Influenza Virus Subtype H5N1 Research Articles

Longitudinal serological measures of common infection in the Avon Longitudinal Study of Parents and Children cohort

Antibodies against pathogens provide information on exposure to infectious agents and are meaningful measures of past and present infection. Antibodies were measured in the plasma of children that are the offspring in a population-based birth cohort, the Avon Longitudinal Study of Parents and Children (ALSPAC). Plasma was collected during clinics at age 5, 7, 11 and 15 years. The antigens examined include: fungal (Saccharomyces cerevisiae); protozoan (Toxoplasma gondii and surface antigen 1 of T. gondii); herpes viruses (cytomegalovirus, Epstein-Barr virus, herpes simplex virus type 1); common colds (influenza virus subtypes H1N1 and H3N2); other antigens (measles); animal (feline herpes virus, Theiler’s virus); bacteria (Helicobacter pylori); dietary antigens (bovine casein alpha protein, bovine casein beta protein). Alongside the depth of data available within the ALSPAC cohort, this longitudinal resource will enable the investigation of the association between infections and a wide variety of outcomes.

Development of a real time reverse transcription loop-mediated isothermal amplification method (RT-LAMP) for detection of a novel swine acute diarrhea syndrome coronavirus (SADS-CoV)

A novel swine acute diarrhea syndrome Coronavirus (SADS-CoV) that causes severe diarrhea in suckling piglets was identified in southern China in 2017. A simple and rapid detection test was developed for this virus using real-time RT-LAMP based on the conserved N gene of the virus. The method had a detection limit of 1.0 × 101 copies/μL with no cross-reactions with classical swine fever virus, porcine and respiratory syndrome virus NA, porcine and respiratory syndrome virus EU, transmissible gastroenteritis coronavirus, foot and mouth disease virus, porcine epidemic diarrhea virus (S-INDEL and non-S-INDEL), swine influenza virus subtype H1N1, porcine circovirus type 2, seneca valley virus, porcine parvovirus, porcine deltacoronavirus and rotavirus. This method was also reproducible. Twenty of 24 clinical samples were identified as SADS-CoV RNA-positive by the real-time RT-LAMP and the results were consistent with that of the real time RT-PCR method. This new method for detecting SADS-CoV is specific and sensitive for the detection of SADS-CoV.

Avian Influenza Virus Subtype H9N2 Affects Intestinal Microbiota, Barrier Structure Injury, and Inflammatory Intestinal Disease in the Chicken Ileum.

Avian influenza virus subtype H9N2 (H9N2 AIV) has caused significant losses to the poultry industry due to the high mortality associated with secondary infections attributable to E. coli. This study tries to address the underlying secondary mechanisms after H9N2 AIV infection. Initially, nine day-old specific pathogen-free chickens were assigned to control (uninfected) and H9N2-infected groups, respectively. Using Illumina sequencing, histological examination, and quantitative real-time PCR, it was found that H9N2 AIV caused intestinal microbiota disorder, injury, and inflammatory damage to the intestinal mucosa. Notably, the genera Escherichia, especially E. coli, significantly increased (p < 0.01) at five days post-infection (dpi), while Lactobacillus, Enterococcus, and other probiotic organisms were significantly reduced (p < 0.01). Simultaneously, the mRNA expression of tight junction proteins (ZO-1, claudin 3, and occludin), TFF2, and Muc2 were significantly reduced (p < 0.01), indicating the destruction of the intestinal epithelial cell tight junctions and the damage of mucin layer construction. Moreover, the mRNA expression of proinflammatory cytokines IFN-γ, IL-22, IFN-α, and IL-17A in intestinal epithelial cells were significantly upregulated, resulting in the inflammatory response and intestinal injury. Our findings may provide a theoretical basis for observed gastroenteritis-like symptoms such as diarrhea and secondary E. coli infection following H9N2 AIV infection.

