of the construct, as determined by mortality, clinical severity, and feed and water intake. This enhancement of protective immunity was further confirmed by reduced rectal shedding and replication of AIV H9N2 in different tissues of challenged chickens.ConclusionsOur results indicate the value of combined administration of chIFN-α and chIL-18 using a Salmonella vaccine strain to generate an effective immunization strategy in chickens against LPAI H9N2.

Co-infection of broilers with Ornithobacterium rhinotracheale and H9N2 avian influenza virus

BackgroundSince 2008, a progressive pneumonia has become prevalent in broilers and laying hens. This disease occurrs the first day after hatching and lasts more than 30 days, resulting in approximately 70% morbidity and 30% mortality in broilers. The objective of this study was to isolate and identify the pathogens that are responsible for the progressive pneumonia and establish an animal model for drug screening.Results193 serum samples were collected from 8 intensive farms from 5 provinces in China and analysed in the current research. Our clinical survey showed that 65.2% to 100% of breeding broilers, breeding layers, broilers and laying hens were seropositive for ORT antibodies. From 8 intensive farms, six ORT isolates were identified by PCR and biochemical assays, and two H9N2 viruses were isolated. Newcastle Disease Virus (NDV) and Infectious BronchitisVirus (IBV) were excluded. Typical pneumonia and airsacculitis were observed both in broilers inoculated intraperitoneally with an ORT isolate alone and in those co-infected with ORT and H9N2 virus isolates. Specifically, the survival rate was 30%, 20%, 70%, 50% and 90% in birds inoculated with ORT+H9N2 virus, ORT followed by H9N2 virus, H9N2 virus followed by ORT, and ORT or H9N2 virus alone, respectively.ConclusionsThe results of this study suggest that ORT infections of domestic poultry have been occurring frequently in China. ORT infection can induce higher economic losses and mortality if H9N2 AIV is also present. Although the isolation of ORT and H9N2 virus has been reported previously, there have been no reported co-infections of poultry with these two pathogens. This is the first report of co-infection of broilers with ORT and H9N2 virus, and this co-infection is probably associated with the outbreak of broiler airsacculitis in China, which has caused extensive economic losses.

A Single E627K Mutation in the PB2 Protein of H9N2 Avian Influenza Virus Increases Virulence by Inducing Higher Glucocorticoids (GCs) Level

While repeated infection of humans and enhanced replication and transmission in mice has attracted more attention to it, the pathogenesis of H9N2 virus was less known in mice. PB2 residue 627 as the virulent determinant of H5N1 virus is associated with systemic infection and impaired TCR activation, but the impact of this position in H9N2 virus on the host immune response has not been evaluated. In this study, we quantified the cellular immune response to infection in the mouse lung and demonstrate that VK627 and rTsE627K infection caused a significant reduction in the numbers of T cells and inflammatory cells (Macrophage, Neutrophils, Dendritic cells) compared to mice infected with rVK627E and TsE627. Further, we discovered (i) a high level of thymocyte apoptosis resulted in impaired T cell development, which led to the reduced amount of mature T cells into lung, and (ii) the reduced inflammatory cells entering into lung was attributed to the diminished levels in pro-inflammatory cytokines and chemokines. Thereafter, we recognized that higher GCs level in plasma induced by VK627 and rTsE627K infection was associated with the increased apoptosis in thymus and the reduced pro-inflammatory cytokines and chemokines levels in lung. These data demonstrated that VK627 and rTsE627K infection contributing to higher GCs level would decrease the magnitude of antiviral response in lung, which may be offered as a novel mechanism of enhanced pathogenicity for H9N2 AIV.

An experimental study on broiler chicken co-infected with the specimens containing avian influenza (H9 subtype) and infectious bronchitis (4/91 strain) viruses.

