Stop H5N1 influenza in US cattle now

The relentless march of a highly pathogenic avian influenza virus(HPAIV)strain,known as H5N1,to become an unprecedented panzootic continues unchecked.The leap of H5N1 clade 2.3.4.4b from Eurasia and Africa to North America in 2021 and its further spread to South America and the Antarctic have exposed new avian and mammalian populations to the virus and led to outbreaks on an unrivaled scale.The virus has infected wild birds across vast geographic regions and caused wildlife deaths in some of the world's most biodiverse ecosystems.

H5N1 influenza viruses： outbreaks and biological properties

All known subtypes of influenza A viruses are maintained in wild waterfowl, the natural reservoir of these viruses. Influenza A viruses are isolated from a variety of animal species with varying morbidity and mortality rates. More importantly, influenza A viruses cause respiratory disease in humans with potentially fatal outcome. Local or global outbreaks in humans are typically characterized by excess hospitalizations and deaths. In 1997, highly pathogenic avian influenza viruses of the H5N1 subtype emerged in Hong Kong that transmitted to humans, resulting in the first documented cases of human death by avian influenza virus infection. A new outbreak started in July 2003 in poultry in Vietnam, Indonesia, and Thailand, and highly pathogenic avian H5N1 influenza viruses have since spread throughout Asia and into Europe and Africa. These viruses continue to infect humans with a high mortality rate and cause worldwide concern of a looming pandemic. Moreover, H5N1 virus outbreaks have had devastating effects on the poultry industries throughout Asia. Since H5N1 virus outbreaks appear to originate from Southern China, we here examine H5N1 influenza viruses in China, with an emphasis on their biological properties.

An Overview of the Highly Pathogenic H5N1 Influenza Virus

Since the first human case of H5N1 avian influenza virus infection was reported in 1997, this highly pathogenic virus has infected hundreds of people around the world and resulted in many deaths. The ability of H5N1 to cross species boundaries, and the presence of polymorphisms that enhance virulence, present challenges to developing clear strategies to prevent the pandemic spread of this highly pathogenic avian influenza (HPAI) virus. This review summarizes the current understanding of, and recent research on, the avian influenza H5N1 virus, including transmission, virulence, pathogenesis, clinical characteristics, treatment and prevention.

Antiviral activity of five Asian medicinal pant crude extracts against highly pathogenic H5N1 avian influenza virus

Objective: To study the antiviral properties of the five Asian medicinal plants against in vitro infection by the highly pathogenic avian influenza virus(H5N1).Methods: Crude extracts of Andrographis paniculata, Curcuma longa(C. longa),Gynostemma pentaphyllum, Kaempferia parviflora(K. parviflora), and Psidium guajava obtained by both water and ethanol extractions were investigated for their cytotoxicity in the Madin–Darby canine kidney cells. Thereafter, they were investigated in vitro for antiviral activity and cytokine response upon H5N1 virus infection.Results: The results revealed that both water and ethanol extracts of all the five studied plants showed significant antiviral activity against H5N1 virus. Among these plants,C. longa and K. parviflora showed strong anti-H5N1 activity. Thus, they were selected for further studies on their cytokine response upon virus infection. It was found that ethanol and water crude extracts of C. longa and K. parviflora induced significant upregulation of TNF-a and IFN-b m RNA expressions, suggesting their roles in the inhibition of H5N1 virus replication.Conclusions: To the best of the authors' knowledge, this study is among the earliest reports to illustrate the antiviral property of these Asian medicinal plants against the highly pathogenic avian H5N1 influenza virus. The results of this study shed light on alternative therapeutic sources for treatment of H5N1 influenza virus infection in the future.

