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.

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.

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.

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.

Antibody Dynamic Changes in SPF Chickens and SPF Ducks Immuned with Recombinant Avian Influenza Virus H5 Subtype Bivalent Inactivated Vaccine

To study the immune effect of recombinant avian influenza virus H5 subtype bivalent inactivated vaccine （ HSN1, Re-6 strain ＋ Re-4 strain） and to provide the basis for formulating reasonable immune procedure of avian influenza vaccine in clinical practice. A total of 12 batches of vaccines from three companies were used for the iannune of SPF chickens and SPF ducks. Each chicken or duck serum was separately collected every 3 weeks until the immunization up to the 24^th week. The serum antibody titers of Re-6 and Re-4 were detected. The results showed that the HI titers of the inoculated SPF chickens and SPF ducks roached the peak when the immune time were the 6^th and 3^rd week after the first immunization respectively; then the titer decreased gradually as time prolonged; the highest titer of SPF chickens was greater than that of SPF ducks; the high titer duration of SPF chickens were longer than that of SPF ducks ; and all the vaccines from the three companies showed a good immune effect.

Prevention of Highly Pathogenic Avian Influenza A/H5N1 Infection by Passive Immunotherapy Using Antiserum

The rapid epidemic of highly pathogenic A/H5N1 avian influenza virus by transmission from poultry to humans triggered global unrest in the pandemic of novel influenza. If a human trophic strain of avian influenza viruses replicates in livestock including pigs and chickens, it may have high infectivity and pathogenicity to humans. The most effective method of reducing the outbreaks of influenza would be prophylaxis with an effective vaccine as well as anti-viral drugs including Oseltamivir and Zanamivir hydrate. In this study, chicken antiserum against A/H5N1 virus was produced: the antisera from immunized adult chicken had a strong binding activity to A/H5N1 viral antigens by ELISA. Furthermore, the antiserum strongly inhibited hemaggregation of erythrocytes and cytopathic effects in MDCK cells, indicating a strong neutralization activity against A/H5N1 infections. Interestingly, the mortality rate of chicks inoculated with A/H5N1 virus was dramatically decreased with the antiserum injection. These results suggest that antiserum may be a potentially effective protective and therapeutic modality for A/H5N1 infection.

Substrate specificity of avian influenza H5N1 neuraminidase

AIM: To characterise neuraminidase(NA) substrate specificity of avian influenza H5N1 strains from humans and birds comparing to seasonal influenza virus.METHODS: Avian influenza H5N1 strains from humans and birds were recruited for characterising their NA substrate specificity by using a modified commercial fluorescence Amplex Red assay. This method can identify the preference of α2,6-linked sialic acid or α2,3-linked sialic acid. Moreover, to avoid the bias of input virus, reverse genetic virus using NA gene from human isolated H5N1 were generated and used to compare with the seasonal influenza virus. Lastly, the substrate specificity profile was further confirmed by high-performance liquid chromatography(HPLC) analysis of the enzymatic product. RESULTS: The H5N1 NA showed higher activity on α2,3-linked sialic acid than α2,6-linked(P < 0.0001). To compare the NA activity between the H5N1 and seasonal influenza viruses, reverse genetic viruses carrying the NA of H5N1 viruses and NA from a seasonal H3N2 virus was generated. In these reverse genetic viruses, the NA activity of the H5N1 showed markedly higher activity against α2,3-linked sialic acid than that of the H3N2 virus, whereas the activities on α2,6-linkage were comparable. Interestingly, NA from an H5N1 human isolate that was previously shown to have heamagglutinin(HA) with dual specificity showed reduced activity on α2,3-linkage. To confirm the substrate specificity profile, HPLC analytic of enzymatic product was performed. Similar to Amplex red assay, H5N1 virus showed abundant preference on α2,3-linked sialic acid.CONCLUSION: H5N1 virus maintains the avian specific NA and NA changes may be needed to accompany changes in HA receptor preference for the viral adaptation to humans.

Suppression of Highly Pathogenic Avian Influenza A/H5N1 Infection Using Migratory Antibody Passed from Mother to Chick

Avian influenza is the most contagious disease not only in poultry, but also in humans. Avian influenza in humans occurs mainly in Southeast Asia, but no human-to-human pandemic has occurred. Meanwhile, outbreaks of avian influenza in poultry occur on a global scale and cause a large economic loss. Migration antibodies passed from mother birds via eggs are said to be an important component of the immune system that protects birds from infection. Thus, the immunity status of mother birds can determine the ability of offspring to defend against infection. In this study, we investigated the presence of anti-avian influenza virus antibody in chickens hatched on a poultry farm in Indonesia and examined the involvement of migratory antibodies in protecting against virus infection by infectious experiments of highly pathogenic avian influenza in chickens. Blood was collected from randomly selected chicks, and antibodies against avian influenza virus were evaluated in all birds. Since these young birds had no history of vaccination, the antibodies were deemed to have been transferred from the mother birds. The enzyme-linked immunosorbent assay antibody titer in each bird varied. Infection of these birds with highly pathogenic avian influenza virus A/H5N1 intra-nasally resulted in a high mortality rate in chicks with low antibody titers but a low mortality rate in chicks with high antibody titers. These findings indicate that migratory antibody prevented highly pathogenic avian influenza A/H5N1 infection in chicks, suggesting that such a preventive effect could also be expected with outdoor natural infection.

