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.

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.

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.

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.

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.

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.

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

Objective In China, 24 cases of human infection with highly pathogenic avian influenza(HPAI) H5 N6 virus have been confirmed since the first confirmed case in 2014. Therefore, we developed and assessed two H5 N6 candidate vaccine viruses(CVVs).Methods In accordance with the World Health Organization(WHO) recommendations, we constructed two reassortant viruses using reverse genetics(RG) technology to match the two different epidemic H5 N6 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.Results The 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.Conclusion We obtained two genetically stable CVVs of HPAI H5N6 with high growth characteristics,which may aid in our preparedness for a potential H5N6 pandemic.

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.

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.

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.

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.

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.

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.

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.

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.

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.

International chicken trade and increased risk for introducing or reintroducing highly pathogenic avian influenza A (H5N1) to uninfected countries

Every year billions of chickens are shipped thousands of miles around the globe in order to meet the ever increasing demands for this cheap and nutritious protein source.Unfortunately,transporting chickens internationally can also increase the chance for introducing zoonotic viruses,such as highly pathogenic avian influenza A(H5N1)to new countries.Our study used a retrospective analysis of poultry trading data from 2003 through 2011 to assess the risk of H5N1 poultry infection in an importing country.We found that the risk of infection in an importing country increased by a factor of 1.3(95%CI:1.1e1.5)for every 10-fold increase in live chickens imported from countries experiencing at least one H5N1 poultry case during that year.These results suggest that the risk in a particular country can be significantly reduced if imports from countries experiencing an outbreak are decreased during the year of infection or if biosecurity measures such as screening,vaccination,and infection control practices are increased.These findings show that limiting trade of live chickens or increasing infection control practices during contagious periods may be an important step in reducing the spread of H5N1 and other emerging avian influenza viruses.