Longitudinal serological measures of common infection in the Avon Longitudinal Study of Parents and Children cohort

Antibodies against pathogens provide information on exposure to infectious agents and are meaningful measures of past and present infection. Antibodies were measured in the plasma of children that are the offspring in a population-based birth cohort, the Avon Longitudinal Study of Parents and Children (ALSPAC). Plasma was collected during clinics at age 5, 7, 11 and 15 years. The antigens examined include: fungal ( Saccharomyces cerevisiae); protozoan ( Toxoplasma gondii and surface antigen 1 of T. gondii); herpes viruses (cytomegalovirus, Epstein-Barr virus, herpes simplex virus type 1); common colds (influenza virus subtypes H1N1 and H3N2); other antigens (measles); animal (feline herpes virus, Theiler's virus); bacteria ( Helicobacter pylori); dietary antigens (bovine casein alpha protein, bovine casein beta protein). Alongside the depth of data available within the ALSPAC cohort, this longitudinal resource will enable the investigation of the association between infections and a wide variety of outcomes.

Analysis of antigen conservation and inactivation of gamma-irradiated avian influenza virus subtype H9N2.

Avian influenza (AI) A subtype H9N2 virus belongs to Orthomyxoviridae family and causes low-pathogenic disease AI. The use of gamma-irradiated viral antigens has been developed in the production of effective vaccines. In this research, LPAIV H9N2 strain, A/Chicken/IRN/Ghazvin/2001, was multiplied on SPF eggs and irradiated by a Nordian gamma cell instrument. Irradiated and non-irradiated AI virus (AIV) samples were titrated by EID50 method and hemagglutinin (HA) antigen was analyzed by HA test as the WHO pattern method. Infectivity of irradiated virus was determined by egg inoculation method during four blind cultures. The results showed that after increasing the dose of gamma radiation, virus titer gradually decreased. D10 value and optimum dose for complete virus inactivation were calculated by dose/response curve, 3.36 and 29.52kGy, respectively. In addition, HA antigenicity of gamma-irradiated virus samples from 0 to 30kGy was not changed. The results of safety test for gamma-irradiated AIV samples showed complete inactivation with gamma ray doses 30 and 35kGy, without any multiplication on eggs after four blind cultures. According to the results of HA antigen assay and safety test, the gamma-irradiated and complete inactivated AIV subtype H9N2 is a good candidate as an inactivated immunogenic agent for poultry vaccination.

Evaluation of the antiviral effects of aqueous extracts of red and yellow onions (Allium Cepa) against avian influenza virus subtype H9N2

Avian influenza virus subtype H9N2 causes important economic losses in industrial poultry worldwide. Biosecurity and vaccination have not completely prevented the outbreak of avian influenza virus subtype H9N2 in poultry, and there are no appropriate medicines available. Onion is one of the plants used from the ancient times both as food and medicine. The purpose of this study was to evaluate the antiviral effects of aqueous extract of red and yellow onion against avian influenza virus subtype H9N2. First, a study was performed to evaluate the toxic effects of the extracts on the embryonated chicken eggs. For antiviral evaluation, three mixtures were prepared: mixture of the virus and the red onion extract, mixture of the virus and the yellow onion extract, and mixture of the virus and PBS, as a control group. The mixtures were separately inoculated to the chorioallantoic sac of the embryonated eggs after 2, 8 and 24 hours incubation at room temperature. Mortality rate and hemagglutination assay titers were recorded. The results indicated that the red onion extract decreases mortality of the embryos and the yellow onion extract increases the life of the embryos, and both of the extracts decrease HA titers. In conclusion, it seems that both extracts especially aqueous extract of the red onion not only destroys the avian influenza virus subtype H9N2, but also they probably decrease the propagation of the virus in the embryonated chicken eggs.

A rapid and transient innate immune response to avian influenza infection in mallards

The vertebrate innate immune system provides hosts with a rapid, non-specific response to a wide range of invading pathogens. However, the speed and duration of innate responses will be influenced by the co-evolutionary dynamics of specific host-pathogen combinations. Here, we show that low pathogenic avian influenza virus (LPAI) subtype H1N1 elicits a strong but extremely transient innate immune response in its main wildlife reservoir, the mallard (Anas platyrhynchos). Using a series of experimental and methodological improvements over previous studies, we followed the expression of retinoic acid inducible gene 1 (RIG-I) and myxovirus resistance gene (Mx) in mallards semi-naturally infected with low pathogenic H1N1. One day post infection, both RIG-I and Mx were significantly upregulated in all investigated tissues. By two days post infection, the expression of both genes had generally returned to basal levels, and remained so for the remainder of the experiment. This is despite the fact that birds continued to actively shed viral particles throughout the study period. We additionally show that the spleen plays a particularly active role in the innate immune response to LPAI. Waterfowl and avian influenza viruses have a long co-evolutionary history, suggesting that the mallard innate immune response has evolved to provide a minimum effective response to LPAIs such that the viral infection is brought under control while minimising the damaging effects of a sustained immune response.