Cross-neutralizing Anti-hemagglutinin Antibodies Isolated from Patients Infected with Avian Influenza A(H5N1) Virus

Objective To recover broad-neutralizing monoclonal antibodies(Bn Abs)from avian influenza A(H5N1)virus infection cases and investigate their genetic and functional features.Methods We screened the Abs repertoires of expanded B cells circulating in the peripheral blood of H5N1 patients.The genetic basis,biological functions,and epitopes of the obtained Bn Abs were assessed and modeled.Results Two Bn Abs,2-12 D5,and 3-37 G7.1,were respectively obtained from two human H5N1 cases on days 12 and 21 after disease onset.Both Abs demonstrated cross-neutralizing and Ab-dependent cellular cytotoxicity(ADCC)activity.Albeit derived from distinct Ab lineages,i.e.,V^H1-69-D2-15-JH^4(2-12D5)and V^H1-2-D3-9-JH^5(3-32 G7.1),the Bn Abs were directed toward CR6261-like epitopes in the HA stem,and HA2 I45 in the hydrophobic pocket was the critical residue for their binding.Signature motifs for binding with the HA stem,namely,IFY in VH1-69-encoded Abs and LXYFXW in D3-9-encoded Abs,were also observed in 2-12D5 and 3-32 G7.1,respectively.Conclusions Cross-reactive B cells of different germline origins could be activated and re-circulated by avian influenza virus.The HA stem epitopes targeted by the Bn Abs,and the two Ab-encoding genes usage implied the VH1-69 and D3-9 are the ideal candidates triggered by influenza virus for vaccine development.

Gene Expression Profiles Comparison between 2009 Pandemic and Seasonal H1N1 Influenza Viruses in A549 Cells

Objective To perform gene expression profiles comparison so that to identify and understand the potential differences in pathogenesis between the pandemic and seasonal A （H1N1） influenza viruses. Methods A549 cells were infected with A/California/07/09 （H1N1） and A/GuangdongBaoan/51/08 （H1N1） respectively at the same MOI of 2 and collected at 2, 4, 8, and 24 h post infection （p.i.）. Gene expression profiles of A549 cells were obtained using the 22 K Human Genome Oligo Array, and differentially expressed genes were analyzed at selected time points. Results Microarrays results indicated that both of the viruses suppressed host immune response related pathways including cytokine production while pandemic H1N1 virus displayed weaker suppression of host immune response than seasonal H1N1 virus. Observation on similar anti-apoptotic events such as activation of apoptosis inhibitor and down-regulation of key genes of apoptosis pathways in both infections showed that activities of promoting apoptosis were different in later stage of infection. Conclusion The immuno-suppression and anti-apoptosis events of pandemic H1N1 virus were similar to those seen by seasonal H1N1 virus. The pandemic H1N1 virus had an ability to inhibit biological pathways associated with cytokine responses, NK activation and macrophage recognition .

Cloning of M and NP Gene of H5N1 Avian Influenza Virus and Immune Efficacy of their DNA Vaccines

H5N1 鸟的流行性感冒病毒(A/chicken/Hubei/489/2004 ) 的 M 和 NP 基因被 RT-PCR 从病毒的 RNA 放大，并且分别地克隆向量进 pMD18-T。包含 M 基因(pHM6-m ) 或 NP 基因(pHM6-np ) 的表示 plasmid 然后被把 M 或 NP 基因插入到 pHM6 优核质表示向量构造；构造 plasmid 然后被定序。32 只 BALB/c 老鼠(6-week-old ) 在随机被划分成四个组。三组 BALB/c 老鼠被接种一次有 plasmid pHM6-m， plasmid pHM6-np 的 30 渭 g 或 plasmid pHM6-m (15 渭 g ) 和 pHM6-np (15 渭 g ) 的混合的任何一个 30 渭 g 的肌内的线路分别地。老鼠的一个另外的组作为控制与 100 渭 l PBS 被注射。二个星期以后，所有老鼠与相应 H5N1 鸟的流行性感冒病毒被质问，并且在下列 12 天内观察了。在 pHM6-m 组， pHM6-np 组和混合 plasmids 组的老鼠的幸存率分别地是 62.5% ， 25.0% 和 50.0% 。结果证明有效保护能被 pHM6-m 或 pHM6-np 提供，但是 pHM6-m 比 pHM6-np 提供了更好保护的效果。关键词 H5N1 流行性感冒病毒 - M 基因 - NP 基因 - 克隆 - DNA 疫苗的 CLC 数字 S852.65 基础条款：国家基本科学才能训练资助(NSFC J0630648 )

