HA Gene Research Articles

Mutations during the adaptation of H7N9 avian influenza virus to mice lungs enhance human-like sialic acid binding activity and virulence in mice

The first avian H7N9 influenza outbreak in spring of 2013 emerged in an unprecedented transmission from infected poultry to humans in the Yangtze delta area, eastern China, posing a dual challenge to public health and poultry industry. However, the mechanism for how avian H7N9 influenza virus adapts to mammalian hosts has not been clearly understood. Here, to identify adaptive changes that confer enhanced virulence of H7N9 virus in mammals, we generated a mouse-adapted H7N9 variant virus (S8) by serial lung-to-lung passages of the wild-type SDL124 virus in mice and compared their phenotype in vivo and in vitro. Sequence analysis showed that the two viruses differed by 27 amino acids distributed among six genes, containing changes in PB2 (E627K, D701N) and HA (Q226L) genes. The 50% mouse lethal dose (MLD50) of S8 reduced about 500 folds, to be moderately pathogenic to mice when compared to that of low pathogenic wild-type SDL124. Moreover, S8 replicated efficiently in mouse lungs and displayed expanded tissue tropism, and induced a greater degree of pulmonary edema and higher level of inflammatory cell infiltration in bronchoalveolar lavage fluids than SDL124 did. Interestingly, the mouse adapted S8 virus obtained strong affinity for human-like (SAα-2,6 Gal) receptor during the adaptation in mice. Correspondingly, compared with SDL124 virus, S8 virus showed higher replication efficiency in mammalian cells, whereas lower replication ability in avian cells. Taken together, these findings suggest that these mutations synergistically elevate the ability of H7N9 virus to disseminate to multiple organs and subsequently enhance the virulence of H7N9 virus in mammalian hosts.

Functional Characterization of Two Thioredoxin Proteins of Toxoplasma gondii Using the CRISPR-Cas9 System.

Toxoplasmosis caused by infection with Toxoplasma gondii is an important parasitic zoonosis with a worldwide distribution. In this study, we examined the functions of two thioredoxins (namely CTrp26 and CTrx1) of T. gondii tachyzoites by generation of HA tag strains or gene deficient parasites in Type I RH strain (ToxoDB#10). Immunofluorescence analysis (IFA) was used to investigate the subcellular localization of the thioredoxins (Trxs). Results of IFA showed that both CTrp26 and CTrx1 were located in the cytoplasm of T. gondii. Functional characterizations of CTrp26 and CTrx1-deficient parasites were performed by plaque assay, intracellular replication, egress, H2O2 resistance, detection of reactive oxygen species (ROS) level and total antioxidant capacity (T-AOC) assays in vitro, as well as mouse infection in vivo. Our results showed that deletion of CTrp26 or CTrx1 did not influence the ability of T. gondii RH strain to replicate, egress, form plaque, resist H2O2 exposure, maintain the ROS level, and T-AOC, and also did not serve as virulence factors in Kunming mice. Taken together, these results provide new properties of the two Trxs. Although they are not essential for RH strain, they may have roles in other strains of this parasite due to their different expression patterns, which warrants future research.

RECENT UPDATES ON MOLECULAR DETECTION OF H9N2 AS LOW PATHOGENIC STRAIN OF AVIAN INFLUENZA VIRUS FROM POULTRY FARMS OF LAHORE, PAKISTAN

H9N2 avian influenza outbreaks have caused great economic losses to the poultry industry in recent decades due to a decrease of egg production, high morbidity, and mortality. Due to different antigenic variants, Influenza virus has become problematical because it has the ability to cross the species barrier. As it is highly pathogenic so its diagnosis and vaccines are of high importance. Hemagglutination inhibition (HI) test is mostly used for subtyping and detection of antibody titer against the virus. Furthermore, its continuous mutations in the HA gene transforms AIV subtype H9N2 (a low pathogenic subtype) into high pathogenic virus subtypes like H5N2 and H7N7 that may have pandemic potential. Thus, it is necessary to identify various antigenic variants of Influenza virus, so it is direly needed to study the HA gene, its attachment to host receptors, the release of genetic material and pathogenicity. In the present study, virus samples from poultry were isolated. Both serological and molecular confirmation was done for 100 samples collected from the different area. They were properly labeled and prepared for the process of egg inoculation in embryonated eggs. The virus was grown in amnioallantoic membrane of embryonated eggs and harvested fluid is then proceeded for confirmatory testing. Hemagglutination and Hemagglutination inhibition testing was done. RNA was extracted by the kit method and cDNA was synthesized. Reverse transcriptase (RTPCR) was performed using specific primer sets and then the PCR product was run on agarose gel. The bands obtained were sent for sequencing.

