Avian Influenza H5N1 Virus Research Articles

Analysis of a highly pathogenic avian influenza (H5N1) virus causing the first outbreak in domestic poultry in Bulgaria in January 2015

This study documents the clinical signs, necropsy findings and viral antigen distribution of the highly pathogenic avian influenza (HPAI) H5N1 virus infection in domestic poultry (a backyard farm) and the phylogenetic analysis of the virus. On January 29, 2015, an outbreak of HPAI H5N1 in domestic poultry was reported on a backyard farm in Bulgaria. Out of the twenty-two chickens with clinical signs, twenty died while the remaining two were destroyed. The morbidity was 100%, whereas the overall mortality and lethality were 90.91%. The clinical observations made were sudden death, high mortality, weakness, and recumbency. Although multisystemic lesions were observed occasionally, the main pathologic findings were observed in the nervous, circulatory, respiratory, and gastrointestinal systems. An influenza virus nucleocapsid protein was identified by an immunohistochemical analysis in all the analysed organs: brain 3/3, trachea 3/3, lung 3/3, intestine 3/3, heart 3/3, which confirmed the systemic infection. The phylogenetic analyses of the virus showed a close genetic relationship with the H5N1 viruses of Asian origin, isolated in 2012 and 2013, belonging to the clade 2.3.2.1c. The HA-gene genetically clusters with HPAI H5N1 viruses isolated from wild pelicans in Romania and Bulgaria, thereby demonstrating the link between wild and domestic birds in the epidemiology of avian influenza. The contact between the affected chickens and migrating water birds over Bulgaria’s territory was suspected as a reason for the outbreak in the backyard farm. In addition, the detection of the virus in wild bird populations in Bulgaria three days earlier strongly supports the hypothesis of migrating wild birds spreading HPAI H5N1.

Development and Assessment of Two Highly Pathogenic Avian Influenza (HPAI) H5N6 Candidate Vaccine Viruses for Pandemic Preparedness

ObjectiveIn China, 24 cases of human infection with highly pathogenic avian influenza (HPAI) H5N6 virus have been confirmed since the first confirmed case in 2014. Therefore, we developed and assessed two H5N6 candidate vaccine viruses (CVVs). MethodsIn accordance with the World Health Organization (WHO) recommendations, we constructed two reassortant viruses using reverse genetics (RG) technology to match the two different epidemic H5N6 viruses. We performed complete genome sequencing to determine the genetic stability. We assessed the growth ability of the studied viruses in MDCK cells and conducted a hemagglutination inhibition assay to analyze their antigenicity. Pathogenicity attenuation was also evaluated in vitro and in vivo. ResultsThe results showed that no mutations occurred in hemagglutinin or neuraminidase, and both CVVs retained their original antigenicity. The replication capacity of the two CVVs reached a level similar to that of A/Puerto Rico/8/34 in MDCK cells. The two CVVs showed low pathogenicity in vitro and in vivo, which are in line with the WHO requirements for CVVs. ConclusionWe obtained two genetically stable CVVs of HPAI H5N6 with high growth characteristics, which may aid in our preparedness for a potential H5N6 pandemic.

Haemagglutinin displayed on the surface of Lactococcus lactis confers broad cross-clade protection against different H5N1 viruses in chickens

BackgroundThe highly pathogenic avian influenza (HPAI) H5N1 virus poses a potential threat to the poultry industry. The currently available avian influenza H5N1 vaccines for poultry are clade-specific. Therefore, an effective vaccine for preventing and controlling H5N1 viruses belonging to different clades needs to be developed.ResultsRecombinant L. lactis/pNZ8148-Spax-HA was generated, and the influenza virus haemagglutinin (HA) protein of A/Vietnam/1203/2004 (H5N1) was displayed on the surface of Lactococcus lactis (L. lactis). Spax was used as an anchor protein. Chickens vaccinated orally with unadjuvanted L. lactis/pNZ8148-Spax-HA could produce significant humoral and mucosal responses and neutralizing activities against H5N1 viruses belonging to different clades. Importantly, unadjuvanted L. lactis/pNZ8148-Spax-HA conferred cross-clade protection against lethal challenge with different H5N1 viruses in the chicken model.ConclusionThis study provides insights into the cross-clade protection conferred by unadjuvanted L. lactis/pNZ8148-Spax-HA, and the results might help the establishment of a promising platform for the development of a safe and effective H5N1 cross-clade vaccine for poultry.