The Evidence of Clade 7.1 Avian Influenza Virus (H5N1) in Qinghai Lake

The highly pathogenic influenza A virus subtype H5N1 spread throughout Asia since 2003, reached to Europe in 2005, and the Middle East, as well as Africa and caused a global concern for a potential pandemic threat last decade. A Clade 2.3.2 H5N1 virus became dominate in the Qinghai Lake region in 2009 with sporadic mammal cases of infection and transferred to Russia and Europe through wild migratory birds. Currently, HPAI H5N1 of clades 2.3.4, 2.3.2, and 7 are the dominant co-circulating H5N1 viruses in poultry in Asia. 2.3.2 Clade is dominant in wild birds through the world whereas there is no evident data about Clade 7 circulation in wild birds. We detected HPAI H5N1 virus of Clade 7.1 in Qinghai Lake, that closely related to Shanxi-like and Vietnam viruses co-circulating in poultry. This is the first report of Clade 7.1 H5N1 in wild birds. Based on phylogenetic analyses, the virus can be originated from Clade 7.1 virus gene pool that spread in Vietnam and Chinese poultry and could spread with migratory birds to Qinghai Lake. The Qinghai Lake continues to be significant hotspot for H5N1 surveillance since the regular outbreaks occurred there in wild birds and mammals. Based on these facts and findings, the related researchers should pay more attention to the Qinghai Lake basin as significant hotspot for H5N1 avian influenza surveillance since the regular H5N1 outbreaks occurred there in wild birds with sporadic mammal cases of infection.

Histopathology of Ducks Experimentally Infected with a Highly Pathogenic Avian Influenza Virus,A/duck/Guangdong/220/2004(H5N1)

Ducks inoculated intravenously or via the ocular-nasal-oral-cloacal routes with a highly pathogenic avian influenza virus,A/duck/Guangdong/220/2004(H5N1),developed systemic hyperemia,congestion,hemorrhage,thrombosis and edema in various organs,as well as necrosis or apoptosis in the parenchyma of the heart,liver,spleen,lungs,kidneys,pancreas,brain,thymus and bursa of Fabricius.The main manifestations were angiitis,necrotic pancreatitis,atrophic necrotic thymitis and bursitis Fabricii,splenitis,tracheitis,hemorrhagic bronchointerstitial pneumonia,viral myocarditis,nonsuppurative encephalitis,focal viral hepatitis,ulcerative enteritis,renal tubule interstitial nephritis,and intraglomerular mesangial cell hyperplastic glomerular nephritis.The results demonstrated that the mechanism of pathogenesis involved cellular necrosis and apoptosis,and that death of the ducks was caused by severe pathologic trauma occurring in multiple visceral organs.

Transcriptomic analyses reveal new genes and networks response to H5N1 influenza viruses in duck(Anas platyrhynchos)

H5N1 influenza represents one of the great challenges to public health.Some H5N1 viruses(i.e.,A/goose/Hubei/65/05,GS/65) are weakly pathogenic,while the others(i.e.,A/duck/Hubei/49/05,DK/49) are highly pathogenic to their natural hosts.Here,we performed brain and spleen transcriptomic analyses of control ducks and ones infected by the DK/49 or the GS/65 H5N1 virus.We demonstrated that,compared to the GS/65 virus,the DK/49 virus infection changed more numerous immune genes’ expression and caused continuous increasing of immune pathways(i.e.,RIG-I and MDA5) in ducks.We found that both H5N1 virus strains might escape or subvert host immune response through affecting alternative translation of immune genes,while the DK/49 virus seemed to induce alternative translation of more immune genes than the GS/65 virus.We also identified five co-expressional modules associated with H5N1 virus replication through the weight correlation network analysis(WGCNA).Moreover,we first demonstrated that the duck BCL2 L15 and DCSTAMP in one of these five modules inhibited both the highly pathogenic and weakly pathogenic H5N1 virus replication efficiently.These analyses,in combination with our comprehensive transcriptomic data,provided global view of the molecular architecture for the interaction between host and H5N1 viruses.