Summary This experimental study was carried out to induce a co-infection of H9 AIV and IBV with inoculums prepared from tracheal scrubbed epithelium tissues in natural co-infected birds to rule out the effect of field undesired environmental conditions and also other infectious causative agents. Eighty 1-day-old broilers were randomly divided into four equal groups. At 21 days of age, three test groups were inoculated intranasally with prepared inoculums containing IBV and H9 AIV alone and a co-infected group. The fourth group remained uninfected as a control group. The results showed that experimental co-infection of AIV and IBV increased the severity of clinical signs, mortality rate and gross lesions. The HI titer against AIV infection in the co-infected group was significantly higher than the HI titer of other groups, which may indicate that IBV could promote the propagation of H9N2 AIV or stimulate the immune response.

Detection of H9N2 avian influenza virus in various organs of experimentally infected chickens

H9N2 Avian influenza virus (AVI) infection is a major cause of economic losses in poultry industry. Therefore further study to explain the virus pathogenesis is necessary. In this study tissue tropism and dissemination of A/chicken/Iran/11T/99(H9N2) virus in various organs of specific pathogen free (SPF) chickens were investigated. Fifty 2-week-old chickens hatched from SPF eggs were divided randomly into two groups. Forty chicks in the experimental and ten chicks in the control group. Experimental chicks were inoculated intranasally-intraorally with the virus. Samples of lung, trachea, pancreas, thymus, spleen, brain, bursa of fabricius, proventriclus, cloaca and kidney were aseptically collected at 1, 3, 5, 7, 9 and 10 day post inoculation (DPI). A reverse transcriptase polymerase chain Reaction (RT-PCR) test was performed for virus detection. Viral RNA was detected in the respiratory system on days 3, 5 and 7 PI. The virus was also found in the kidney on days 3,5,7,9 PI and in the pancreas on days 3 and 5 PI. Viral RNA was observed only on day 5 PI in cloaca. The virus was not detected in the blood, brain and immune system. The virus was not found from any organs on day 10 PI. These results suggest that H9N2 AIV has tropism for respiratory, digestive and urinary system following intranasal/intraoral inoculation. Key words: Avian influenza, H9N2, SPF, RT-PCR.

The interaction between the PARP10 protein and the NS1 protein of H5N1 AIV and its effect on virus replication

BackgroundDuring the process that AIV infect hosts, the NS1 protein can act on hosts, change corresponding signal pathways, promote the translation of virus proteins and result in virus replication.ResultsIn our study, we found that PARP domain and Glu-rich region of PARP10 interacted with NS1, and the presence of NS1 could induce PARP10 migrate from cytoplasm to nucleus. NS1 high expression could reduce the endogenous PARP10 expression. Cell cycle analysis showed that with inhibited PARP10 expression, NS1 could induce cell arrest in G2-M stage, and the percentage of cells in G2-M stage rise from the previous 10%-45%, consistent with the cell proliferation result. Plague forming unit measurement showed that inhibited PARP10 expression could help virus replication.ConclusionsIn a word, our results showed that NS1 acts on host cells and PARP10 plays a regulating role in virus replication.

Development of rHA1-ELISA for specific and sensitive detection of H9 subtype influenza virus

A recombinant antigen-based single serum dilution ELISA was developed for simultaneous detection and subtyping of influenza viruses. Recombinant baculovirus encoding the hemagglutinin (HA 1 subunit) of H9N2 virus was generated. To evaluate the rHA1-ELISA, microplates were coated with purified HA1 protein and tested with reference control sera. Subsequently, 92 field sera collected from chickens suspected to be infected with H9N2 AIV were employed to test the efficacy of the rHA1-ELISA. The sera were tested simultaneously by HI and a commercial AIV ELISA kit. The rHA1-ELISA appeared to be highly specific and sensitive for direct detection of H9N2 antibodies in serum samples.