Molecular Characterization, Pathogenesis and Immunological Effect of Avian Influenza H9N2 Virus Isolated From Broiler Chickens in Egypt During 2019

Many commercial flocks in Egypt were infected with the low pathogenic avian influenza virus (LPAIV) subtype H9N2, resulting in severe economic losses, particularly when combined with other viral and bacterial diseases. The current study aimed to investigate the genetic evolution of LPAIV (H9N2) in broiler farms in Egypt during 2019 and evaluate its effect on the viral pathogenesis, cytokine response, innate immunity and oxidative state in specific pathogen free (SPF) chicks. Sixty tracheal swabs were collected from broilers and tested by RRT-PCR and the HA gene were sequenced for the genetic evolution. Then this isolated virus was experimentally studied in a total of 20 SPF chicks (21 days old) that were divided into two equal groups. Group-1(G1) was negative control and group-2 (G2) was experimentally infected with the isolated LPAIV (H9N2). The obtained results revealed that all isolated LPAIV (H9N2) in this study were belong to G1-like Eurasian sub-lineages (A/quail/Hong Kong/G1/97-like) and clustered with Israeli strains in group B with specific mutation specific for Egyptian AIV-H9N2 isolated in 2018-2019 forming new subgroup-I. The experimental infection with LPAIV (H9N2) (G2) in SPF chickens, induced mild clinical signs, moderate histopathological changes in lung and spleen, shedding of higher mean LPAIV (H9N2) titre in trachea, up regulation of both the pro-inflammatory (IL-6) and the anti-viral (IFN-γ) cytokines, significant decrease of the serum levels of lysozyme and disturbance of the oxidative state where the serum nitric oxide (NO) and the malondialdehyde (MDA) levels were significantly increased and the antioxidants glutathione (GSH) and superoxide dismutase (SOD) were significantly decreased. In conclusion, the LPAIV (H9N2) was continually circulated in broiler chicken farms and highly evolved. The induced oxidative stress in LPAIV (H9N2) infection could be contributed to the viral pathogenesis, up-regulation of pro-inflammatory and anti-viral cytokines and down-regulation of the innate immune response.

Genetic Characterization of Two Human Cases Infected with the Avian Influenza A (H5N6) Viruses - Guangxi Zhuang Autonomous Region, China, 2021.

What is known about this topic?H5N6 has replaced H5N1 as a dominant avian influenza virus (AIV) subtype in southern China. The increasing genetic diversity and geographical distribution of H5N6 pose a serious threat to the poultry industry and human health.What is added by this report?A total of 2 cases of H5N6 that occurred from February 2021 to July 2021 in Guangxi, China were reported in this study. Phylogenetic analysis of gene was constructed, and some mutations of HA gene, PB2 gene, PA gene, M1 gene, NS1 gene, the receptor-binding site were detected. The evolutionary origins of the internal genes were different.What are the implications for public health practice?As a multi-source reassortant virus, the H5N6 highly pathogenic AIV is continuously evolving. There is an urgent need to strengthen the surveillance of drug-resistant strains and novel variants.

A single-amino-acid mutation at position 225 in hemagglutinin attenuates H5N6 influenza virus in mice

The highly pathogenic avian influenza H5N6 viruses are widely circulating in poultry and wild birds, and have caused 38 human infections including 21 deaths; however, the key genetic determinants of the pathogenicity of these viruses have yet to be fully investigated. Here, we characterized two H5N6 avian influenza viruses––A/duck/Guangdong/S1330/2016 (GD/330) and A/environment/Fujian/S1160/2016 (FJ/160)––that have similar viral genomes but differ markedly in their lethality in mice. GD/330 is highly pathogenic with a 50% mouse lethal dose (MLD50) of 2.5 log10 50% egg infectious doses (EID50), whereas FJ/160 exhibits low pathogenicity with an MLD50 of 7.4 log10 EID50. We explored the molecular basis for the difference in virulence between these two viruses. By using reverse genetics, we created a series of reassortants and mutants in the GD/330 background and assessed their virulence in mice. We found that the HA gene of FJ/160 substantially attenuated the virulence of GD/330 and that the mutation of glycine (G) to tryptophan (W) at position 225 (H3 numbering) in HA played a key role in this function. We further found that the amino acid mutation G225W in HA decreased the acid and thermal stability and increased the pH of HA activation, thereby attenuating the H5N6 virus in mice. Our study thus identifies a novel molecular determinant in the HA protein and provides a new target for the development of live attenuated vaccines and antiviral drugs against H5 influenza viruses.

