Influenza Virus H5N1 Research Articles

Designing a novel multi-epitopes pan-vaccine against SARS-CoV-2 and seasonal influenza: in silico and immunoinformatics approach

The merger of COVID-19 and seasonal influenza infections is considered a potentially serious threat to public health. These two viral agents can cause extensive and severe lower and upper respiratory tract infections with lung damage with host factors. Today, the development of vaccination has been shown to reduce the risk of hospitalization and mortality from the COVID-19 virus and influenza epidemics. Therefore, this study contributes to an immunoinformatics approach to producing a vaccine that can elicit strong and specific immune responses against COVID-19 and influenza A and B viruses. The NCBI, GISAID, and Uniprot databases were used to retrieve sequences. Linear B cell, Cytotoxic T lymphocyte, and Helper T lymphocyte epitopes were predicted using the online servers. Population coverage of MHC I epitopes worldwide for SARS-CoV-2, Influenza virus H3N2, H3N2, and Yamagata/Victoria were 99.93%, 68.67%, 68.38%, and 85.45%, respectively. Candidate epitopes were linked by GGGGS, GPGPG, and KK linkers. Different epitopes were permutated several times to form different peptides and then screened for antigenicity, allergenicity, and toxicity. The vaccine construct was analyzed for physicochemical properties, conformational B-cell epitopes, interaction with Toll-like receptors, and IFN-gamma-induced. Immune stimulation response of final construct was evaluated using C-IMMSIM. Eventually, the final construct sequence was codon-optimized for Escherichia coli K12 and Homo sapiens to design a multi-epitope vaccine and mRNA vaccine. In conclusion, due to the variable nature of SARS-CoV-2 and influenza proteins, the design of a multi-epitope vaccine can protect against all their standard variants, but laboratory validation is required. Communicated by Ramaswamy H. Sarma

Hylin-a1: A Pan-Inhibitor against Emerging and Re-Emerging Respiratory Viruses.

Pandemic and epidemic outbreaks of respiratory viruses are a challenge for public health and social care system worldwide, leading to high mortality and morbidity among the human populations. In light of the limited efficacy of current vaccines and antiviral drugs against respiratory viral infections and the emergence and re-emergence of new viruses, novel broad-spectrum antiviral drugs are needed for the prevention and treatment of these infections. Antimicrobial peptides with an antiviral effect, also known as AVPs, have already been reported as potent inhibitors of viral infections by affecting different stages of the virus lifecycle. In the present study, we analyzed the activity of the AVP Hylin-a1, secreted by the frog Hypsiboas albopunctatus, against a wide range of respiratory viruses, including the coronaviruses HCoV-229E and SARS-CoV-2, measles virus, human parainfluenza virus type 3, and influenza virus H1N1. We report a significant inhibitory effect on infectivity in all the enveloped viruses, whereas there was a lack of activity against the naked coxsackievirus B3. Considering the enormous therapeutic potential of Hylin-a1, further experiments are required to elucidate its mechanism of action and to increase its stability by modifying the native sequence.

Lactiplantibacillus pentoses CCFM1227 Produces Desaminotyrosine to Protect against Influenza Virus H1N1 Infection through the Type I Interferon in Mice.

Microbiota-derived desaminotyrosine (DAT) protects the host from influenza by modulating the type I interferon (IFN) response. The aim of this study was to investigate the antivirus effects of a DAT-producing bacteria strain. A comparative genomics analysis and UHPLC Q-Exactive MS were used to search for potential strains and confirm their ability to produce DAT, respectively. The anti-influenza functions of the DAT producer were evaluated using an antibiotic-treated mouse model by orally administering the specific strain before viral infection. The results showed the Lactiplantibacillus pentosus CCFM1227 contained the phy gene and produced DAT by degrading phloretin. In vivo, L. pentosus CCFM1227 re-inoculation increased the DAT level in feces, and protected from influenza through inhibiting viral replication and alleviating lung immunopathology. Furthermore, CCFM1227-derived DAT was positively correlated with the IFN-β level in the lung. The transcriptome results showed that CCFM1227 activated gene expression in the context of the defense response to the virus, and the response to interferon-beta. Moreover, CCFM1227 treatment upregulated the expression of MHC-I family genes, which regulate the adaptive immune response. In conclusion, L. pentosus CCFM1227 exerted antiviral effects by producing DAT in the gut, and this may provide a potential solution for creating effective antiviral probiotics.