Intranasal administration of inactivated avian influenza virus of H5N1 subtype vaccine-induced systemic immune response in chicken and mice.

Aim:The need for non-parenteral administration of inactivated avian influenza virus of H5N1 subtype (AIV-H5N1) vaccine is paramount. Here, we provide preliminary data on the immune response of chicken and mice after intranasal administration of AIV-H5N1-inactivated vaccine with ISCOMS, Inmunair (INM), and combined ISCOMS and INM as an adjuvant.Materials and Methods:The AIV isolate of A/Chicken/Denpasar/01/2004 (H5N1) was cultivated in specific pathogen-free chicken eggs and inactivated with formaldehyde. The vaccine preparation was added with those adjuvants for intranasal administration and aluminum hydroxide for subcutaneous injection. The chicken and mouse were vaccinated at the age of 3 weeks or 1 month and repeated 2 weeks thereafter. In one experiment, chicken was injected with Newcastle disease virus (NDV) at the same time with AIV vaccine. The sera were collected at one (serum 1) and 2 w (serum 2) after booster vaccination. The anti-AIV-H5 and NDV antibodies in chicken sera were detected using hemagglutination inhibition (HI) assay. Mouse IgG anti-AIV-H5N1 antibody was detected using ELISA.Results:The result shows that the geometric mean titers (GMTs) of chicken sera of intranasal vaccinated with inactivated AIV-H5N1 vaccine with mixed ISCOM- INM as adjuvant were <20.0 and 22.7 unit HI-unit (HIU) in serum 1 and serum 2, respectively. The GMTs of the positive control group were 23.7 and 25.7 HIU in serum 1 and serum 2, respectively. The result of the second experiment shows that IgG anti-AIV-H5N1 was detected in mouse sera. In the third experiment, the GMTs of anti-NDV in chicken vaccinated subsequently with inactivated NDV vaccine and AIV-H5N1 with mixed ISCOMS-INM administrated intranasally and aluminum hydroxide adjuvant administrated through subcutaneous injection as well as positive control group receiving NDV vaccine only were 28.0, 28.0, and 27.4 HIU in serum 1 while were 29.6, 29.2, and 28.2 HIU in serum 2, respectively.Conclusion:Intranasal administration of inactivated AIV-H5N1 vaccine-induced a systemic immune response in chicken and mice after adding ISCOMS and/or INM as adjuvants. The adjuvant and the intranasal administration caused no immunosuppressive effect on the chicken immune response to NDV vaccine.

Gut microbiota modulates type I interferon and antibody-mediated immune responses in chickens infected with influenza virus subtype H9N2.

Commensal gut microbes play a critical role in shaping host defences against pathogens, including influenza viruses. The current study was conducted to assess the role and mechanisms of action of commensal gut microbiota on the innate and antibody-mediated responses of layer chickens against influenza virus subtype H9N2. A total of 104 one-day-old specific pathogen free chickens were assigned to either of the four treatments, which included two levels of antibiotics treatment (ABX- and ABX+) and two levels of H9N2 virus infection (H9N2- and H9N2+). At day 17 of age, chickens in the H9N2+ group were infected via the oral-nasal route with 400 μl of 107 TCID50/ml (200 μl/each route). Oropharyngeal and cloacal swabs at days 1, 3, 5, 7 and 9 post-infection (p.i.) for virus shedding, tissue samples at 12 h, 24 h and 36 h p.i. for mRNA measurement, and serum samples at days 7 and 14 p.i. for hemagglutination inhibition (HI) assay and IgG antibodies were collected. Virus shedding analysis showed that antibiotic treated (depleted)-H9N2 virus infected chickens showed a significantly higher oropharyngeal virus shedding at all time points, and cloacal shedding at days 3 and 5 p.i. compared to control treated (undepleted)-H9N2 infected chickens. Analysis of mRNA expression showed that infection of depleted chickens with H9N2 virus resulted in significantly down-regulated type I interferon responses both in the respiratory and gastrointestinal tracts compared to undepleted-H9N2 infected chickens. However, antibody-mediated immune response analysis showed a significantly higher HI antibody titre and IgG levels in the serum of chickens depleted with antibiotics and infected with H9N2 virus compared to undepleted-H9N2 infected chickens. In conclusion, findings from the current study suggest that the gut microbiota of chickens plays an important role in the initiation of innate responses against influenza virus infection, while the antibody-mediated immune response remains unaffected.