Novel reassortant 2.3.4.4B H5N6 highly pathogenic avian influenza viruses circulating among wild, domestic birds in Xinjiang, Northwest China.

BackgroundThe H5 avian influenza viruses (AIVs) of clade 2.3.4.4 circulate in wild and domestic birds worldwide. In 2017, nine strains of H5N6 AIVs were isolated from aquatic poultry in Xinjiang, Northwest China.ObjectivesThis study aimed to analyze the origin, reassortment, and mutations of the AIV isolates.MethodsAIVs were isolated from oropharyngeal and cloacal swabs of poultry. Identification was accomplished by inoculating isolates into embryonated chicken eggs and performing hemagglutination tests and reverse transcription polymerase chain reaction (RT-PCR). The viral genomes were amplified with RT-PCR and then sequenced. The sequence alignment, phylogenetic, and molecular characteristic analyses were performed by using bioinformatic software.ResultsNine isolates originated from the same ancestor. The viral HA gene belonged to clade 2.3.4.4B, while the NA gene had a close phylogenetic relationship with the 2.3.4.4C H5N6 highly pathogenic avian influenza viruses (HPAIVs) isolated from shoveler ducks in Ningxia in 2015. The NP gene was grouped into an independent subcluster within the 2.3.4.4B H5N8 AIVs, and the remaining six genes all had close phylogenetic relationships with the 2.3.4.4B H5N8 HPAIVs isolated from the wild birds in China, Egypt, Uganda, Cameroon, and India in 2016–2017, Multiple basic amino acid residues associated with HPAIVs were located adjacent to the cleavage site of the HA protein. The nine isolates comprised reassortant 2.3.4.4B HPAIVs originating from 2.3.4.4B H5N8 and 2.3.4.4C H5N6 viruses in wild birds.ConclusionsThese results suggest that the Northern Tianshan Mountain wetlands in Xinjiang may have a key role in AIVs disseminating from Central China to the Eurasian continent and East African.

Effect of Antigenic Drift on Influenza Vaccine Effectiveness in the United States-2019-2020.

At the start of the 2019-2020 influenza season, concern arose that circulating B/Victoria viruses of the globally emerging clade V1A.3 were antigenically drifted from the strain included in the vaccine. Intense B/Victoria activity was followed by circulation of genetically diverse A(H1N1)pdm09 viruses that were also antigenically drifted. We measured vaccine effectiveness (VE) in the United States against illness from these emerging viruses. We enrolled outpatients aged ≥6 months with acute respiratory illness at 5 sites. Respiratory specimens were tested for influenza by reverse-transcriptase polymerase chain reaction (RT-PCR). Using the test-negative design, we determined influenza VE by virus subtype/lineage and genetic subclades by comparing odds of vaccination in influenza cases versus test-negative controls. Among 8845 enrollees, 2722 (31%) tested positive for influenza, including 1209 (44%) for B/Victoria and 1405 (51%) for A(H1N1)pdm09. Effectiveness against any influenza illness was 39% (95% confidence interval [CI]: 32-44), 45% (95% CI: 37-52) against B/Victoria and 30% (95% CI: 21-39) against A(H1N1)pdm09-associated illness. Vaccination offered no protection against A(H1N1)pdm09 viruses with antigenically drifted clade 6B.1A 183P-5A+156K HA genes (VE 7%; 95% CI: -14 to 23%) which predominated after January. Vaccination provided protection against influenza illness, mainly due to infections from B/Victoria viruses. Vaccine protection against illness from A(H1N1)pdm09 was lower than historically observed effectiveness of 40%-60%, due to late-season vaccine mismatch following emergence of antigenically drifted viruses. The effect of drift on vaccine protection is not easy to predict and, even in drifted years, significant protection can be observed.

Intranasal Delivery of Inactivated Influenza Virus and Poly(I:C) Adsorbed Corn-Based Nanoparticle Vaccine Elicited Robust Antigen-Specific Cell-Mediated Immune Responses in Maternal Antibody Positive Nursery Pigs.