Naturally occurring highly pathogenic avian influenza virus H5N1 clade 2.3.4.4b infection in three domestic cats in North America during 2023

The Eurasian strain of highly pathogenic avian influenza (HPAI) H5N1 is a devastating pathogen for birds that also has the capacity to infect mammals. This report describes the presentation, clinical case findings (including haemogram and serum biochemistry), gross and microscopic lesions and virus detection in three HPAI H5N1-infected domestic cats from the USA in 2023. All three cats presented with neurological abnormalities and were euthanized due to a poor prognosis within 2 days (two cats) or 10 days (one cat) of known clinical disease onset. Necropsy consistently revealed pulmonary congestion and oedema, and cerebrocortical malacia with haemorrhage was also seen in the cat that survived for 10 days. On histology, all cats had necrotizing encephalitis and interstitial pneumonia with pulmonary congestion, oedema, vasculitis and vascular thrombosis. One cat also had microscopic multifocal necrosis in the liver, pancreas and an adrenal gland. To our knowledge, this report is the first to detail pathological findings in HPAI H5N1 naturally-infected cats during the widespread outbreak in North America beginning in 2021, and that describes a cat surviving for 10 days after onset of HPAI H5N1 encephalitis.

Low Susceptibility of Pigs against Experimental Infection with HPAI Virus H5N1 Clade 2.3.4.4b.

We found that nasal and alimentary experimental exposure of pigs to highly pathogenic avian influenza virus H5N1 clade 2.3.4.4b was associated with marginal viral replication, without inducing any clinical manifestation or pathological changes. Only 1 of 8 pigs seroconverted, pointing to high resistance of pigs to clade 2.3.4.4b infection.

Inhibition of Cyclin-Dependent Kinases 8/19 Restricts Bacterial and Virus-Induced Inflammatory Responses in Monocytes.

Hyperactivation of the immune system remains a dramatic, life-threatening complication of viral and bacterial infections, particularly during pneumonia. Therapeutic approaches to counteract local and systemic outbreaks of cytokine storm and to prevent tissue damage remain limited. Cyclin-dependent kinases 8 and 19 (CDK8/19) potentiate transcriptional responses to the altered microenvironment, but CDK8/19 potential in immunoregulation is not fully understood. In the present study, we investigated how a selective CDK8/19 inhibitor, Senexin B, impacts the immunogenic profiles of monocytic cells stimulated using influenza virus H1N1 or bacterial lipopolysaccharides. Senexin B was able to prevent the induction of gene expression of proinflammatory cytokines in THP1 and U937 cell lines and in human peripheral blood-derived mononuclear cells. Moreover, Senexin B substantially reduced functional manifestations of inflammation, including clustering and chemokine-dependent migration of THP1 monocytes and human pulmonary fibroblasts (HPF).

Rapid and reliable ultrasensitive detection of pathogenic H9N2 viruses through virus-binding phage nanofibers decorated with gold nanoparticles

The rapid and sensitive detection of pathogenic viruses is important for controlling pandemics. Herein, a rapid, ultrasensitive, optical biosensing scheme was developed to detect avian influenza virus H9N2 using a genetically engineered filamentous M13 phage probe. The M13 phage was genetically engineered to bear an H9N2-binding peptide (H9N2BP) at the tip and a gold nanoparticle (AuNP)-binding peptide (AuBP) on the sidewall to form an engineered phage nanofiber, M13@H9N2BP@AuBP. Simulated modelling showed that M13@H9N2BP@AuBP enabled a 40-fold enhancement of the electric field enhancement in surface plasmon resonance (SPR) compared to conventional AuNPs. Experimentally, this signal enhancement scheme was employed for detecting H9N2 particles with a sensitivity down to 6.3 copies/mL (1.04 × 10−5 fM). The phage-based SPR scheme can detect H9N2 viruses in real allantoic samples within 10 min, even at very low concentrations beyond the detection limit of quantitative polymerase chain reaction (qPCR). Moreover, after capturing the H9N2 viruses on the sensor chip, the H9N2-binding phage nanofibers can be quantitatively converted into plaques that are visible to the naked eye for further quantification, thereby allowing us to enumerate the H9N2 virus particles through a second mode to cross-validate the SPR results. This novel phage-based biosensing strategy can be employed to detect other pathogens because the H9N2-binding peptides can be easily switched with other pathogen-binding peptides using phage display technology.