Comparison of the Effectiveness of Two Different Vaccination Regimes for Avian Influenza H9N2 in Broiler Chicken.

Simple SummaryThe low pathogenic avian influenza H9N2 virus has been associated with severe economic losses in broiler chicken flocks. Problems associated with its control strategy are potential early infection and level of immune response that may be high in one-day-old chicks due to maternally derived antibodies. Herein, two vaccination regimes were evaluated in commercial broilers kept under either field or laboratory conditions. Two different vaccine types and concentrations were used, and the results highlighted a significantly higher protection against early infection when a homologous vaccine of high antigenic mass was applied at 7 days of life. Shedding was significantly reduced in this regime.Low pathogenic avian influenza virus is one of the major threats that has been affecting the poultry industry in the Middle East region for decades. Attempts to eradicate this disease have failed. Currently, there are commercial vaccines that are either imported or produced locally from recently circulating isolates of H9N2 in Egypt and Middle Eastern countries. This present work focused on comparing the effectiveness of two vaccines belonging to these categories in Egypt. Two commercial broiler flocks (Cobb-500 Broiler) with maternally derived immunity (MDA) against H9N2 virus were employed and placed under normal commercial field conditions or laboratory conditions. Immunity was evaluated on the basis of detectable humoral antibodies against influenza H9N2 virus, and challenge was conducted at 28 days of life using a recent wild H9N2 virus. The results showed that vaccination on the 7th day of life provided significantly higher immune response in both vaccine types, with significantly lower virus shedding compared to vaccination at day 1 of life, regardless of field or laboratory conditions. In addition, the vaccine produced from a recent local H9N2 isolate (MEFLUVAC-H9-16) provided a significantly higher humoral immune response under both field and laboratory conditions, as measured by serology and virus shedding (number of shedders and amount of shedding virus), being significantly lower following challenge on the 28th day of life, contrary to the imported H9 vaccine. In conclusion, use of H9N2 vaccine at 7 days of life provided a significantly higher protection than vaccination at day 1 of life in birds with MDA, suggesting vaccination regimes between 5–8-days of life for broiler chicks with MDA. Moreover, use of a vaccine prepared from a recently circulating H9N2 virus showed significantly higher protection and was more suitable for birds in the Middle East.

Protective efficacy of a polyvalent influenza A DNA vaccine against both homologous (H1N1pdm09) and heterologous (H5N1) challenge in the ferret model

This study describes the protective efficacy of a novel influenza plasmid DNA vaccine in the ferret challenge model. The rationally designed polyvalent influenza DNA vaccine encodes haemagglutinin and neuraminidase proteins derived from less glycosylated pandemic H1N1 (2009) and H3N2 (1968) virus strains as well as the nucleoprotein (NP) and matrix proteins (M1 and M2) from a different pandemic H1N1 (1918) strain. Needle-free intradermal immunisation with the influenza DNA vaccine protected ferrets against homologous challenge with an H1N1pdm09 virus strain, demonstrated by restriction of viral replication to the upper respiratory tract and reduced duration of viral shedding post-challenge. Breadth of protection was demonstrated in two heterologous efficacy experiments in which animals immunised with the influenza DNA vaccine were protected against challenge with a highly pathogenic avian influenza H5N1 virus strain with reproducible survival and clinical outcomes.

Immune Escape Adaptive Mutations in the H7N9 Avian Influenza Hemagglutinin Protein Increase Virus Replication Fitness and Decrease Pandemic Potential.