Construction and Immunogenicity of a Recombinant Adenovirus Expressing the HA Gene of H5N1 Subtype Swine Influenza Virus

To construct a recombinant adenovirus shuttle plasmid pDC315-H5HA-EGFP,the HA gene of A/Swine/Fujian/1/2001(H5N1) was amplified by RT-PCR and then inserted into adenovirus shuttle plasmid pDC315.A replication-defective recombinant adenovirus expressing the HA gene(rAd-H5HA-EGFP) was generated by co-transfecting the recombinant shuttle plasmid pDC315-H5HA-EGFP and the genomic plasmid pBHGlox△E1,E3Cre in HEK293 cells.The recombinant adenovirus was confirmed by PCR,RT-PCR and Western blot assay.These results demonstrated that HA protein was properly expressed by the rAd-H5HA-EGFP in HEK293 cells and had natural biological activities.The TCID50 of the rAd-H5HA- EGFP was assessed to be 2.26×1010/mL after propagation and purification.Immunization of BALB/ c mice indicated that rAd-H5HA-EGFP induced HI antibodies and protected mice from replication of the challenge virus in their lungs.

Cloning and Phylogenetic Analysis of NS1 Genes from Different Isolates of H9N2 Subtype Duck Influenza Virus

[ Objective] The study aimed to lay a foundation for the further studies on function mechanism of NS1 protein in the interspecies transmission of waterfowl influenza virus. [Method] Using the serologic assay and the specific RT-PCR method, some strains of H9 subtype waterfowl influenza virus were isolated from the 12 to 20 day-old muscovy duck flocks without any clinical symptoms in different areas of Guangdong Province. Four of these strains, including A/duck/ZQ/303/2007（H9N2） （A3 for short）, A/Duck/FJ/301/2007 （H9N2） （C1 for short）, A/Duck/NH/306/2007（H9N2） （ D6 for short）, A/duck/SS/402/2007（H9N2） （ E2 for short）, and a strain named A/duck/ZC/2007（H9N2） （L1 for short） from a muscovy duck died of avian influenza virus （AIV）, were used for NSl gene cloning and sequencing. Subsequently, the obtained NSl gene sequences were compared with other NS1 sequences registered in GenBank, and the phylogenetic analysis was also conducted. [Result] When compared with the H9N2 AIV NS1 sequences in GenBank, the NSl genes of the four AIV strains A3, C1, 136 and E2 displayed homologies ranging from 99% to 100% at nucleotide level, and 95% to 100% at amino acid level; while the NSl gene of L1 strain displayed homology ranging from 94% to 97% at nucleotide level, and 93% to 98% at amino acid level. The phylogenetic tree demonstrated that A3, C1, D6 and E2 were highly resemblant, and L1 was closest to AY66473 （chicken, 2003）. By comparison with the NS1 gene sequences of L1, AF523514 （duck）, AY664743 （chicken） and EF155262.1 （quail） using DNAstar, A3, C1, D6 and E.2 presented nucleotide variations at site 21 （ R→Q）, 70, 71 （ KE→EG）, 86 （ A→S）, 124 （V→M） and 225 （ S→N）, and amino acid variations at site 21,70, 71 and 86 in dsRNA- dependent protein kinase （PKR） binding domain of NSl gene, which induced the evident variations of antigenic determinant and surface proba- bility plot of NS1 protein. [ Conclusion] This study suggested that the amino acid sequence variation in PKR binding domain of NS1 protein had something to do with the virus pathogenicity.

Therapeutic Trial of an Endothelin Receptor Agonist for the Highly Pathogenic Avian Influenza A/H5N1 Virus Infection in Chicks

The rapid spread of the highly pathogenic A/H5N1 avian influenza virus among domestic birds and its transmission to humans has induced world-wide fears of a new influenza pandemic. A/H5N1 has infected over 300 people since 1997, and has shown a mortality rate of over 50%. The high mortality in human cases is thought to be enhanced by the excessive secretion of various endogenous factors, including cytokines and interleukins, stimulated by viral infections. Chickens infected with A/H5N1 viruses experience sudden death without showing severe clinical symptoms or inflammation. However, severe hemorrhage and congestion are seen in various tissues in sporadic chicken cases of A/H5N1-infections, especially in the pulmonary tissues, thus indicating that there is ischemia due to vascular abnormalities. Our previous studies have focused on the expression pattern of endothelin-1, which modulates the vascular tone via endothelin receptors. An Indonesian sporadic strain of A/H5N1 virus was intranasally administered to 10-day-old chicks, and the expression of endothelin was examined in the infected birds. All birds died within five days of inoculation, and had moderate inflammation accompanied by severe hemorrhage and congestion in the lungs. Immunohistochemical studies showed enhanced expression of endothelin-1 in the infected lungs. In addition, the real-time PCR analyses revealed that endothelin-1 and endothelin receptor A mRNA were significantly elevated in the birds with A/H5N1 infections. Subsequently, H5N1-infected birds were inoculated with bosentan hydrate, a competitive antagonist of endothelin receptors. Interestingly, the mortality rate of the infected birds was dramatically decreased in a dose-dependent manner by the administration of bosentan hydrate. The pathological lesions, including congestion and hemorrhage in the pulmonary tissues, were clearly inhibited. These findings are promising, and suggest that endothelin receptor antagonists are a potential treatment for the highly pathogenic avian flu.