Host-targeted self-attenuated influenza virus as an effective nasal spray vaccine in aged population

Abstract The elderly are at high risk for influenza infection and its complication. However, current inactivated influenza vaccines are often modestly immunogenic and show low protective immunity in the elderly, due to immunosenescence. New approaches are urgently needed for developing effective vaccines against influenza for the elderly. In current study, we have constructed new recombinant influenza viruses, named host-targeted self-attenuated influenza viruses (SAIVs), which can express functional mammalian species-specific artificial microRNAs (amiRNAs). The expression of these amiRNAs can inhibit expression of some host factors critical for influenza replication, and therefore the resultant recombinant influenza viruses are replication restricted and attenuated in the host cells. One of these SAIVs, which can express an amiRNA that inhibits expression of the host cellular Cdc2-like kinase 1 (CLK1), was produced in embryonic chicken eggs and evaluated in an aged mouse model of influenza infection. It elicited robust antibody and T cell responses against influenza virus and demonstrated significantly protective efficacy against lethal infection with wild type influenza virus H1N1 PR8 after single dose of intranasal vaccination. Our research finding provided a proof of concept that the new host targeted self-attenuated influenza virus can be further used as an effective vaccine against influenza in aged population. Supported by a research grant from National Institute of Allergy and Infectious Diseases (AI133207).

The changing dynamics of highly pathogenic avian influenza H5N1: Next steps for management & science in North America

Highly pathogenic avian influenza virus (HPAIV) H5N1 was introduced in North America in late 2021 through trans-Atlantic pathways via migratory birds. These introductions have resulted in an unprecedented epizootic, a widespread disease event in animals, heavily affecting poultry, wild birds, and recently mammals. The North American incursions occurred during the largest epidemic season (2021–2022) in Europe where H5N1 may now be endemic (i.e., continuously present). The continuing outbreak includes expansion into Mexico, Central and South America beginning in late 2022. Here, we provide an overview of the Eurasian origin H5N1 introduction to the Americas, including a significant shift in virus dynamics and severe disease in wild birds. Then, to investigate the global changes in confirmed detections in wild birds and poultry across time and geographic regions, we analyzed FAO's EMPRES-i + database. To examine the 2021 introduction and spread in North American wild birds and poultry, we collated publicly available data across USA and Canadian federal sources. Based on our assessment, the unique magnitude of the North American H5N1 spread indicates the need for effective decision framing to prioritize management needs and scientific inquiry, particularly for species at risk and interface areas for wildlife, poultry, and humans. We illustrate the rapidly occurring and likely increasing detrimental effects that this One Health issue has on wildlife, agriculture, and potentially human health, and we offer a reframing of HPAIV disease response towards a decision analytical context to guide scientific prioritization as a potentially valuable change in focus.

Detecting the Neuraminidase R294K Mutation in Avian Influenza A (H7N9) Virus Using Reverse Transcription Droplet Digital PCR Method.

The R294K mutation in neuraminidase (NA) causes resistance to oseltamivir in the avian influenza virus H7N9. Reverse transcription droplet digital polymerase chain reaction (RT-dd PCR) is a novel technique for detecting single-nucleotide polymorphisms. This study aimed to develop an RT-dd PCR method for detecting the R294K mutation in H7N9. Primers and dual probes were designed using the H7N9 NA gene and the annealing temperature was optimized at 58.0 °C. The sensitivity of our RT-dd PCR method was not significantly different from that of RT-qPCR (p = 0.625), but it could specifically detect R294 and 294K in H7N9. Among 89 clinical samples, 2 showed the R294K mutation. These two strains were evaluated using a neuraminidase inhibition test, which revealed that their sensitivity to oseltamivir was greatly reduced. The sensitivity and specificity of RT-dd PCR were similar to those of RT-qPCR and its accuracy was comparable to that of NGS. The RT-dd PCR method had the advantages of absolute quantitation, eliminating the need for a calibration standard curve, and being simpler in both experimental operation and result interpretation than NGS. Therefore, this RT-dd PCR method can be used to quantitatively detect the R294K mutation in H7N9.