H7N9 avian influenza viruses (AIVs) continue to evolve and remain a huge threat to human health and the poultry industry. Previously, serially passaging the H7N9 A/Anhui/1/2013 virus in the presence of homologous ferret antiserum resulted in immune escape viruses containing amino acid substitutions alanine to threonine at residues 125 (A125T) and 151 (A151T) and leucine to glutamine at residue 217 (L217Q) in the hemagglutinin (HA) protein. These HA mutations have also been found in field isolates in 2019. To investigate the potential threat of serum escape mutant viruses to humans and poultry, the impact of these HA substitutions, either individually or in combination, on receptor binding, pH of fusion, thermal stability, and virus replication were investigated. Our results showed the serum escape mutant formed large plaques in Madin-Darby canine kidney (MDCK) cells and grew robustly in vitro and in ovo They had a lower pH of fusion and increased thermal stability. Of note, the serum escape mutant completely lost the ability to bind to human-like receptor analogues. Further analysis revealed that N-linked glycosylation, as a result of A125T or A151T substitutions in HA, resulted in reduced receptor-binding avidity toward both human and avian-like receptor analogues, and the A125T+A151T mutations completely abolished human-like receptor binding. The L217Q mutation enhanced the H7N9 acid and thermal stability while the A151T mutation dramatically decreased H7N9 HA thermal stability. To conclude, H7N9 AIVs that contain A125T+A151T+L217Q mutations in the HA protein may pose a reduced pandemic risk but remain a heightened threat for poultry.IMPORTANCE Avian influenza H7N9 viruses have been causing disease outbreaks in poultry and humans. We previously determined that propagation of H7N9 virus in virus-specific antiserum gives rise to mutant viruses carrying mutations A125T+A151T+L217Q in their hemagglutinin protein, enabling the virus to overcome vaccine-induced immunity. As predicted, these immune escape mutations were also observed in the field viruses that likely emerged in the immunized or naturally exposed birds. This study demonstrates that the immune escape mutants also (i) gained greater replication ability in cultured cells and in chicken embryos as well as (ii) increased acid and thermal stability but (iii) lost preferences for binding to human-type receptor while maintaining binding for the avian-like receptor. Therefore, they potentially pose reduced pandemic risk. However, the emergent virus variants containing the indicated mutations remain a significant risk to poultry due to antigenic drift and improved fitness for poultry.

Incidence and Seroprevalence of Avian Influenza in a Cohort of Backyard Poultry Growers, Egypt, August 2015-March 2019.

Currently enzootic avian influenza H5N1, H9N2, and H5N8 viruses were introduced into poultry in Egypt in 2006, 2011, and 2016, respectively. Infections with H5N1 and H9N2 were reported among poultry-exposed humans. We followed 2,402 persons from households raising backyard poultry from 5 villages in Egypt during August 2015–March 2019. We collected demographic, exposure, and health condition data and annual serum samples from each participant and obtained swab samples from participants reporting influenza-like illness symptoms. We performed serologic and molecular analyses and detected 4 cases of infection with H5N1 and 3 cases with H9N2. We detected very low seroprevalence of H5N1 antibodies and no H5N8 antibodies among the cohort; up to 11% had H9 antibodies. None of the exposure, health status, or demographic variables were related to being seropositive. Our findings indicate that avian influenza remains a public health risk in Eqypt, but infections may go undetected because of their mild or asymptomatic nature.

Self-Assembly M2e-Based Peptide Nanovaccine Confers Broad Protection Against Influenza Viruses.

The extracellular domain of influenza M2 protein (M2e) is highly conserved and is a promising target for development of universal influenza vaccines. Here, we synthesized a peptide vaccine consisting of M2e epitope linked to a fibrillizing peptide, which could self-assemble into nanoparticle in physiological salt solutions. When administrated into mice without additional adjuvant, the influenza A M2e epitope-bearing nanoparticles induced antibodies against M2e of different influenza subtypes. Comparing with other M2e-based vaccine, these M2e nanoparticles did not induce immune response against the fibrillizing peptide, demonstrating minimal immunogenicity of vaccine carrier. Furthermore, vaccination with M2e-based nanoparticles did not only protect mice against homologous challenge of influenza PR8 H1N1 virus, but also provide protection against heterologous challenge of highly pathogenic avian influenza H7N9 virus. These results indicated that M2e-based self-assembled nanoparticle vaccine is safe and can elicit cross-protection, therefore is a promising candidate of universal influenza vaccines.