Variation and evolution of NP genes of human avian H_5N_1 virus strains

In order to reveal variation and revolution of NP genes of human avian H5 N1 influenza virus strains, the NP gene of a human avian H5 N1 influenza virus strain in Guangdong was sequenced and the global NP genes of strains were retrieved. The sequences were analyzed by DNAStar 5.0, and the evolutionary speed was studied with reference to the epidemiological data. It was found that NP genes of 45 strains during 1997-2006 were homologically classified into three groups： strains in 1997-1998, strains in 2004-2005 and strains from 2003 to 2006. There were 35 substitutions in NPs in all strains accounting for a ratio of 7.03% （35/498）. An additional glycoprotein domain （NGT430-432） was found in NP genes in the strains of 2003-2006, the mutation of N370S in GD-01-06 resulted in occurrence of one more glycoprotein domain （NES368-370）. In the synonymous variation, Ks values in NP were 2.03 × 10^-5-2.55 × 10^-5 Nt/d and K. values in NP were 1.58 × 10^-6-3.10 × 10^-6 Nt/d. There didn＇t exist obviously selective pressure. An additional glycoprotein domain in every strain of 2003-2006 and one more in strain GD-01-06 might change the antigenicity of human avian H5 N1 influenza virus. The variation on human avian H5 N1 influenza strains occurred frequently in the natural world, which would result in high probability of human-human transmission along with the natural evolution of the virus.

Close Relationship between the 2009 H1N1 Virus and South Dakota AIV Strains

Although previous publications suggest the 2009 pandemic influenza A (H1N1) virus was reassorted from swine viruses of North America and Eurasia,the immediate ancestry still remains elusive due to the big evolutionary distance between the 2009 H1N1 virus and the previously isolated strains. Since the unveiling of the 2009 H1N1 influenza,great deal of interest has been drawn to influenza,consequently a large number of influenza virus sequences have been deposited into the public sequence databases. Blast analysis demonstrated that the recently submitted 2007 South Dakota avian influenza virus strains and other North American avian strains contained genetic segments very closely related to the 2009 H1N1 virus,which suggests these avian influenza viruses are very close relatives of the 2009 H1N1 virus. Phylogenetic analyses also indicate that the 2009 H1N1 viruses are associated with both avian and swine influenza viruses circulating in North America. Since the migrating wild birds are preferable to pigs as the carrier to spread the influenza viruses across vast distances,it is very likely that birds played an important role in the inter-continental evolution of the 2009 H1N1 virus. It is essential to understand the evolutionary route of the emerging influenza virus in order to find a way to prevent further emerging cases. This study suggests the close relationship between 2009 pandemic virus and the North America avian viruses and underscores enhanced surveillance of influenza in birds for understanding the evolution of the 2009 pandemic influenza.

Endemicity of H9N2 and H5N1 avian influenza viruses in poultry in China poses a serious threat to poultry industry and public health

The H9N2 and H5N1 avian in fluenza viruses(AIVs) have been circulating in poultry in China and become endemic since 1998 and 2004, respectively.Currently, they are prevalent in poultry throughout China. This endemicity makes them actively involved in the emergence of the novel lineages of other subtypes of in fluenza viruses, such as the well-known viruses of the highly pathogenic avian in fluenza(HPAI) H5N2 and the2013 novel H7N7, H7N9 and H10N8 subtypes, thereby threatening both the poultry industry and public health.Here, we will review brie fly the prevalence and evolution,pathogenicity, transmission, and disease control of these two subtypes and also discuss the possibility of emergence of potentially virulent and highly transmissible AIVs to humans.

Human avian influenza A (H5N1) virus infection in China

Highly pathogenic influenza A (H5N1) virus causes a widespread poultry deaths worldwide. The first human H5N1 infected case was reported in Hong Kong Special Administrative Region of China in 1997. Since then, the virus re-emerged in 2003 and continues to infect people worldwide. Currently, over 400 human infections have been reported in more than 15 countries and mortality rate is greater than 60%. H5N1 viruses still pose a potential pandemic threat in the future because of the continuing global spread and evolution. Here, we summarize the epidemiological, clinical and virological characteristics of human H5N1 infection in China monitored and identified by our national surveillance systems.