We designed the killed swine influenza A virus (SwIAV) H1N2 antigen (KAg) with polyriboinosinic:polyribocytidylic acid [(Poly(I:C)] adsorbed corn-derived Nano-11 particle based nanovaccine called Nano-11-KAg+Poly(I:C), and evaluated its immune correlates in maternally derived antibody (MDA)-positive pigs against a heterologous H1N1 SwIAV infection. Immunologically, in tracheobronchial lymph nodes (TBLN) detected enhanced H1N2-specific cytotoxic T-lymphocytes (CTLs) in Nano-11-KAg+Poly(I:C) vaccinates, and in commercial vaccinates detected CTLs with mainly IL-17A+ and early effector phenotypes specific to both H1N2 and H1N1 SwAIV. In commercial vaccinates, activated H1N2- and H1N1-specific IFNγ+&TNFα+, IL-17A+ and central memory T-helper/Memory cells, and in Nano-11-KAg+Poly(I:C) vaccinates H1N2-specific central memory, IFNγ+ and IFNγ+&TNFα+, and H1N1-specific IL-17A+ T-helper/Memory cells were observed. Systemically, Nano-11-KAg+Poly(I:C) vaccine augmented H1N2-specific IFNγ+ CTLs and H1N1-specific IFNγ+ T-helper/Memory cells, and commercial vaccine boosted H1N2- specific early effector CTLs and H1N1-specific IFNγ+&TNFα+ CTLs, as well as H1N2- and H1N1-specific T-helper/Memory cells with central memory, IFNγ+&TNFα+, and IL-17A+ phenotypes. Remarkably, commercial vaccine induced an increase in H1N1-specific T-helper cells in TBLN and naive T-helper cells in both TBLN and peripheral blood mononuclear cells (PBMCs), while H1N1- and H1N2-specific only T-helper cells were augmented in Nano-11-KAg+Poly(I:C) vaccinates in both TBLN and PBMCs. Furthermore, the Nano-11-KAg+Poly(I:C) vaccine stimulated robust cross-reactive IgG and secretory IgA (SIgA) responses in lungs, while the commercial vaccine elicited high levels of serum and lung IgG and serum hemagglutination inhibition (HI) titers. In conclusion, despite vast genetic difference (77% in HA gene identity) between the vaccine H1N2 and H1N1 challenge viruses in Nano-11-KAg+Poly(I:C) vaccinates, compared to over 95% identity between H1N1 of commercial vaccine and challenge viruses, the virus load and macroscopic lesions in the lungs of both types of vaccinates were comparable, but the Nano-11-KAg+Poly(I:C) vaccine cleared the virus from the nasal passage better. These data suggested the important role played by Nano-11 and Poly(I:C) in the induction of polyfunctional, cross-protective cell-mediated immunity against SwIAV in MDA-positive pigs.

Detection and genetic characterization of avian influenza A (H5N6) virus clade 2.3.4.4 in isolates from house crow and poultry in Bangladesh, 2017

Background: Bangladesh has experienced repeated house crow (Corvus splendens) mortality events due to highly pathogenic avian influenza (HPAI) viruses since 2011. We investigated one such event in Dhaka in 2017 to detect and molecular characterize the virus in crows and poultry from nearby live poultry markets (LPMs). Methods and materials: We collected cloacal and oropharyngeal swab samples from dead and moribund crows (N = 166) and environmental samples (N = 149) from 19 nearby LPMs. Samples were tested by qRT-PCR for influenza A (M gene) and H5/H7/H9/N1/N6. H5 positive samples were inoculated into embryonated eggs for virus culture. We performed conventional RT-PCR and Sanger sequencing of the isolated viruses and used MEGA7 for phylogenetic analysis. Results: 91.6% (n = 152; 95%Confidence Interval (CI): 86.3–95.3) of crows were positive with influenza A/H5 including 77.6% for H5N1 (n = 118), 2% for H5N6 (n = 3) and 20.4% H5Nx (n = 31). Of the 149 LPM samples, 46.3% (n = 69; 95%CI: 38.1–54.7) were positive for influenza A/H5 among which 85.5% for H5N1, 5.8% for H5N6 and 9% for H5Nx. We successfully isolated 2 H5N6; 1 from chicken and 1 from crow. Here we describe molecular characteristics of H5N6 only. Phylogeny of the HA gene showed 99.0% nucleotide similarity with the H5N8 virus belonging to clade 2.3.4.4B, isolated from Muscovy duck in Congo, 2017. NA gene showed 98.4% similarity with that of H4N6, isolated from duck in Mongolia, 2015. The PB1 and PA gene had high nucleotide identity, 98.0% and 99.0% respectively, with an H3N1 isolated from duck in Bangladesh, 2017. NP, M and NS genes of the two H5N6 isolates are closely related to H5N8. The isolates were identified as HPAIV based on multiple basic amino acids at the HA proteolytic cleavage site. Molecular markers in the HA protein suggested predicted binding to human receptor α2,6 linkage and increased pandemic potential. Conclusion: This investigation detected and characterized a novel H5N6 reassortant in wild birds for the first time and highlighted the potential for the exchange of HPAI between poultry and wild birds. Continued surveillance of both poultry and wild birds is needed for early detection of novel viral introductions, to trace the transmission route and to reduce public health risks.