Immune-engineered H7N9 influenza hemagglutinin improves protection against viral influenza virus challenge

ABSTRACT The influenza hemagglutinin (HA) isolated from avian H7N9 influenza virus strains elicit weak immune responses. This low immunogenicity may be due to a regulatory T cell (Treg)–stimulating epitopes in HA from the H7N9 isolate A/Anhui/1/2013 (Anh/13). In this report, this Treg stimulating sequence was removed from the wild-type (WT) H7 HA amino acid sequence and replaced with a conserved CD4 + T cell stimulating sequences from human seasonal H3N2 strains and designed OPT1 H7 HA. The effectiveness of this optimized H7 HA protein was determined using a humanized mouse (HLA-DR3) expressing the human leukocyte antigen (HLA) DR3 allele. HLA-DR3 mice were pre-immunized by infecting with H3N2 influenza virus, A/Hong Kong/4108/2014 and then vaccinated intramuscularly with either the WT H7 HA from Anh/13 or the OPT1 H7 HA antigen without adjuvant. The OPT1 H7 HA vaccination group elicited higher H7 HA-specific IgG titers that resulted in a lower mortality, weight loss, and lung viral titer following lethal challenge with the H7N9 Anh/13 influenza virus compared to WT-vaccinated mice. Overall, T-cell epitope-engineered vaccines can improve the immunogenicity of H7 HA antigens resulting in enhanced survival and lower morbidity against H7N9 influenza virus challenge.

Profile and generation of reduced neuraminidase inhibitor susceptibility in highly pathogenic avian influenza H7N9 virus from human cases in Mainland of China, 2016–2019

Human infections with highly pathogenic avian influenza (HPAI) H7N9 virus were detected in late 2016. We examined the drug resistance profile of 30 HPAI H7N9 isolates from Mainland of China (2016–2019). Altogether, 23% (7/30) carried neuraminidase inhibitors (NAIs) - resistance mutations, and 13% (4/30) displayed reduced susceptibility to NAIs in neuraminidase (NA) inhibition test. An HPAI H7N9 reassortment virus we prepared was passaged with NAIs for 10 passages. Passage with zanamivir induced an E119G substitution in NA, whereas passage with oseltamivir induced R292K and E119V substitutions that simulated that seen in oseltamivir -treated HPAI H7N9 cases, indicating that the high frequency of resistant strains in the HPAI H7N9 isolates is related to NAIs use. In presence of NAIs, R238I, A146E, G151E and G234T substitutions were found in HA1 region of HA. No amino acid mutations were found in the internal genes of the recombinant virus.

Toward Developing Therapies against Corona Virus: Synthesis and Anti-Avian Influenza Virus Activity of Novel Cytosine Thioglycoside Analogues.

A series of pyrimidine and pyrimidine thioglycoside derivatives were newly synthesized using sodium 2-cyano-3-(arylamino)prop-1-ene-1,1-bis(thiolates) and urea to give the corresponding sodium 6-amino-5-aryl-2-oxo-1,2-dihydropyrimidine-4-thiolates. Stirring of the latter with peracetylated α-d-gluco- and galacto-pyranosyl bromides in DMF afforded the corresponding pyrimidine thioglycosides. On the other hand, treatment of 6-amino-5-aryl-2-oxo-1,2-dihydropyrimidine-4-thiolate salts with hydrochloric acid produced the corresponding pyrimidine-4-thioles, which on stirring with α-d-gluco- and galacto-pyranosyl bromides in sodium hydride and DMF gave the corresponding pyrimidine thioglycosides. Deacetylation of the protected pyrimidine thioglycosides gave the corresponding free pyrimidine thioglycosides. The synthesized compounds have been characterized by 13C NMR, 1H NMR, and IR spectroscopy. The pyrimidine thioglycosides and free pyrimidine thioglycosides were tested against avian influenza H5N1 virus strain and exhibited highest to moderate activity.