Highly pathogenic avian influenza H5N1 Clade 2.3.2.1c virus in migratory birds,2014–2015

A novel Clade 2.3.2.1c H5N1 reassortant virus caused several outbreaks in wild birds in some regions of China from late 2014 to 2015.Based on the genetic and phylogenetic analyses,the viruses possess a stable gene constellation with a Clade 2.3.2.1c HA,a H9N2-derived PB2 gene and the other six genes of Asian H5N1-origin.The Clade 2.3.2.1c H5N1 reassortants displayed a high genetic relationship to a human H5N1 strain(A/Alberta/01/2014).Further analysis showed that similar viruses have been circulating in wild birds in China,Russia,Dubai(Western Asia),Bulgaria and Romania(Europe),as well as domestic poultry in some regions of Africa.The affected areas include the Central Asian,East Asian-Australasian,West Asian-East African,and Black Sea/Mediterranean flyways.These results show that the novel Clade 2.3.2.1c reassortant viruses are circulating worldwide and may have gained a selective advantage in migratory birds,thus posing a serious threat to wild birds and potentially humans.

Reference Gene Selection for Normalization of PCR Analysis in Chicken Embryo Fibroblast Infected with H5N1 AIV

Chicken embryo fibroblasts (CEFs) are among the most commonly used cells for the study of interactions between chicken hosts and H5N1 avian influenza virus (AIV).In this study,the expression of eleven housekeeping genes typically used for the normalization of quantitative real-time PCR (QPCR) analysis in mammals were compared in CEFs infected with H5N1 AIV to determine the most reliable reference genes in this system.CEFs cultured from 10-day-old SPF chicken embryos were infected with 100 TCID50 of H5N1 AIV and harvested at 3,12,24 and 30 hours post-infection.The expression levels of the eleven reference genes in infected and uninfected CEFs were determined by real-time PCR.Based on expression stability and expression levels,our data suggest that the ribosomal protein L4 (RPL4) and tyrosine 3-monooxygenase tryptophan 5-monooxygenase activation protein zeta polypeptide (YWHAZ) are the best reference genes to use in the study of host cell response to H5N1 AIV infection.However,for the study of replication levels of H5N1 AIV in CEFs,the β-actin gene (ACTB) and the ribosomal protein L4 (RPL4) gene are the best references.

Conserved peptides enhance immune efficiency of inactive vaccines against emerging avian influenza viruses in chicken

Avian influenza viruses(AIVs) such as H5N1 and H7N9 are a great threat to poultry economics and public health. Vaccination can effectively inhibit the spread of AIV in poultry, which is also a viable strategy for controlling virus transmission from poultry to human. Adjuvants that are commonly used in current inactivated vaccines to provide stronger anti-AIV immune responses are often limited in their capacity to quantitatively induce both humoral and cellular immune responses. Herein, we assessed the levels of immune responses generated by a vaccine formulation comprising inactivated H5N1 antigen and synthetic peptides covering conserved CD4^+, CD8^+ T cell, and B cell epitopes. We found that the synthetic peptides enhanced the antibody responses against conserved influenza virus antigen M2 e. Notably, the hemagglutination inhibition test results indicated that the peptides significantly augmented the antibody responses of inactivated H5N1 antigen even in the 1/10 or 1/5 dose group, in the identical antibody level as antigen alone used at the full dose. This indicates that the peptide can significantly reduce the use of inactivated virus, lowering the cost of the vaccine. Moreover, the peptides increased the transcript levels of interleukin-4 and interferon-γcytokines in chicken peripheral blood mononuclear cells, which may facilitate both humoral and cellular immune responses. Our data suggest that this peptide combined with inactivated H5N1 antigen enhances both the humoral and cellular immune responses,which may benefit the prediction and design of synthetic peptide-based adjuvants for vaccines in chicken.

A Mechanism-based 3D-QSAR and DFT Approach for the Prediction of H5N1 Entry Inhibitory Potency of 3-O-β-chacotriosyl Ursolic Acid Derivatives

In this work, 25 3-O-β-chacotriosyl ursolic acid derivatives were employed to achieve the highly reliable and predictive 3 D-QSAR models by Co MFA and Co MSIA methods, respectively. The predictive capabilities of two constructed CoMFA and CoMSIA models were verified by the leave-one-out cross-validation method. The results showed that the cross-validated coefficient（q2） and non-cross-validated coefficient（R2） were 0.559, 0.981 in the CoMFA model and 0.696, 0.978 in the CoM SIA model, respectively, which suggests that these two models are robust and have good exterior predictive capabilities. Furthermore, based on the contour maps information of two models, ten novel inhibitors with higher inhibitory potency were designed, and the quantum chemical calculation of density functional theory（DFT） was performed to investigate the mechanism why the designed molecules have stronger inhibitory activity than the lead compound. The calculations show that the C-50 position of lead compound is a key active site for the enhancement of inhibitory activity, and it should be introduced into the large electron withdrawing group, which would result in generating potent and selective H5 N1 entry inhibitors. We expect that the results in this paper could provide important information to develop new potent H5 N1 entry inhibitors.

