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.

Emergence of H5N1 highly pathogenic avian influenza in Democratic People's Republic of Korea

In the past decade,there has been extensive global surveillance for highly pathogenic avian influenza(HPAI)infection in both animals and humans,however,few studies on epidemiology of avian influenza in Democratic People’s Republic of Korea(DPRK)were published.During the period 2013–2014,HPAI H5N1 viruses were detected with outbreaks in domestic poultry in DPRK.Phylogenetic analysis revealed that the hemagglutinin gene of all samples belonged to clade 2.3.2.1c with high homology.The HPAI H5N1 virus found in ducks at the Tudan Duck Farm in 2013 was might introduced by migratory birds and then led to the outbreaks on neighboring chicken farms in 2014.These data provide direct evidence for the transmission of avian influenza viruses from wild birds to waterfowl to terrestrial birds.Therefore,the monitoring and control of influenza virus in ducks must be given top priority,which are essential components to prevent and control HPAI.

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 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.

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.

Avian Influenza H5N1 Surveillance in Geese of Qinghai Province, China (2012)

The aim of study was to detect H5N1 virus in wild geese in Qinghai Province in 2012. The work was provided according to WHO and OIE guidelines. In 2012, we collected 532 samples from wild geese of two species: Bar-headed Goose (Anser indicus) and Graylag Goose (Anser anser). We analyzed samples by chicken embryo inoculation and PCR. No avian influenza viruses were isolated. History of HPAI H5N1 shows obvious importance of Central Asian region in its spreading. The outbreaks of the H5N1 Highly Pathogenic Avian Influenza (HPAI H5N1) were reported in wild birds at the Qinghai Lake since 2005. This area seems to be key point for H5N1 avian influenza surveillance in wild birds. We did not find viruses although H5N1 cases in poultry were reported from 5 provinces of China in 2012. Annual surveillance is required for early AIV detection in this region.

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.

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.

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.

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.

Improvement of the efficacy of influenza vaccination (H5N1) in chicken by using extract of Cochinchina momordica seed (ECMS)

Seeds of a Chinese traditional medicine plant, Cochinchina momordica were used in the present study for the improvement of influenza vaccine （HSN 1） in chicken. Crude extraction from Cochinchina momordica seed （ECMS） was obtained by ethanol extraction method. In experiment No. 1, two weeks old chickens were immunized with influenza vaccine （HSN1） alone or combined with ECMS （5, 10, 20, 40 and 80 μg/dose）. Serum IgG antibody levels （by ELISA） as well as effects on dally weight gain were measured on 0, 7, 14 and 28th day after immunization. Results revealed that all ECMS groups numerically increased the antibody levels while 10 and 20 μg/dose groups significantly （P〈0.05） enhanced total IgG antibody on day 28, when compared with control. Average daily weight gain was also significantly higher in 20 μg/dose ECMS group. Adjuvant effect was also confirmed in experiment No. 2 when chickens were immunized with 20 μg/dose ECMS and antibody titer was measured through hemagglutination inhibition （HI）. It is concluded that ECMS has potential to improve the immune responses and deserve further study as an adjuvant.

Risk Analysis of Wild Birds in Spread of Highly Pathogenic Avian Influenza

Outbreaks of highly pathogenic avian influenza （HPAI） H5N 1 have taken place in 15 countries in Asia, Europe and Africa since 2003, and have caused great economic losses. Much likelihood has been considered as risk factors, of which wild birds are attributed as one of the main factors. This is related to the environmental deterioration in the wetland and expanse of human＇s activities in production. The risk analysis in this paper only focused on the effect of wild birds to HPAI, and confirmed the high risk of wild birds in the spread of AIVs.