Virulence and transmissibility of highly pathogenic avian influenza H5N6 viruses in the ferret model

Background: Highly pathogenic avian influenza (HPAI) H5 viruses have exhibited substantial geographic spread throughout Asia, Europe, Africa, and recently North America since their first detection in 1996. Accumulation of mutations in the HA gene has resulted in several phylogenetic clades, while reassortment with other avian influenza viruses has led to the emergence of new virus subtypes (H5Nx), notably H5N2, H5N6, and H5N8. H5Nx viruses represent a threat to both the poultry industry and human health. As reassortment and accumulation of mammalian adaptations may increase virus fitness and virulence, continuous in vivo evaluation of H5Nx viruses is necessary in aiding pandemic preparedness efforts. Methods and materials: Ferrets were inoculated with HPAI H5N6 virus (A/Sichuan/26221/2014, A/Yunnan/14563/2015, or A/duck/Bangladesh/19D770/2017) and evaluated for clinical signs and symptoms of infection, replication throughout the respiratory tract, extra pulmonary virus spread, and virus transmission. A North American HPAI H5N2 virus (A/turkey/Minnesota/10915/2015) was used for comparison. Levels of virus released into the air by infected ferrets was evaluated using a cyclone bioaerosol sampler. Replication kinetics of all viruses in the human bronchial epithelial cell line, Calu-3, was evaluated. Results: Tested H5N6 viruses were more pathogenic in ferrets compared to the H5N2 virus. However, pathogenicity profiles of the H5N6 viruses varied by strain, from a mild infection with only sporadic virus dissemination beyond the respiratory tract, to a severe and fatal infection. In vitro evaluation of H5Nx virus replication in Calu-3 cells and the presence of mammalian adaptation markers in key genes supported these findings. Limited transmission between co-housed ferrets was observed with the H5N6 viruses but not the H5N2 virus, which is in agreement with generally low levels of aerosolized virus exhaled from virus-inoculated ferrets. Conclusion: HPAI H5 viruses bearing the N6 subtype neuraminidase and mammalian adaptation markers in the polymerase genes are capable of causing lethal infection in ferrets as well as transmitting to uninfected animals in a direct contact setting. The heterogeneity in mammalian virulence among H5N6 viruses underscores the necessity of in vivo evaluation of H5Nx viruses as they continue to evolve and pose a threat to human health.

A single dose of inactivated oil-emulsion bivalent H5N8/H5N1 vaccine protects chickens against the lethal challenge of both highly pathogenic avian influenza viruses

In this study, two highly pathogenic avian influenza (HPAI) H5N8 viruses were isolated from chicken and geese in 2018 and 2019 (Chicken/ME-2018 and Geese/Egypt/MG4/2019). The hemagglutinin and neuraminidase gene analyses revealed their close relatedness to the clade-2.3.4.4b H5N8 viruses isolated from Egypt and Eurasian countries. A monovalent inactivated oil-emulsion vaccine containing a reassortant virus with HA gene of the Chicken/ME-2018/H5N8 strain and a bivalent vaccine containing same reassortant virus plus a previously generated reassortant H5N1 strain (CK/Eg/RG-173CAL/17). The safety of both vaccines was evaluated in specific-pathogen-free (SPF) chickens. To evaluate the efficacy of the prepared vaccines, 2-week-old SPF chickens were vaccinated with 0.5 mL of a vaccine formula containing 108/EID50 /dose from each strain via the subcutaneous route. Vaccinated birds were challenged with either wild-type HPAI-H5N8 or H5N1 viruses separately at 3 weeks post-vaccine. Results revealed that both vaccines induced protective hemagglutination-inhibiting (HI) antibody titers as early as 2 weeks PV (≥5.0 log2). Vaccinated birds were protected clinically against both subtypes (100 % protection). HPAI-H5N1 virus shedding was significantly reduced in birds that were vaccinated with the bivalent vaccine; meanwhile, HPAI-H5N8 virus shedding was completely neutralized in both tracheal and cloacal swabs after 3 days post-infection in birds that had been vaccinated with either vaccine. In conclusion, the developed bivalent vaccine proved to be efficient in protecting chickens clinically and reduced virus shedding via the respiratory and digestive tracts. The applicability of the multivalent avian influenza vaccines further supported their value to facilitate vaccination programs in endemic countries.