Environmental Samples Test Negative for Avian Influenza Virus H5N1 Four Months after Mass Mortality at A Seabird Colony.

High pathogenicity avian influenza (HPAI) profoundly impacted several seabird populations during the summers of 2021 and 2022. Infection spread rapidly across colonies, causing unprecedented mortality. At Foula, Shetland, 1500 breeding adult great skuas Stercorarius skua, totalling about two tonnes of decomposing virus-laden material, died at the colony in May-July 2022. Carcasses were left where they died as Government policy was not to remove dead birds. The factors influencing risk of further spread of infection are uncertain, but evidence suggests that HPAI can persist in water for many months in cool conditions and may be a major transmission factor for birds living in wetlands. We investigated risk of further spread of infection from water samples collected from under 45 decomposing carcasses and in three freshwater lochs/streams by sampling water in October 2022, by which time the great skua carcasses had rotted to bones, skin, and feathers. No viral genetic material was detected four months after the mortality, suggesting a low risk of seabird infection from the local environment when the seabirds would return the next breeding season. These findings, although based on a relatively small number of water samples, suggest that the high rainfall typical at Shetland probably washed away the virus from the decomposing carcasses. However, limitations to our study need to be taken on board in the design of environmental monitoring at seabird colonies during and immediately after future outbreaks of HPAI.

Evaluation of the antiviral potential of gemini surfactants against influenza virus H1N1

Influenza A virus (IAV) affects human health worldwide as a high-risk disease. It can neither be easily controlled by current vaccines and nor be treated by conventional drugs. Gemini surfactants (GS) have shown several properties including antiviral activity. In this study, the antiviral capacity of some GS compounds with different levels of hydrophobicity was examined. The 50% cytotoxic (CC50) and non-cytotoxic (NCTC) concentrations of the compounds were determined by MTT method. The NCTCs, the same as effective concentrations (EC50s), were tested for the antiviral capacity against IAV in different combination treatments for 1h incubation on MDCK cells. The HA and MTT assays were used to evaluate the virus titer and cell viabilities, respectively. The hemolytic activity of the compounds was also assessed using an HA inhibition assay. To evaluate the apoptotic effect of GS compounds, Annexin V-PI kit was used. The HA titers decreased between 1-6.5 logs, 1-4.5 logs, and 1-5.5 logs in simultaneous, pre- and post-penetration combination treatments, respectively. The cell viability values in all combination treatments were favorable. The HI assay indicated the hemolytic potential of GSs and their physical interaction with viral HA. The apoptosis test results highlighted anti-apoptotic capacity of the GS compounds alone and in the presence of influenza virus especially for the hydrophobic ones. Gemini surfactants were generally more efficacious in simultaneous treatment. Their antiviral potential may be attributed to their physical interaction with viral membrane or HA glycoprotein that disrupts viral particle or blocks viral entry to the cell and inhibits its propagation.

The beneficial effects of spraying of probiotic Bacillus and Lactobacillus bacteria on broiler chickens experimentally infected with avian influenza virus H9N2

This study investigated the clinical, antiviral, and immunological effects of spraying Bacillusspp. andLactobacillus spp. as a single or mixture probiotic compound on experimentally infected broiler chickens with AIV H9N2. Two hundred and forty 1-day-old broilers were randomly assigned to 6 groups as follows: Ctrl- (no challenge AIV; no spray probiotic), Ctrl+ (AIV challenged; no spray probiotic), AI+B (AIV challenged; daily spraying of probiotic Bacillusspp.), AI+L group (AIV challenged; daily spraying of probioticLactobacillusspp.), AIV+BL (AIV challenged; daily spraying of probioticBacillusspp. andLactobacillusspp.), and G-DW (daily spraying of normal saline; no AIV challenged). The birds were reared for 35 d. On the 22nd day of age, broiler chickens were challenged by AIV H9N2. The probiotics were sprayed at 9×109 CFU/m2 daily for 35 d. Growth performance, clinical signs, virus shedding, macroscopic and microscopic lesions were evaluated at various days in all groups. Spraying with probiotics improved the body weight gain and food conversion ratio in the AI+B, AI+L, and AI+BL groups compared to the Ctrl+. The severity of clinical signs, gross lesions, pathological lesions and viral shedding in the probiotic treatment groups was lower than in the Ctrl+ group. The findings of this study suggest the daily spraying of Lactobacillus and Bacillus probiotics alone or in combination during the rearing period reduce clinical and nonclinical aspects of H9N2 virus infection; so, it can be effective as a preventive protocol for controlling the severity of AIV H9N2 infection in broilers.