Anti-influenza H7 human antibody targets antigenic site in hemagglutinin head domain interface.

Although broadly protective, stem-targeted Abs against the influenza A virus hemagglutinin (HA) have been well studied, very limited information is available on Abs that broadly recognize the head domain. We determined the crystal structure of the HA protein of the avian H7N9 influenza virus in complex with a pan-H7, non-neutralizing, protective human Ab. The structure revealed a B cell epitope in the HA head domain trimer interface (TI). This discovery of a second major protective TI epitope supports a model in which uncleaved HA trimers exist on the surface of infected cells in a highly dynamic state that exposes hidden HA head domain features.

FluConvert and IniFlu: a suite of integrated software to identify novel signatures of emerging influenza viruses with increasing risk

BackgroundThe pandemic threat of influenza has attracted great attention worldwide. To assist public health decision-makers, new suites of tools are needed to rapidly process and combine viral information retrieved from public-domain databases for a better risk assessment.ResultsUsing our recently developed FluConvert and IniFlu software, we automatically processed and rearranged sequence data by standard viral nomenclature, determined the group-related consensus sequences, and identified group-specific polygenic signatures. The software possesses powerful ability to integrate viral, clinical, and epidemiological data. We demonstrated that both multiple basic amino acids at the cleavage site of the HA gene and also at least 11 more evidence-based viral amino acid substitutions present in global highly pathogenic avian influenza H5N2 viruses during the years 2009–2016 that are associated with viral virulence and human infection.ConclusionsFluConvert and IniFlu are useful to monitor and assess all subtypes of influenza viruses with pandemic potential. These programs are implemented through command-line and user-friendly graphical interfaces, and identify molecular signatures with virological, epidemiological and clinical significance. FluConvert and IniFlu are available at https://apps.flutures.com or https://github.com/chinrur/FluConvert_IniFlu

Patterns of Avian Influenza A (H5) and A (H9) virus infection in backyard, commercial broiler and layer chicken farms in Bangladesh.

In order to control Highly Pathogenic Avian Influenza (HPAI) H5N1 and Low Pathogenic Avian Influenza (LPAI) H9N2 virus spread in endemically infected countries, a detailed understanding of infection patterns is required. We conducted cross-sectional studies in Bangladesh in 2016 and 2017, on 144 backyard, 106 broiler and 113 layer chicken farms. Although all sampled birds were negative for H5 virus by RT-PCR, H5 antibodies were detected in unvaccinated birds on all three farming systems. Higher H5 antibody prevalence was observed in ducks raised on backyard farms, 14.2% (95% CI: 10.0%-19.8%), compared to in-contact backyard chickens, 4.2% (95% CI: 2.8%-6.1%). The H5 antibody prevalence was lower in broiler chickens, 1.5% (95% CI: 0.9%-2.5%), compared to layer chickens, 7.8% (95% CI: 6.1%-9.8%). H9 viruses were detected by RT-PCR in 0.5% (95% CI: 0.2%-1.3%) and 0.6% (95% CI: 0.3%-1.5%) of broilers and layers, respectively, and in 0.2% (95% CI: 0.0%-1.2%) of backyard chickens. Backyard chickens and ducks showed similar H9 antibody prevalence, 16.0% (95% CI: 13.2%-19.2%) and 15.7% (95% CI: 11.3%-21.4%), which was higher compared to layers, 5.8% (95% CI: 4.3%-7.6%), and broilers, 1.5% (95% CI: 0.9%-2.5%). Over the course of a production cycle, H5 and H9 antibody prevalence increased with the age of backyard and layer chickens. Usually, multiple ducks within a flock were H5 antibody positive, in contrast to backyard chickens, broilers and layers where only individual birds within flocks developed H5 antibodies. Our findings highlight low virus circulation in healthy chickens of all production systems in Bangladesh, which is in contrast to high virus circulation reported from live bird markets. Data generated in this project can be used to adopt risk-based surveillance approaches in different chicken production systems in Bangladesh and to inform mathematical models exploring HPAI infection dynamics in poultry from the source of production.