H5N1 influenza outbreak during March 2006 in Jordan

The highly pathogenic avian influenza H5N1 pandemic motivated countries around the World to be prepared for outbreaks within their borders. Well ahead of the outbreak, in Jordan contingency plans were prepared involving all relevant bodies in both the private and public sectors and a practice was simulated to test the effectiveness of the task force and the applicability of the plan. The highly pathogenic H5N1 influenza virus was detected and confirmed in one turkey in a backyard flock comprising 12 turkeys and 25 chickens in Kufranjah, 6 km from Ajloun city, on 23 March 2006. The samples were confirmed by the following day. Control measures were implemented immediately according to the national contingency plan and complete clean up achieved by 27 March 2006. A 3 km area around the index case was designated as a control zone, with a 10 km area around the holding further designated as a surveillance zone. The objective was to stamp out the infection and approximately 20,000 birds were destroyed including all commercial and backyard flocks in the control zone. All the human contacts were examined, treated and discharged from the hospital. The immediate response from all Jordanian sectors involved in the control strategy was positive as a result of increasing awareness and training programs that were implemented six months before the occurrence of the outbreak. A total estimated economic loss posed by this outbreak including compensation was almost $US169 million. Following the outbreak, passive surveil-lance for the virus was undertaken and training and capacity building continued. Efforts made in the preparation for an outbreak paid dividends and the need for a department responsible for monitoring migratory and wild birds in Jordan was recognized. Legislation regarding biosecurity of poultry farms and poultry transport vehicles should be revised.

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.

一种新型H5N1禽流感病毒血凝素抗原快速检测试剂的建立

利用5株广谱特异性抗H5亚型血凝素单克隆抗体和酶联免疫渗滤技术成功地建立了一种适于现场检测H5亚型禽流感病毒血凝素蛋白的抗原快速检测试剂H5-HA(Ag)Dot-ELISA。该试剂对41株代表当前亚洲地区流行的各种遗传变异亚系H5N1禽流感病毒检测均为阳性,对多数毒株的分析灵敏度优于0.1个血凝滴度(HA titer),其中部分优于0.01个血凝滴度;比较该试剂与早期开发的同类ELISA试剂,发现前者对后者未能检出的H5N1新变异株检测均为阳性;利用该试剂和商品化Directigen Flu A(BD)试剂检测两株H5N1病毒株,提示前者灵敏度高于后者;该试剂对一株H5N1病毒的检测灵敏度与标准RT-PCR相当;该试剂对24株非H5亚型病毒检测均为阴性,显示出良好特异性。以上结果提示,此研究建立的H5N1病毒抗原快速检测试剂在H5禽流感现场检测上具有较好的应用前景。

H5N1高致病性禽流感病毒的危害及生态问题

高致病性禽流感(HPAI)H5N1病毒亚型已对人类健康、养殖业发展、野生鸟类及生态环境带来极大危害,引起国内外广泛关注。研究发现,禽流感病毒通过发生重组或者突变,可产生感染人类或其他生物的新病毒亚型,或产生更高的致病性,而人类亦具有丰富的与H5N1结合的受体。对候鸟迁徙停歇地禽流感调查表明,湿地、湖泊可能是HPAI病毒存活、散播的疫源地,病毒可随着鸟类的迁徙到处传播。因此,野生鸟类及其赖以生存的主要湿地环境处于感染HPAI的风险之中。

H5N1亚型禽流感病毒NP基因的克隆与表达

根据已知H5N1亚型禽流感病毒NP基因序列设计、合成PCR克隆引物。自H5N1阳性病料中提取总RNA,反转录后采用高可信度DNA聚合酶,经PCR扩增NP基因,采用Invitrogen定向表达系统进行克隆表达,纯化获得N末端携带多聚组氨酸标签的重组NP蛋白,分子质量约66.0ku。采用单克隆抗体和阳性血清经ELISA、免疫印迹分析重组蛋白的免疫反应性。结果表明:所获得的重组NP蛋白在ELISA分析中均能与阳性血清和单克隆抗体发生特异性结合,但在免疫印迹分析中仅与阳性血清发生特异性结合。

Highly pathogenic avian influenza A(H5N1)virus outbreak in Peru in 2022-2023

Background:An epizootic of highly pathogenic avian influenza A(H5N1)has spread worldwide since 2022.Even though this virus has been extensively studied for many decades,little is known about its evolution in South America.Methods:Here,we describe the sequencing and characterization of 13 H5N1 genomes collected from wild birds,poultry,and wild mammals in Peru during the genomic surveillance of this outbreak.Results:The samples belonged to the highly pathogenic avian influenza(H5N1)2.3.4.4b clade.Chilean and Peruvian samples clustered in the same group and therefore share a common ancestor.An analysis of the hemag-glutinin and neuraminidase genes detected new mutations,some dependent upon the host type.Conclusions:The genomic surveillance of highly pathogenic avian influenza is necessary to promote the One Health policy and to overcome the new problems entailed by climate change,which may alter the habitats of resident and migratory birds.