Human death due to H5N1 amid the COVID-19 pandemic and Mpox outbreak: A call for action.

Human death due to H5N1 amid the COVID-19 pandemic and Mpox outbreak: A call for action.

Whole-genome sequence and genesis of an avian influenza virus H5N1 isolated from a healthy chicken in a live bird market in Indonesia: accumulation of mammalian adaptation markers in avian hosts.

Influenza A viruses are a major pathogen that causes significant clinical and economic harm to many animals. In Indonesia, the highly pathogenic avian influenza (HPAI) H5N1 virus has been endemic in poultry since 2003 and has caused sporadic deadly infections in humans. The genetic bases that determine host range have not yet been fully elucidated. We analyzed the whole-genome sequence of a recent H5 isolate to reveal the evolution toward its mammalian adaptation. We determined the whole-genome sequence of A/chicken/East Java/Av1955/2022 (hereafter, "Av1955") from a healthy chicken in April 2022 and conducted phylogenetic and mutational analysis. Phylogenetic analysis revealed that Av1955 belonged to the H5N1 clade 2.3.2.1c (Eurasian lineage). The six gene segments (PB1, PB2, HA, NP, NA, and NS) out of the eight segments derived from viruses of H5N1 Eurasian lineage, one (PB2) from the H3N6 subtype and the remaining one (M) from the H5N1 clade 2.1.3.2b (Indonesian lineage). The donor of the PB2 segment was a reassortant among three viruses of H5N1 Eurasian and Indonesian lineages and the H3N6 subtype. The HA amino acid sequence contained multiple basic amino acids at the cleavage site. Mutation analysis revealed that Av1955 possessed the maximal number of mammalian adaptation marker mutations. Av1955 was a virus of H5N1 Eurasian lineage. The HA protein contains an HPAI H5N1-type cleavage site sequence, while the virus was isolated from a healthy chicken suggesting its low pathogenicity nature. The virus has increased mammalian adaptation markers by mutation and intra- and inter-subtype reassortment, gathering gene segments possessing the most abundant maker mutations among previously circulating viruses. The increasing mammalian adaptation mutation in avian hosts suggests that they might be adaptive to infection in mammalian and avian hosts. It highlights the importance of genomic surveillance and adequate control measures for H5N1 infection in live poultry markets.

Incidence and population immunity to influenza and ARVI in the context of the COVID-19 pandemic

The pandemic of the new COVID-19 infection has had an impact on the epidemic process of most known infectious diseases. Given the high pandemic potential and the economic damage caused by influenza and SARS, the assessment of the prevalence of these infections in modern realities seems relevant. Aim: to assess the current manifestations of the epidemic process of SARS and influenza during the COVID-19 pandemic. Official statistics data were used to study the manifestations of epidemic processes of influenza and SARS. For serological examination, blood serum samples from conditionally healthy adults (N=270) collected in February 2022 were used. The titer of IgG antibodies to strains of influenza virus H1N1 pnm09, influenza H3N2, influenza B (Phuket), influenza B (Washington) was determined using a hemagglutination inhibition reaction. The samples were examined by enzyme immunoassay for the presence of IgG antibodies to adenovirus, respiratory syncytial (RS) virus, metapneumovirus, rhinovirus and parainfluenza viruses of types 1 and 3. Against the background of the development of the COVID-19 pandemic, an increase in the incidence of influenza has been established to a maximum value in 2021 (65.99 per 100 thousand population). The prevalence of influenza in the capital was 4.6 times higher than the average in Russia (14.96 per 100 thousand population). A significant proportion of persons immune to the influenza virus (more than 60 %) with a maximum value for the influenza B virus was identified, which is consistent with the data on the prevalence of this variant of the pathogen in the etiological interpretation of the incidence in 2021. Among the pathogens of ARVI, the maximum proportion of seropositive was detected to rhinovirus (77.8 %), which corresponds to the data on the greatest prevalence of this pathogen in the etiological structure of this group of infections. The active circulation of the influenza virus and the vaccination of the population became the basis for the formation of a high level of population immunity to the corresponding virus, which was shown by the results of the assessment of the prevalence of specific IgG antibodies.