Evolution of an avian H5N1 influenza A virus escape mutant

AIM: To investigate the genetic constitution of an escape mutant H5N1 strain and to screen the presence of possible amino acid signatures that could differentiate it from other Egyptian H5N1 strains.METHODS: Phylogenetic, evolutionary patterns and amino acid signatures of the genes of an escape mutant H5N1 influenza A virus isolated in Egypt on 2009 were analyzed using direct sequencing and multisequence alignments.RESULTS: All the genes of the escape mutant H5N1 strain showed a genetic pattern potentially related to Eurasian lineages. Evolution of phylogenetic trees of different viral genes revealed the absence of reassortment in the escape mutant strain while confirming close relatedness to other H5N1 Egyptian strains from human and avian species. A variety of amino acid substitutions were recorded in different proteins compared to the available Egyptian H5N1 strains. The strain displayed amino acid substitutions in different viral alleles similar to other Egyptian H5N1 strains without showing amino acid signatures that could differentiate the escape mutant from other Egyptian H5N1.CONCLUSION: The genetic characteristics of avian H5N1 in Egypt revealed evidence of a high possibility of inter-species transmission. No amino acid signatures were found to differentiate the escape mutant H5N1 strain from other Egyptian H5N1 strains.

The cloning of non-structural-1 (NS1) gene of H9N2 subtype of avian influenza virus in pGEX-4T-1 and pMAL-c2X plasmids and expression in <i>Escherichia coli</i>DH5<i>α</i>strain

Avian influenza is a viral contagious disease that affects poultry industry and human health. Vaccination has been considered as a preventive tool in the eradication of AI, but it causes some limitations including trade embargoes and interfering with serologic surveillance in differentiation between infected and vaccinated animals (DIVA strategy). Several distinct DIVA strategies have been presented to conquer these limitations. In this study, the open reading frame of NS1 gene of a H9N2 subtype of AI virus was amplified by polymerase chain reaction. After extraction and purification of NS1 gene from agarose gel, it was inserted into two different pGEX-4T-1 and pMAL-c2X plasmids and transferred in DH5α strain of Escherichia coli by using electroporation procedure. The E. coli colonies possessing recombinant NS1 gene were screened using PCR, restriction mapping and sequencing analysis. The expressed rNS1 protein was purified using affinity chromatography based on MBP (pMAL- c2X) and GST (pGEX-4T-1). The MBP-NS1 and GST- NS1 proteins on SDS-PAGE had bands with molecular weight of 68 and 52 kDa respectively. Western blotting with MBP-NS1 protein showed positive reaction using antisera obtained from chickens challenged with a H9N2 subtype strain. But, the most sera prepared from H9N2 vaccinated chickens were negative in WB. These findings indicated that the MBP-rNS1 protein of 26 kDa expressed by pMAL-c2X plasmid can be used in a DIVA for differentiation of AI infected and vaccinated chickens.

Molecular Features of Highly Pathogenic Avian and Human H5N1 Influenza A Viruses in Asia