Virucidal Action Against Avian Influenza H5N1 Virus and Immunomodulatory Effects of Nanoformulations Consisting of Mesoporous Silica Nanoparticles Loaded with Natural Prodrugs.

BackgroundCombating infectious diseases caused by influenza virus is a major challenge due to its resistance to available drugs and vaccines, side effects, and cost of treatment. Nanomedicines are being developed to allow targeted delivery of drugs to attack specific cells or viruses.Materials and MethodsIn this study, mesoporous silica nanoparticles (MSNs) functionalized with amino groups and loaded with natural prodrugs of shikimic acid (SH), quercetin (QR) or both were explored as a novel antiviral nanoformulations targeting the highly pathogenic avian influenza H5N1 virus. Also, the immunomodulatory effects were investigated in vitro tests and anti-inflammatory activity was determined in vivo using the acute carrageenan-induced paw edema rat model.ResultsProdrugs alone or the MSNs displayed weaker antiviral effects as evidenced by virus titers and plaque formation compared to nanoformulations. The MSNs-NH2-SH and MSNs-NH2-SH-QR2 nanoformulations displayed a strong virucidal by inactivating the H5N1 virus. They induced also strong immunomodulatory effects: they inhibited cytokines (TNF-α, IL-1β) and nitric oxide production by approximately 50% for MSNs-NH2-SH-QR2 (containing both SH and QR). Remarkable anti-inflammatory effects were observed during in vivo tests in an acute carrageenan-induced rat model.ConclusionOur preliminary findings show the potential of nanotechnology for the application of natural prodrug substances to produce a novel safe, effective, and affordable antiviral drug.

Phenotypic Effects of Substitutions within the Receptor Binding Site of Highly Pathogenic Avian Influenza H5N1 Virus Observed during Human Infection.

Highly pathogenic avian influenza (HPAI) viruses are enzootic in wild birds and poultry and continue to cause human infections with high mortality. To date, more than 850 confirmed human cases of H5N1 virus infection have been reported, of which ∼60% were fatal. Global concern persists that these or similar avian influenza viruses will evolve into viruses that can transmit efficiently between humans, causing a severe influenza pandemic. It was shown previously that a change in receptor specificity is a hallmark for adaptation to humans and evolution toward a transmittable virus. Substantial genetic diversity was detected within the receptor binding site of hemagglutinin of HPAI A/H5N1 viruses, evolved during human infection, as detected by next-generation sequencing. Here, we investigated the functional impact of substitutions that were detected during these human infections. Upon rescue of 21 mutant viruses, most substitutions in the receptor binding site (RBS) resulted in viable virus, but virus replication, entry, and stability were often impeded. None of the tested substitutions individually resulted in a clear switch in receptor preference as measured with modified red blood cells and glycan arrays. Although several combinations of the substitutions can lead to human-type receptor specificity, accumulation of multiple amino acid substitutions within a single hemagglutinin during human infection is rare, thus reducing the risk of virus adaptation to humans.IMPORTANCE H5 viruses continue to be a threat for public health. Because these viruses are immunologically novel to humans, they could spark a pandemic when adapted to transmit between humans. Avian influenza viruses need several adaptive mutations to bind to human-type receptors, increase hemagglutinin (HA) stability, and replicate in human cells. However, knowledge on adaptive mutations during human infections is limited. A previous study showed substantial diversity within the receptor binding site of H5N1 during human infection. We therefore analyzed the observed amino acid changes phenotypically in a diverse set of assays, including virus replication, stability, and receptor specificity. None of the tested substitutions resulted in a clear step toward a human-adapted virus capable of aerosol transmission. It is notable that acquiring human-type receptor specificity needs multiple amino acid mutations, and that variability at key position 226 is not tolerated, reducing the risk of them being acquired naturally.