Penentuan Secara Imunopatologi Organ Target Virus Flu Burung Menggunakan Streptavidin Biotin (DETERMINATION OF TARGET ORGANS OF AVIAN INFLUENZA VIRUS USING IMMUNOPATHOLOGICAL IMMUNOHISTOCHEMISTRY STREPTAVIDIN-BIOTIN)

Avian influenza is a viral disease in poultry caused by avian influenza virus (AIV) subtype H5N1 with varying clinical signs are often similar to the clinical signs of other viral infections, such as Newcastle disease virus (NDV). The mechanism of disease pathogenesis to express clinical signs tightly correlated to the determination of the target organ seen from AIV H5N1 antigens distribution in organs, such as respiratory tract, brain and gastrointestinal tract. Immunopathological immunohistochemistry streptavidin-biotin (IHC SB) is a method for sensitive and accurate in detecting antigens of AIV on the tissues. In the present study, it was determined whether in laying hens with clinical signs of torticollis and curled toe paralysis, and pathologic anatomic lesions in the form of petechial and foci necrotic hemorrhages tested with immunopathological IHC SB is positive AIV H5N1 infection. IHC SB study results showed that the AIV H5N1 antigen were found in tissues of the lung, brain, duodenum and proventriculus. Based on these results, we can conclude that the IHC SB is a method that is highly sensitive and accurate to detect H5N1 antigens and its distribution in the host.

Optimizing the process of inactivating influenza virus subtype H9N2 by formalin in the production of killed avian influenza vaccine

Avian influenza is one of the most important diseases in avian industry, which also threats human population. Thus, vaccination is necessary for controlling this viral disease. In this study, killed vaccine of avian influenza subtype H9N2 and formalin solution (for virus inactivation) were used. It is necessary to study the effect of different factors such as formalin concentration, as well as incubation temperature and duration on inactivation process. For this purpose, after preparation and measurement of antibody titers of vaccinal strains of avian influenza, 27 experimental samples of H9N2 avian influenza virus were prepared at different formalin concentrations (0.1%, 0.05%, and 0.025%), at different incubation temperatures (4 °C, 25 °C, and 37 °C), and in different incubation durations (12, 18, and 24 h). In addition, three control samples were prepared at three different test temperatures without adding formalin. All the samples were evaluated by inactivation test, hemagglutination assay, and measurement of free formaldehyde. All the experiments were repeated within three consecutive periods. Considering the findings and destructive effects of long incubation durations at 37°C on antigens, the temperature of 25 °C was more suitable. Furthermore, the free formaldehyde amounts at different concentrations at 25 °C were slightly different in comparison with 37 °C. Therefore, formalin concentration of 0.1% at 25 °C completely inactivated the virus within 24 h and was proposed as the optimal condition.

Immune-Related Gene Expression Patterns in GPV- or H9N2-Infected Goose Spleens.

Goose parvovirus (GPV) and avian influenza virus subtype H9N2 are single-stranded DNA (ssDNA) and single-stranded RNA (ssRNA) viruses, respectively, both of which can spread in goslings and cause a significant economic loss. To explore the comprehensive transcriptome of GPV- or H9N2-infected goose spleens and to understand the immune responses induced by a DNA virus (GPV) or a RNA virus (H9N2), RNA-seq was performed on the spleens of goslings at the fifth day post infection. In the present study, 2604 and 2409 differentially expressed unigenes were identified in the GPV- and H9N2-infected groups, respectively. Through KEGG pathway enrichment analyses, the up-regulated transcripts in the two virus-infected groups were mainly involved in immune-related pathways. In addition, the two virus-infected groups displayed similar expression patterns in the immune response pathways, including pattern-recognition receptor signaling pathways, the antigen processing and presentation pathway, the NF-κB signaling pathway and the JAK-STAT signaling pathway, as well as cytokines. Furthermore, most of the immune-related genes, particularly TLR7, TRAF3, Mx, TRIM25, CD4, and CD8α, increased in response to GPV and H9N2 infection. However, the depression of NF-κB signaling may be a mechanism by which the viruses evade the host immune system or a strategy to achieve immune homeostasis.