The variable codons of H5N1 avian influenza A virus haemagglutinin genes

We investigated the selection pressures on the haemagglutinin genes of H5N1 avian influenza viruses using fixed effects likelihood models. We found evidence of positive selection in the sequences from isolates from 1997 to 2007, except viruses from 2000. The haemagglutinin sequences of viruses from southeast Asia, Hong Kong and China's Mainland were the most polymorphic and had similar nonsyn-onymous profiles. Some sites were positively selected in viruses from most regions and a few of these sites displayed different amino acid patterns. Selection appeared to produce different outcomes in vi-ruses from Europe, Africa and Russia and from different host types. One position was found to be positively selected for human isolates only. Although the functions of some positively selected posi-tions are unknown, our analysis provided evidence of different temporal, spatial and host adaptations for H5N1 avian influenza viruses.

Different infection routes of avian influenza A (H5N1) virus in mice

The continuing outbreaks of avian influenza A H5N1 virus infection in Asia and Africa have caused worldwide concern because of the high mortality rates in poultry,suggesting its potential to become a pandemic influenza virus in humans.The transmission route of the virus among either the same species or different species is not yet clear.Broilers and BABL/c mice were inoculated with the H5N1 strain of influenza A virus isolated from birds.The animals were inoculated with 0.1 mL 106.83 TCID50 of H5N1 virus oronasally,intraperitoneally and using eye drops.The viruses were examined by virological and pathological assays.In addition,to detect horizontal transmission,in each group,healthy chicks and mice were mixed with those infected.Viruses were detected in homogenates of the heart,liver,spleen,kidney and blood of the infected mice and chickens.Virus antigen was not detected in the spleen,kidney or gastrointestinal tract,but detected by Plaque Forming Unit(PFU)assay in the brain,liver and lung without degenerative change in these organs(in the group inoculated using eye drops.The detection results for mice inoculated using eye drops suggest that this virus might have a different tissue tropism from other influenza viruses mainly restricted to the respiratory tract in mice.All chicken samples tested positive for the virus,regardless of the method of inoculation.Avian influenza A H5N1 viruses are highly pathogenic to chickens,but its virulence in other animals is not yet known.To sum up,the results suggest that the virus replicates not only in different animal species but also through different routes of infection.In addition,the virus was detection not only in the respiratory tract but also in multiple extra-respiratory tissues.This study demonstrates that H5N1 virus infection in mice can cause systemic disease and spread through potentially novel routes within and between mammalian hosts.

New light shed on global epidemiology of avian influenza A H5N1 virus infection in humans,1997—2015

With the support by the National Natural Science Foundation of China,the research team led by Prof.Yu Hongjie(余宏杰)at the School of Public Health,Fudan University,Key Laboratory of Public Health Safety,Ministry of Education,and the Key Laboratory of Surveillance and Early Warning on Infectious Disease,Chinese Center for Disease Control and Prevention,has published the paper entitled“Global epi-

Analysis of human infectious avian influenza virus:Hemagglutinin genetic characteristics in Asia and Africa from 2004 to 2009

In the present study,we used nucleotide and protein sequences of avian influenza virus H5N1,which were obtained in Asia and Africa,analyzed HA proteins using ClustalX1.83 and MEGA4.0,and built a genetic evolutionary tree of HA nucleotides.The analysis revealed that the receptor specificity amino acid of A/HK/213/2003,A/Turkey/65596/2006 and etc mutated into QNG,which could bind withá-2,3 galactose andá-2,6 galactose.A mutation might thus take place and lead to an outbreak of human infections of avian influenza virus.The mutations of HA protein amino acids from 2004 to 2009 coincided with human infections provided by the World Health Organization,indicating a“low–high–highest–high–low”pattern.We also found out that virus strains in Asia are from different origins:strains from Southeast Asia and East Asia are of the same origin,whereas those from West Asia,South Asia and Africa descend from one ancestor.The composition of the phylogenetic tree and mutations of key site amino acids in HA proteins reflected the fact that the majority of strains are regional and long term,and virus diffusions exist between China,Laos,Malaysia,Indonesia,Azerbaijan,Turkey and Iraq.We would advise that pertinent vaccines be developed and due attention be paid to the spread of viruses between neighboring countries and the dangers of virus mutation and evolution.