Assessment of Pathogenic Potential of Two Indian H5N1 Highly Pathogenic Avian Influenza Virus Isolates by Intravenous Pathogenicity Index Test

Intravenous Pathogenicity Index (IVPI) test was conducted to assess the pathogenicity of two H5N1 (A/Ck/Ind/7966/06 and A/Ck/Ind/7972/06) AIV isolates. Both the H5N1 virus isolates were isolated from natural outbreaks during 2006, both isolates caused death of all birds with in 48 h after inoculation experimentally, birds showed typical clinical signs, gross and microscopic lesions of Avian influenza. IVPI of A/Ck/Ind/7966/06 and A/Ck/Ind/7972/06 was 2.96 and 2.95 respectively. This test showed that both H5N1 isolates are highly pathogenic. Virus could be re-isolated from all the organs of infected chickens and it was reconfirmed by RT-PCR using WHO and Lee primers.

Priming with a DNA vaccine delivered by attenuated Salmonella typhimurium and boosting with a killed vaccine confers protection of chickens against infection with the H9 subtype of avian influenza virus

Control of the circulation of H9 low-pathogenic avian influenza virus (LPAIV) is a major concern for both animal and public health. To improve vaccine efficacy against H9 LPAIV, we have utilized a novel prime–boost vaccination strategy. Specific-pathogen free (SPF) chickens were first orally immunized with a hemagglutinin (HA) DNA vaccine delivered by attenuated Salmonella typhimurium, followed by boosting with a killed avian influenza (AI) vaccine. Chickens in this combined vaccination group were completely protected against both oropharyngeal and cloacal virus shedding after intranasal challenge with H9N2 AIV, while viruses were detected from these sites in other vaccination groups. Prior to challenge, chickens in the prime–boost group also had higher (P<0.05) serum hemagglutination inhibition (HI) titers and intestinal mucosal IgA ELISA titers against AIV, and higher lymphoproliferation stimulation indices than those from other groups. Thus, we have demonstrated the efficacy of a novel prime–boost vaccination strategy against H9N2 avian influenza virus, which could be also applied for the development of vaccines against other mucosally infectious pathogens.

An indirect sandwich ELISA for the detection of avian influenza H5 subtype viruses using anti-hemagglutinin protein monoclonal antibody

A sandwich ELISA test using AIV H5 subtype specific monoclonal antibody (clone 2H4) to an epitope of hemagglutinin protein has been developed. The monoclonal antibody was used to capture the antigen from clinical samples (swabs and tissues). Captured antigens from clinical samples were detected using polyclonal sera, purified AIV H5N1 particles were titrated in the sandwich ELISA and the limit of detection was determined to be approximately 1.0 ng of influenza viral protein in virus preparations. Fifteen AIV strains of H1–H15 subtypes and some other pathogens were tested by this system, and the test is specific to H5 subtype viruses as it failed to detect other AIV subtype viruses and other pathogens. Varieties of clinical samples originating from laboratory experiments ( n = 382) and from fields ( n = 288) were employed to test the efficacy of DAS-ELISA test. The test compared very well with the traditional method for detection of influenza virus: virus isolation (VI) in embryonated chicken eggs. In comparison to virus isolation the sensitivity and specificity of sandwich ELISA were found to be 98.6% and 97.6% respectively. In addition, the DAS-ELISA was used to test samples of experimentally infected birds and clinical samples obtained from central China in 2005. The assay proved to be sensitive and specific for the rapid detection of AIV H5 subtype virus form the tissues and swabs in infected animals.

Genetic characterization of two low pathogenic avian influenza virus H5N1 isolates from Ontario, Canada

Genomes of two low pathogenic H5N1 avian influenza (LPAI) viruses, A/Turkey/ON/84/1983 and A/Mallard/ON/499/2005 from Ontario, Canada were cloned and genetically characterized. Phylogenetic analysis showed that the Canadian isolates cluster with other North American AIVs and are distinct from the Euro-Asian H5N1 isolates. Individual gene comparisons demonstrated that the Ontario isolates were most similar to the viruses isolated from around the same time period and geographical area. A long deletion of 22 amino acids was identified in the stalk region of NA of A/Turkey/ON/84/1983 isolate, a characteristic mutation related to its adaptation to domestic birds. To our knowledge A/Turkey/ON/84/1983 genomic sequence is the first and only available entire genomic sequence of a H5N1 AIV from domestic birds in Canada and USA.