Chlamydia psittaci infection increases mortality of avian influenza virus H9N2 by suppressing host immune response.

Avian influenza virus subtype H9N2 (H9N2) and Chlamydia psittaci (C. psittaci) are frequently isolated in chickens with respiratory disease. However, their roles in co-infection remain unclear. We tested the hypothesis that C. psittaci enhances H9N2 infection through suppression of host immunity. Thus, 10-day-old SPF chickens were inoculated intra-tracheally with a high or low virulence C. psittaci strain, and were simultaneously vaccinated against Newcastle disease virus (NDV). Significant decreases in body weight, NDV antibodies and immune organ indices occurred in birds with the virulent C. psittaci infection, while the ratio of CD4+/CD8+ T cells increased significantly compared to that of the lower virulence strain. A second group of birds were inoculated with C. psittaci and H9N2 simultaneously (C. psittaci+H9N2), C. psittaci 3 days prior to H9N2 (C. psittaci/H9N2), or 3 days after H9N2 (H9N2/C. psittaci), C. psittaci or H9N2 alone. Survival rates were 65%, 80% and 90% in the C. psittaci/H9N2, C. psittaci+H9N2 and H9N2/C. psittaci groups, respectively and respiratory clinical signs, lower expression of pro-inflammatory cytokines and higher pathogen loads were found in both C. psittaci/H9N2 and C. psittaci+H9N2 groups. Hence, virulent C. psittaci infection suppresses immune response by inhibiting humoral responses and altering Th1/Th2 balance, increasing mortality in H9N2 infected birds.

High-Performance Three-Dimensional Tubular Nanomembrane Sensor for DNA Detection.

We report an ultrasensitive label-free DNA biosensor with fully on-chip integrated rolled-up nanomembrane electrodes. The hybridization of complementary DNA strands (avian influenza virus subtype H1N1) is selectively detected down to attomolar concentrations, an unprecedented level for miniaturized sensors without amplification. Impedimetric DNA detection with such a rolled-up biosensor shows 4 orders of magnitude sensitivity improvement over its planar counterpart. Furthermore, it is observed that the impedance response of the proposed device is contrary to the expected behavior due to its particular geometry. To further investigate this difference, a thorough model analysis of the measured signal and the electric field calculation is performed, revealing enhanced electron hopping/tunneling along the DNA chains due to an enriched electric field inside the tube. Likewise, conformational changes of DNA might also contribute to this effect. Accordingly, these highly integrated three-dimensional sensors provide a tool to study electrical properties of DNA under versatile experimental conditions and open a new avenue for novel biosensing applications (i.e., for protein, enzyme detection, or monitoring of cell behavior under in vivo like conditions).

Antiviral activity of the oseltamivir and Melissa officinalis L. essential oil against avian influenza A virus (H9N2).

Lemon balm derivatives are going to acquire a novelty as natural and potent remedy for treatment of viral infections since the influenza viruses are developing resistance to the current antivirals widely. Oseltamivir, Melissa officinalis essential oil (MOEO) and their synergistic efficacy against avian influenza virus (AIV) subtype H9N2 were evaluated in vitro in MDCK cells at different time exposure by using TCID50, HA, Real Time PCR and HI assay. The results showed that MOEO could inhibit replication of AVI through the different virus replication phase (P≤0.05). Also the highest antiviral activity of MOEO was seen when AIV incubated with MOEO before cell infection. The TCID50/ml was reduced 1.3-2.1, 2.3-2.8, 3.7-4.5 log 10 than control group (5.6 log 10), HAU/50µl was decreased 85-94, 71.4-94, 71.4-94% and viral genome copy number/µl was brought down 68-95, 90-100, 89.6-99.9% at pre-infection, post-infection and simultaneous stage, respectively. Hemagglutination inhibition result showed the MOEO was not able to inhibit agglutination of the chicken red blood cell (cRBC). Replication of the AVI was suppressed by the different concentration of oseltamivir completely or near 100%. Also oseltamivir showed a synergistic activity with MOEO especially when oseltamivir concentration reduced under 0.005mg/ml. The chemical composition was examined by GC-MS analysis and Its main constituents were identified as monoterpenaldehydes citral a, citral b. In conclusion, the findings of the study showed that lemon balm essential oil could inhibit influenza virus replication through different replication cycle steps especially throughout the direct interaction with the virus particles.