Genotyping and reassortment analysis of highly pathogenic avian influenza viruses H5N8 and H5N2 from Egypt reveals successive annual replacement of genotypes

Highly pathogenic (HP) H5N1, clade 2.2.1, and low pathogenic avian influenza (LPAI) H9N2 viruses, G1-B lineage, are endemic in poultry in Egypt and have co-circulated for almost a decade. Surprisingly, no inter-subtypic reassortment events have been reported from the field during that time. After the introduction of HPAIV H5N8, clade 2.3.4.4b, in Egyptian poultry in 2016, suddenly HP H5N2 reassortants with H9N2 viruses emerged. The current analyses focussed on studying 32 duck flocks, 4 broiler chicken flocks, and 1 turkey flock, suffering from respiratory manifestations with moderate to high mortality reared in two Egyptian governorates during 2019. Real-time RT-PCR substantiated the presence of HP H5N8 in 21 of the 37 investigated flocks with mixed infection of H9N2 in two of them. HP H5N1 was not detected. Full hemagglutinin (HA) sequencing of 10 samples with full-genome sequencing of three of them revealed presence of a single genotype. Very few substituting mutations in the HA protein were detected versus previous Egyptian HA sequences of that clade. Interestingly, amino acid substitutions in the Matrix (M2) and the Neuraminidase (NA) proteins associated with conferring both Amantadine and Oseltamivir resistance were present. Systematic reassortment analysis of all publicly available Egyptian whole genome sequences of HP H5N8 (n = 23), reassortant HP H5N2 (n = 2) and LP H9N2 (n = 53) viruses revealed presence of at least seven different genotypes of HPAI H5Nx viruses of clade 2.3.4.4b in Egypt since 2016. For H9N2 viruses, at least three genotypes were distinguishable. Heat mapping and tanglegram analyses suggested that several internal gene segments in both HP H5Nx and H9N2 viruses originated from avian influenza viruses circulating in wild bird species in Egypt. Based on the limited set of whole genome sequences available, annual replacement patterns of HP H5Nx genotypes emerged and suggested selective advantages of certain genotypes since 2016.

Highly pathogenic H7N9 avian influenza virus infection associated with up-regulation of PD-1/PD-Ls pathway-related molecules

To investigate the main transcriptional and biological changes of human host during low and highly pathogenic avian H7N9 influenza virus infection and to analyze the possible causes of escalated virulence and the systematic progression of H7N9 virus infection, we utilized whole transcriptome sequencing (RNA-chip and RNA-seq) and other biomolecular methods to analyze and verify remarkable changes of host cells during these two subtypes of H7N9 influenza viruses infection. Whole transcriptome analysis showed the global profiles of differentially expressed genes (DEGs) and identified 458 DEGs associated with major changes in biological processes of the host cells after infection with 2017 HPAI H7N9 virus versus 2013 LPAI H7N9 virus, mainly including drastically increased defense responses to viruses (e.g. negative regulation of viral gene replication), IFNs related pathways, immune response/native immune response, and inflammatory response. Genes of programmed cell death 1 (PD-1) pathways were found changed remarkably and several highly correlated non-coding RNAs were identified. The results suggested that HPAI H7N9 virus induces stronger immune response and suppressing response than LPAI H7N9. Meanwhile, PD-1/PD-Ls signaling pathways work together in regulating host responses including antiviral defense, lethal inflammation caused by the virus and immune response, thus contribute to the high pathogenicity of 2017H7N9 virus that can be regulated by non-coding RNAs. The present study represents a comprehensive understanding and good reference of regulation of pathogenicity of H7N9 virus even other fatal viruses and correlated host immune responses.