The highly pathogenic avian H5N1 influenza virus could infect humans with high mortality rate, even though it has not yet become efficiently transmissible among humans. This proteomic study investigated the molecular basis of interspecies transmission and host range of this lethal virus in Asia, due to its potential pandemic threat. Although there are host markers located in previous research between general avian and human influenza viruses, the novelty of our work was to uncover host markers between highly pathogenic avian and human H5N1 viruses in Asia. Many host markers we found were not present in the previous general markers, thus expanding the current repertoire of host markers with these strain-specific host markers. Ranked by their order of importance, the top 10 host markers discovered in this report were PB2_627, HA_325, NS1_205, PB2_524, HA_86, NA_201, NP_373, NS1_7, HA_156, NA_74, confirming our current knowledge that PB2_627 is the most critical site for distinguishing avian and human H5N1. We also identified several naturally-occurred mutations in the HA protein that might shift the receptor binding preference of Asian avian H5N1, since early detection of mutations that might lead to emergence of a new pandemic virus is of prime importance. Finally, we analyzed the distinctive interaction patterns within and between proteins of avian and human H5N1 in Asia at protein level and individual residue level. From multiple viewpoints, our findings reinforced the experimental observation that multiple genes of Asian avian H5N1 are involved in its gradual adaptation to human hosts.

Upregulation of GLE1 and LCP2 Genes in H5N1 Influenza Virus Infected Patients

Previous study showed that the Gle1 RNA export mediator-like (Gle1l) gene and the lymphocyte cytosolic protein 2 (Lcp2) gene were upregulated in response to influenza virus A/Puerto Rico/8/1934 (H1N1) in a mouse mode. To determine whether these two genes were upregulated in humans after influenza A virus infection, nasopharyngeal swabs were collected from eleven patients with flu-like symptoms for viral RNA extraction and PCR amplification. Sequencing analysis revealed that nucleoprotein (NP) gene fragments amplified from nasopharyngeal swabs of four patients shared the highest similarity with the NP gene from avian influenza A (H5N1) virus (A/ goose/Shantou/753/2002). Peripheral blood samples were then collected from four patients for quantitative analysis of GLE1 and LCP2 gene expression. Our results demonstrated that both GLE1 and LCP2 genes were upregulated in H5N1 influenza A virus infected patients, suggesting that upregulation of GLE1 and LCP2 genes may be important for the host defense against influenza A viruses.

Characterization of Highly Pathogenic Avian Influenza H5N1 Viruses Isolated from Domestic Poultry in China

The highly pathogenic avian influenza （HPAI） H5N1 virus has caused several outbreaks in domestic poultry. Despite great efforts to control the spread of this virus, it continues to evolve and poses a substantial threat to public health because of a high mortality rate. In this study, we sequenced whole genomes of eight H5N1 avian influenza viruses isolated from domestic poultry in eastern China and compared them with those of typical influenza virus strains. Phylogenetic analyses showed that all eight genomes belonged to clade 2.3.2.1 and clade 7.2, the two main circulating clades in China. Viruses that clustered in clade 2.3.2.1 shared a high degree of homology with H5N1 isolates located in eastern Asian. Isolates that clustered in clade 7.2 were found to circulate throughout China, with an east-to-west density gradient. Pathogenicity studies in mice showed that these isolates replicate in the lungs, and clade 2.3.2.1 viruses exhibit a notably higher degree of virulence compared to clade 7.2 viruses. Our results contribute to the elucidation of the biological characterization and pathogenicity of HPAI H5N1 viruses.

Antiviral activity of Basidiomycete mycelia against influenza type A(serotype H1N1) and herpes simplex virus type 2 in cell culture

In this study, we investigated the in vitro antiviral activity of the mycelia of higher mushrooms against influenza virus type A(serotype H1N1) and herpes simplex virus type 2(HSV-2), strain BH. All 10 investigated mushroom species inhibited the reproduction of influenza virus strain A/FM/1/47(H1N1) in MDCK cells reducing the infectious titer by 2.0–6.0 lg ID50. Four species, Pleurotus ostreatus, Fomes fomentarius, Auriporia aurea, and Trametes versicolor, were also determined to be effective against HSV-2 strain BH in RK-13 cells, with similar levels of inhibition as for influenza. For some of the investigated mushroom species—Pleurotus eryngii, Lyophyllum shimeji, and Flammulina velutipes—this is the first report of an anti-influenza effect. This study also reports the first data on the medicinal properties of A. aurea, including anti-influenza and antiherpetic activities. T. versicolor 353 mycelium was found to have a high therapeutic index(324.67), and may be a promising material for the pharmaceutical industry as an anti-influenza and antiherpetic agent with low toxicity. Mycelia with antiviral activity were obtained in our investigation by bioconversion of agricultural wastes(amaranth flour after CO2 extraction), which would reduce the cost of the final product and solve some ecological problems.