Homo- and Heterosubtypic Immunity to Low Pathogenic Avian Influenza Virus Mitigates the Clinical Outcome of Infection with Highly Pathogenic Avian Influenza H5N8 Clade 2.3.4.4.b in Captive Mallards (Anas platyrhynchos).

In this study, we investigated the clinical response, viral shedding, transmissibility, pathologic lesions, and tropism of HPAIV Gs/Gd H5N8 subtype (clade 2.3.4.4b), following experimental infection of three groups of captive mallards (Anas platyrhynchos): (i) fully susceptible, (ii) pre-exposed to low pathogenic avian influenza virus (LPAIV) H5N1 subtype, and (iii) pre-exposed to LPAIV H3N8 subtype. Infection of naïve mallards with HPAIV H5N8 resulted in ~60% mortality, neurological signs, abundant shedding, and transmission to contact ducks, who also became sick and died. High amounts of viral RNA were found in all collected organs, with the highest RNA load recorded in the brain. The IHC examinations performed on tissues collected at 4 and 14 days post-infection (dpi) revealed tropism to nervous tissue, myocardium, respiratory epithelium, and hepatic and pancreatic cells. The mallards pre-exposed to LPAIV H5N1 and challenged with HPAIV H5N8 were asymptomatic and showed a significant reduction of viral RNA shedding, yet still sufficient to cause infection (but no disease) in the contact ducks. The AIV antigen was not detected in organs at 4 and 14 dpi, and microscopic lesions were mild and scarce. Similarly, mallards previously inoculated with LPAIV H3N8 remained healthy after challenge with HPAIV H5N8, but viral RNA was detected in large quantities in swabs and organs, particularly in the early phase of infection. However, in contrast to mallards from group I, the IHC staining yielded negative results at the selected timepoints. The virus was transmitted to contact birds, which remained symptomless but demonstrated low levels of viral RNA shedding and mild- to moderate tissue damage despite negative IHC staining. The results indicate that naïve mallards are highly susceptible to HPAIV H5N8 clade 2.3.4.4b and that homo- and heterosubtypic immunity to LPAIV can mitigate the clinical outcomes of infection.

Genetic Characterization and Pathogenesis of Avian Influenza Virus H3N8 Isolated from Chinese pond heron in China in 2021.

In October 2021, a wild bird-origin H3N8 influenza virus-A/Chinese pond heron/Jiangxi 5-1/2021 (H3N8)-was isolated from Chinese pond heron in China. Phylogenetic and molecular analyses were performed to characterize the genetic origin of the H3N8 strain. Phylogenetic analysis revealed that eight gene segments of this avian influenza virus H3N8 belong to Eurasian lineages. HA gene clustered with avian influenza viruses is circulating in poultry in southern China. The NA gene possibly originated from wild ducks in South Korea and has the highest homology (99.3%) with A/Wild duck/South Korea/KNU2020-104/2020 (H3N8), while other internal genes have a complex and wide range of origins. The HA cleavage site is PEKQTR↓GLF with one basic amino acid, Q226 and T228 at HA preferentially bind to the alpha-2,3-linked sialic acid receptor, non-deletion of the stalk region in the NA gene and no mutations at E627K and D701N of the PB2 protein, indicating that isolate A/Chinese pond heron/Jiangxi 5-1/2021 (H3N8) was a typical avian influenza with low pathogenicity. However, there are some mutations that may increase pathogenicity and transmission in mammals, such as N30D, T215A of M1 protein, and P42S of NS1 protein. In animal studies, A/Chinese pond heron/Jiangxi 5-1/2021 (H3N8) replicates inefficiently in the mouse lung and does not adapt well to the mammalian host. Overall, A/Chinese pond heron/Jiangxi 5-1/2021 (H3N8) is a novel wild bird-origin H3N8 influenza virus reassortant from influenza viruses of poultry and wild birds. This wild bird-origin avian influenza virus is associated with wild birds along the East Asian-Australasian flyway. Therefore, surveillance of avian influenza viruses in wild birds should be strengthened to assess their mutation and pandemic risk in advance.