Tropism, replication competence, and innate immune responses of the coronavirus SARS-CoV-2 in human respiratory tract and conjunctiva: an analysis in ex-vivo and in-vitro cultures

BackgroundSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in December 2019, causing a respiratory disease (coronavirus disease 2019, COVID-19) of varying severity in Wuhan, China, and subsequently leading to a pandemic. The transmissibility and pathogenesis of SARS-CoV-2 remain poorly understood. We evaluate its tissue and cellular tropism in human respiratory tract, conjunctiva, and innate immune responses in comparison with other coronavirus and influenza virus to provide insights into COVID-19 pathogenesis.MethodsWe isolated SARS-CoV-2 from a patient with confirmed COVID-19, and compared virus tropism and replication competence with SARS-CoV, Middle East respiratory syndrome-associated coronavirus (MERS-CoV), and 2009 pandemic influenza H1N1 (H1N1pdm) in ex-vivo cultures of human bronchus (n=5) and lung (n=4). We assessed extrapulmonary infection using ex-vivo cultures of human conjunctiva (n=3) and in-vitro cultures of human colorectal adenocarcinoma cell lines. Innate immune responses and angiotensin-converting enzyme 2 expression were investigated in human alveolar epithelial cells and macrophages. In-vitro studies included the highly pathogenic avian influenza H5N1 virus (H5N1) and mock-infected cells as controls.FindingsSARS-CoV-2 infected ciliated, mucus-secreting, and club cells of bronchial epithelium, type 1 pneumocytes in the lung, and the conjunctival mucosa. In the bronchus, SARS-CoV-2 replication competence was similar to MERS-CoV, and higher than SARS-CoV, but lower than H1N1pdm. In the lung, SARS-CoV-2 replication was similar to SARS-CoV and H1N1pdm, but was lower than MERS-CoV. In conjunctiva, SARS-CoV-2 replication was greater than SARS-CoV. SARS-CoV-2 was a less potent inducer of proinflammatory cytokines than H5N1, H1N1pdm, or MERS-CoV.InterpretationThe conjunctival epithelium and conducting airways appear to be potential portals of infection for SARS-CoV-2. Both SARS-CoV and SARS-CoV-2 replicated similarly in the alveolar epithelium; SARS-CoV-2 replicated more extensively in the bronchus than SARS-CoV. These findings provide important insights into the transmissibility and pathogenesis of SARS-CoV-2 infection and differences with other respiratory pathogens.FundingUS National Institute of Allergy and Infectious Diseases, University Grants Committee of Hong Kong Special Administrative Region, China; Health and Medical Research Fund, Food and Health Bureau, Government of Hong Kong Special Administrative Region, China.

A Replication-Defective Influenza Virus Vaccine Confers Complete Protection against H7N9 Viral Infection in Mice.

Avian influenza H7N9 viruses continue to pose a great threat to public health, which is evident by their high case-fatality rates. Although H7N9 was first isolated in humans in China in 2013, to date, there is no commercial vaccine available against this particular strain. Our previous studies developed a replication-defective influenza virus through mutation of the hemagglutinin (HA) cleavage site from a trypsin-sensitive to an elastase-sensitive motif. In this study, we report the development of a reassortant mutant influenza virus derived from the human isolate A/British Columbia/01/2015 (H7N9) [BC15 (H7N9)], which is the QVT virus. The HA gene of this virus possesses three mutations at the cleavage site, Lys-Gly-Arg were mutated to Gln-Thr-Val at amino acid (aa) positions 337, 338, and 339, respectively. We report this virus to rely on elastase in vitro, possess unaltered replication abilities when elastase was provided compared to the wild type virus in vitro, and to be non-virulent and replication-defective in mice. In addition, we report this virus to induce significant levels of antibodies and IFN-γ and IL-5 secreting cells, and to protect mice against a lethal challenge of the BC15 (H7N9) virus. This protection is demonstrated through the lack of body weight loss, 100% survival rate, and the prevention of BC15 (H7N9) viral replication as well as the reduction of proinflammatory cytokines induced in the mouse lung associated with the influenza disease. Therefore, these results provide strong evidence for the use of this reassortant mutant H7N9 virus as a replication-defective virus vaccine candidate against H7N9 viruses.