Low pathogenic avian influenza (H9N2) in chicken: Evaluation of an ancestral H9-MVA vaccine

Modified Vaccinia Ankara (MVA) has proven its efficacy as a recombinant vector vaccine for numerous pathogens including influenza virus. The present study aimed at evaluating a recombinant MVA candidate vaccine against low pathogenic avian influenza virus subtype H9N2 in the chicken model. As the high genetic and antigenic diversity of H9N2 viruses increases vaccine design complexity, one strategy to widen the range of vaccine coverage is to use an ancestor sequence. We therefore generated a recombinant MVA encoding for the gene sequence of an ancestral hemagglutinin H9 protein (a computationally derived amino acid sequence of the node of the H9N2 G1 lineage strains was obtained using the ANCESCON program). We analyzed the genetics and the growth properties of the MVA vector virus confirming suitability for use under biosafety level 1 and tested its efficacy when applied either as an intra-muscular (IM) or an oral vaccine in specific pathogen free chickens challenged with A/chicken/Tunisia/12/2010(H9N2). Two control groups were studied in parallel (unvaccinated and inoculated birds; unvaccinated and non-inoculated birds).IM vaccinated birds seroconverted as early as four days post vaccination and neutralizing antibodies were detected against A/chicken/Tunisia/12/2010(H9N2) in all the birds before challenge. The role of local mucosal immunity is unclear here as no antibodies were detected in eye drop or aerosol vaccinated birds. Clinical signs were not detected in any of the infected birds even in absence of vaccination. Virus replication was observed in both vaccinated and unvaccinated chickens, suggesting the MVA-ancestral H9 vaccine may not stop virus spread in the field. However vaccinated birds showed less histological damage, fewer influenza-positive cells and shorter virus shedding than their unvaccinated counterparts.

Potency of antigenic and serologic tests based on CNTKCQTP linear epitope on H5N1 haemagglutinin for Avian Influenza

&lt;p class="abstrak2"&gt;Rapid diagnostic tools or point-of-care (POC) test is needed in the effort to control and eradicate the high pathogenic avian influenza (HPAI) H5N1 in Indonesia. Accuracy of a POC test is determined by the specificity of antibodies, which is the main component of a POC test. Recently a linear epitope, CNTCKQTP epitope, located at 274-281 amino acid residue of H5 hemagglutinin has been confirmed to be present all clade of H5N1 viruses. This study aimed at producing and evaluating the reactivity of a monospecific, polyclonal antibody against the epitope. The Antibody was produced by immunising a goat with the peptide in the form of multiple antigen peptide (MAP). The specificity of the antibody was estimated by assaying its reactivity against influenza virus subtypes H3N3, H4N4, H5N1, H6N5, H7N7, H9N2, H10N7 and H11N9; and recombinant hemagglutinins H1-H12, H14 and H15 with ELISA and immunoblot. The results of the assay showed that CNTKCQTP antibody was not specific for H5 haemagglutinin because it cross-reacted with other haemagglutinins especially H7, H8 and H9. The potential of the peptide containing the epitope, GNCNTKCQTPMGAINSS. as an ELISA reagent for assaying H5 antibodies in chickens previously vaccinated and challenged with the H5N1 virus was also evaluated in this study. In contrast, the results of previous studies, the ELISA using GNCNTKCQTPMGAINSS as coating antigen was not sensitive in detecting antibody to haemagglutinin H5 in chickens.&lt;/p&gt;&lt;strong&gt;Key Words: &lt;/strong&gt;AI Virus, Hemagglutinin H5, CNTKCQTP Epitope, MAP, Immunoassa&lt;em&gt;y&lt;/em&gt;