Phylogenetic analysis and clinical characteristics of the co-occurring mutations in HA and NA genes of influenza A(H1N1)pdm09 viruses during 2015\u20132017 in Beijing, China

BackgroundInfluenza A(H1N1)pdm09 viruses have undergone rapid evolution, and in recent years the complementary and antagonistic effects of HA and NA have gathered more attentions; however, the effects of co-occurring mutations in HA and NA on the patients’ clinical characteristics are still poorly understood. In this study, we analyzed molecular epidemiology and evolution of A(H1N1) pdm09, explored co-occurring mutations of HA and NA, and investigated effect of co-occurring mutations on patients’ clinical features.MethodsA(H1N1)pdm09 was confirmed by reverse transcription-polymerase chain reaction. HA and NA genes were sequenced and phylogenetically analyzed. Clinical characteristics of the co-occurring mutations were analyzed statistically.ResultsBy analyzing the HA and NA gene sequences of 33 A(H1N1)pdm09 viruses during the 2015–2017 influenza season, we found that all the viruses shared high similarities to each other and the HA genes of these viruses exclusively belonged to subclade 6B.1A. Several unreported substitutions in HA and NA proteins were observed, furthermore, co-occurring mutations of HA-V169T, A278S, E508G, D518E and NA-V67I were detected in 30.3% (10/33) A(H1N1)pdm09 virus strains when comparing with vaccine strains A/California/07/2009 and A/Michigan/45/2015 (H1N1). Sore throat was significantly associated with co-occurring mutations in HA and NA of A(H1N1)pdm09 (χ2, P < 0.05).ConclusionsCo-occurring mutations in HA and NA were detected in A(H1N1)pdm09 isolated during 2015–2017 in Beijing. Symptomatically, sore throat was associated with co-occurring mutations in HA and NA of A(H1N1)pdm09. Therefore, studying the effect and mechanism of co-occurring mutations in HA and NA on patients’ clinical features is of note needed.

Molecular characteristics of H9N2 influenza viruses isolated from farmed raccoon dogs and arctic foxes in China

In this study, eight H9N2 IAVs were isolated from infected diseased, farmed raccoon dogs and arctic foxes. Eight genes shared 98.6%–100% identity among the isolates possessing a PSRSSR/GL motif at the HA cleavage site, which is same as the motif of G1 and Y280 lineages of H9N2 IAVs. The phylogenetic analysis showed that the HA genes of the eight isolates clustered with Y280-like viruses, whereas the NA genes belonged to F/98-like sublineage. Interestingly, the NS, NP, PB2 and PA genes of the isolates were closely related to H7N9 IAVs. This is the first evidence for isolation of H9N2 IAVs from raccoon dogs and arctic foxes. Raccoon dogs and arctic foxes potentially serve as an intermediate host for influenza viruses with pandemic potential toward other animals due to co-expression of both SA α-2,6-Gal and SA α-2,3-Gal receptors in a wide range of their tissues.

Prevalence of Newcastle Disease Virus and Avian Influenza Virus (H7N3) in Poultry at Karachi

Background: Poultry is largest and rapidly growing sector of livestock in Pakistan. It is mainly influenced by viral pathogens such as Newcastle disease virus (NDV) and Avian influenza virus (H7N3). These viruses cause severe disease in poultry and leads to heavy economic losses throughout the world. The outbreaks of these pathogens have been increased in last few decades. Therefore, the study about antigenic prevalence is needed to know about the emergence of these pathogenic viruses, and to get rid of severe ailments associated with reduced poultry production.&#x0D; Objectives: To determine the prevalence of Newcastle disease virus (NDV), Avian influenza virus (H7N3) and co-infections in poultry flocks at Karachi.&#x0D; Methodology: For detection of NDV and H7N3, a total of 200 tracheal swabs were collected and tested through Virus Isolation (V.I); the sample with positive virus isolation were tested through Agar gel precipitation (AGP) and then the RNA was isolated through TRI Reagent, which was further tested through Reverse transcription polymerase chain reaction (RT-PCR).&#x0D; Results: The virus isolation showed that 58% of samples were positive for various viruses. Agar gel precipitation (AGP) revealed that the occurrence of NDV, H7N3 and ND+H7 were 50%, 8% and 38%, respectively. RT-PCR for F and HA gene of NDV and H7N3 confirmed the presence of NDV and H7N3 in the poultry.&#x0D; Conclusion: It is concluded that NDV and H7N3 are circulating in the flocks causing co-infections, therefore it is important to know the field challenge of viruses and to prepare vaccine of circulating serotype of virus to mitigate the rate of infection.