Exosome-mediated delivery of gga-miR-20a-5p regulates immune response of chicken macrophages by targeting IFNGR2, MAPK1, MAP3K5, and MAP3K14.

This study aims to evaluate the target genes of gga-miR-20a-5p and the regulated immune responses in the chicken macrophage cell line, HD11, by the exosome-mediated delivery of miR-20a-5p. Exosomes were purified from the chicken macrophage cell line HD11. Then, mimic gga-miR-20p or negative control miRNA were internalized into HD11 exosomes. HD11 cells were transfected with gga-miR-20a-5p or negative control miRNA containing exosomes. After 44 h of transfection, cells were incubated with or without 5 μg/mL poly(I:C) for 4 h. Then, expression of target genes and cytokines was evaluated by quantitative real-time polymerase chain reaction. Using a luciferase reporter assay, we identified that gga-miR-20a-5p directly targeted interferon gamma receptor 2 (IFNGR2), mitogen-activated protein kinase 1 (MAPK1), mitogen-activated protein kinase kinase kinase 5 (MAP3K5), and mitogen-activated protein kinase kinase kinase 14 (MAP3K14). Moreover, the exosome-mediated delivery of gga-miR-20a-5p successfully repressed the expression of IFNGR2, MAPK1, MAP3K5, and MAP3K14 in HD11 cells. The expressions of interferon-stimulated genes (MX dynamin like GTPase 1 [MX1], eukaryotic translation initiation factor 2A [EIF2A], and oligoadenylate synthase-like [OASL]) and proinflammatory cytokines (interferon-gamma [IFNG], interleukin-1 beta [IL1B], and tumor necrosis factor-alpha [TNFA]) were also downregulated by exosomal miR-20a-5p. In addition, the proliferation of HD11 cells was increased by exosomal miR-20a-5p. The exosome-mediated delivery of gga-miR-20a-5p regulated immune responses by controlling the MAPK and apoptotic signaling pathways. Furthermore, we expected that exosomal miR-20a-5p could maintain immune homeostasis against highly pathogenic avian influenza virus H5N1 infection by regulating the expression of proinflammatory cytokines and cell death.

The role of the major viral pathogens in a respiratory disease outbreak of broiler flocks in Eastern Iran.

Infectious bronchitis virus (IBV), Newcastle disease virus (NDV), and avian influenza virus (AIV) H9N2 are major viral pathogens in broiler respiratory disease. Following a respiratory disease outbreak and economic losses in eastern Iran 2020-2021, we investigated the role of major viral pathogens and the implemented vaccination programs. Thirty-six respiratory disease affected broiler flocks in South Khorasan province were sampled, molecularly tested, and coinfections were investigated. The vaccination programs were obtained and the detected IBV were genotyped. IBV, virulent NDV, and AIV H9N2 were detected in twenty-five, seven, and seven flocks, respectively. IBV+AIV, IBV+NDV, and NDV+AIV coinfections were respectively detected in six, five, and one flocks. Most IBV infected flocks (84%) had been immunized with a live IBV-Mass vaccine. All NDV infected flocks and 14.2% of AIV infected flocks had been vaccinated. IBV genotyping showed a high prevalence of variant 2 (83.3%), followed by Mass-type (12.5%), and Q1-type (4.2%). Variant 2 IB viruses were widely distributed in the province and half of them were mostly similar to the ones that had been detected in northern neighboring province, Khorasan Razavi. Single infection with variant 2 IBV was a major cause of the respiratory disease outbreak in which use of the Mass vaccine was probably not effective. The high coverage and multiple doses of vaccination against Newcastle disease possibly had reduced the prevalence of NDV. Considering the regional origin of IBV strains, strong biosecurity measures should be implemented and vaccination programs using appropriate vaccine strains should be used.