Vaccine escape in 2013–4 and the hydropathic evolution of glycoproteins of A/H3N2 viruses

More virulent strains of influenza virus subtypes H1N1 appeared widely in 2007 and H3N2 in 2011, and especially 2013–4, when the effectiveness of the H3N2 vaccine decreased by more than a factor of two. The amino acid differences of neuraminidase from prior less virulent strains appear to be small (<1%) when tabulated through sequence alignments and counting site identities and similarities. Here we show how analyzing fractal hydropathic forces responsible for neuraminidase globular compaction and modularity quantifies the mutational origins of increased virulence. It also predicts vaccine escape and specifies optimized targets for the 2015 H3N2 vaccine. Unlike some earlier methods based on measuring hemagglutinin antigenic drift, which take several years, cover only a few candidate strains, and are ambiguous, the new methods are timely and can be completed, using NCBI and GISAID amino acid sequences only, in a few days.

Phylogenetic Analysis and Pathogenicity Assessment of Two Strains of Avian Influenza Virus Subtype H9N2 Isolated from Migratory Birds: High Homology of Internal Genes with Human H10N8 Virus.

Two human-infecting avian influenza viruses (AIVs), H7N9 and H10N8, have emerged in China, which further indicate that the H9N2 subtype of AIVs, as an internal gene donor, may have an important role in the generation of new viruses with cross-species transmissibility and pathogenicity. H9N2 viruses that contain such internal genes widely exist in poultry but are rarely reported in migratory birds. In this study, two strains of the H9N2 virus were isolated from fecal samples of migratory birds in 2014: one strain from Caizi Lake in Anhui Province and one from Chen Lake in Hubei Province of China. Nucleotide sequence analysis revealed high homology of all six internal genes of these two strains with the internal genes of the human H10N8 virus in Jiangxi Province, as well as with the human H7N9 virus. Phylogenetic analysis indicated a possible origin of these two strains from poultry in South China. Both of the two viruses tested could replicated in respiratory organs of infective mice without adaption, by both strains of the H9N2 AIVs from wild birds, suggesting their potential capacity for directly infecting mammals. Our findings indicate the existence of H9N2 viruses that contain internal genes highly homologous with human H10N8 or H7N9 viruses. Wild birds can contribute to the spread of the H9N2 virus that contains the “harmful” internal gene complex, leading to gene rearrangement with other influenza viruses and to the generation of new pathogenic viruses. Therefore, strengthening AIV surveillance in wild birds can promote an understanding of the presence and prevalence of viruses and provide scientific evidence for the prevention and control of AIVs and human-infecting AIVs.

Sero-conversion of Avian Influenza Virus Subtype H9N2 in Non-Vaccinated Commercial Poultry Flocks

Sero-conversion of pathogenic subtypes (H5, H7 and H9) of Avian Influenza Virus types was studied in commercial poultry farms in the Province of Punjab, Pakistan during the years 2015-16. Out of 6731 morbid tissues, only 11 samples (4 from broiler, 4 from broiler breeder and 3 from commercial layers farms) were found positive for Avian Influenza Virus (AIV) H9N2 type. Only one isolate was found positive for H9N2 subtype from 17994 tracheal swab samples. All the 37163 cloacal swab samples were found negative. The sero-analysis of 133438 blood sera samples showed sero-conversion in 66219 samples (49.64%) against H9 virus in the non-vaccinated poultry flocks, when tested with Heamagglutination Inhibition test (HI). Highest sero-conversion was recorded in broiler breeder (49.20%) followed by broilers (47.47%), layer (46.25%) and Native/Desi birds (45.59%). Highest HI titres were recorded in the flocks from Muzaffargarh, Rawalpindi, Toba Tek Singh, Sargodha, Gujrat, Faisalabad, Sheikhupura, Jhelum and Pakpattan while Bahawalpur, Gujranwala, Lahore, Jhang, Okara and Multan had relatively lower level of HI antibody titres. In conclusion only one type of AIV subtype H9N2 is prevailing in the province of the Punjab, Pakistan.