Determining Equine Influenza Virus Vaccine Efficacy-The Specific Contribution of Strain Versus Other Vaccine Attributes.

Vaccination is an effective tool to limit equine influenza virus (EIV H3N8) infection, a contagious respiratory disease with potentially huge economic impact. The study assessed the effects of antigenic change on vaccine efficacy and the need for strain update. Horses were vaccinated (V1 and V2) with an ISCOMatrix-adjuvanted, whole inactivated virus vaccine (Equilis Prequenza, group 2, FC1 and European strains) or a carbomer-adjuvanted, modified vector vaccine (ProteqFlu, group 3, FC1 and FC2 HA genes). Serology (SRH, HI, VN), clinical signs and viral shedding were assessed in comparison to unvaccinated control horses. The hypothesis was that group 2 (no FC2 vaccine strain) would be less well protected than group 3 following experimental infection with a recent FC2 field strain (A/equi-2/Wexford/14) 4.5 months after vaccination. All vaccinated horses had antibody titres to FC1 and FC2. After challenge, serology increased more markedly in group 3 than in group 2. Vaccinated horses had significantly lower total clinical scores and viral shedding. Unexpectedly, viral RNA shedding was significantly lower in group 2 than in group 3. Vaccination induced protective antibody titres to FC1 and FC2 and reduced clinical signs and viral shedding. The two tested vaccines provided equivalent protection against a recent FC2 EIV field strain.

Amino acid replacements of the polymorphic locuses of the hemaglutinin, neuraminidasa and nucleoprotein genes of the influenza virus A strains H1N1 and H7N9

Aim. Significant variability contributing to the formation of highly virulent variants, widespread distribution in natural reservoirs, the ability to overcome interspecific barriers and the ease of the airborne pathway contribute to the development of epidemics and pandemics of respiratory infections. Determination of polymorphic sites and type of polymorphism of the investigated virus subtype gene sequences will allow to determine amino acid substitutions and to establish RNA targets for genotyping. Methods. The analysis was performed on the nucleotide sequences of genes HA, NA and NP of strains H1N1 and H7N9 of avian influenza A virus obtained from the NCBI base. Amino acid sequences of genes were obtained by translation of nucleotide gene sequences using MEGA 6. Polymorphic loci and cluster analysis were performed using MEGA 6 using the ClustalW algorithm.Results. The nucleotide sequences of HA, NA and NP genes were investigated, as well as their translation products inavian influenza A strains H1N1 and H7N9. The polymorphic regions and type of polymorphism of the sequences were determined. Conclusions. Polymorphism regions were detected on the nucleotide sequences of the HA, NA, and NP genes (H1N1 and H7N9 strains) and their translation products. The polymorphism of the HA, NA, and NP genes (strain H1N1) is shown to be higher than the polymorphism of HA, NA, and NP (strain H7N9) due to the presence in the first significant number of synonymous triplets.&#x0D; Keywords: influenza A virus, hemagglutinin, neuraminidase, nucleoprotein, polymorphism.

Novel Protein Sequence Comparison Method Based on Transition Probability Graph and Information Entropy.

Aim and Objective: Sequence analysis is one of the foundations in bioinformatics. It is widely used to find out the feature metrics hidden in the sequence. Otherwise, the graphical representation of the biologic sequence is an important tool for sequencing analysis. This study is undertaken to find out a new graphical representation of biosequences. The transition probability is used to describe amino acid combinations of protein sequences. The combinations are composed of amino acids directly adjacent to each other or separated by multiple amino acids. The transition probability graph is built up by the transition probabilities of amino acid combinations. Next, a map is defined as a representation from the transition probability graph to transition probability vector by the k-order transition probability graph. Transition entropy vectors are developed by the transition probability vector and information entropy. Finally, the proposed method is applied to two separate applications, 499 HA genes of H1N1, and 95 coronaviruses. By constructing a phylogenetic tree, it was found that the results of each application are consistent with other studies. The graphical representation proposed in this article is a practical and correct method.

Application of a Biologically Contained Reporter System To Study Gain-of-Function H5N1 Influenza A Viruses with Pandemic Potential.

Natural adaptation of an antigenically novel avian influenza A virus (IAV) to be transmitted efficiently in humans has the potential to trigger a devastating pandemic. Understanding viral genetic determinants underlying adaptation is therefore critical for pandemic preparedness, as the knowledge gained enhances surveillance and eradication efforts, prepandemic vaccine design, and efficacy assessment of antivirals. However, this work has risks, as making gain-of-function substitutions in fully infectious IAVs may create a pathogen with pandemic potential. Thus, such experiments must be tightly controlled through physical and biological risk mitigation strategies. Here, we applied a previously described biological containment system for IAVs to a 2009 pandemic H1N1 strain and a highly pathogenic H5N1 strain. The system relies on deletion of the essential viral hemagglutinin (HA) gene, which is instead provided in trans, thereby restricting multicycle virus replication to genetically modified HA-complementing cells. In place of HA, a Renilla luciferase gene is inserted within the viral genome, and a live-cell luciferase substrate allows real-time quantitative monitoring of viral replication kinetics with a high dynamic range. We demonstrate that biologically contained IAV-like particles exhibit wild-type sensitivities to approved antivirals, including oseltamivir, zanamivir, and baloxavir. Furthermore, the inability of these IAV-like particles to genetically acquire the host-encoded HA allowed us to introduce gain-of-function substitutions in the H5 HA gene that promote mammalian transmissibility. Biologically contained "transmissible" H5N1 IAV-like particles exhibited wild-type sensitivities to approved antivirals, to the fusion inhibitor S20, and to neutralization by existing H5 monoclonal and polyclonal sera. This work represents a proof of principle that biologically contained IAV systems can be used to safely conduct selected gain-of-function experiments.IMPORTANCE Understanding how animal influenza viruses can adapt to spread in humans is critical to prepare for, and prevent, new pandemics. However, working safely with pathogens that have pandemic potential requires tight regulation and the use of high-level physical and biological risk mitigation strategies to stop accidental loss of containment. Here, we used a biological containment system for influenza viruses to study strains with pandemic potential. The system relies on deletion of the essential HA gene from the viral genome and its provision by a genetically modified cell line, to which virus propagation is therefore restricted. We show that this method permits safe handling of these pathogens, including gain-of-function variants, without the risk of generating fully infectious viruses. Furthermore, we demonstrate that this system can be used to assess virus sensitivity to both approved and experimental drugs, as well as the antigenic profile of viruses, important considerations for evaluating prepandemic vaccine and antiviral strategies.

Analysis on genetic characteristics of H9N2 avian influenza virus isolated from human infection and external environment in Gansu province

Objective: To analyze the genomic characteristics of human infection with H9N2 avian influenza virus in Gansu province. Methods: The etiological analysis was conducted for human infection with H9N2 avian influenza virus detected in influenza like illness cases in northwestern China in 2016. Molecular bioinformatics Mega 7.0 software was used to analyze the full genomic sequences of the viral isolate. Results: The gene fragments of HA, NA, MP, NP, NS, PA, PB1 and PB2 of the isolate were highly similar (>90%) to those of H9N2 avian influenza virus strain isolated in external environment in Gansu from 2014 to 2019. The HA gene belonged to BJ/94-like branch, PB2 and MP belonged to G1/97-like branch, and the PB1, PA, NS, and NP genes belonged to F/98-like branch. MP and PB2 were closely related to H7N9, H10N8 and H5N6 viruses. Amino acid sequence alignment showed that the HA cleavage site was arranged in PSRSSR ↓ GLF, H183N and Q226L mutated which included 7 HA glycosylated sites; 62-64 sites of NA absented 3 amino acids (ITE); and M2-31N, NS1-42S, PA-356R, and PA-409N mutated. Conclusions: Apparently, this case of human infection with human infection with H9N2 avian influenza virus was an incidental. However, the isolates of H9N2 influenza virus in external environment of Gansu had a series of mammalian adaptive molecular markers, suggesting that the risk of human infection is higher. It is necessary to strengthen the surveillance by multi departments to deal with influenza pandemic.