The first specific detection of a highly pathogenic avian influenza virus (H5N1) in Ivory Coast

The Virology Laboratory of the Central Laboratory of Animal Diseases in Ivory Coast at Bingerville received samples of wild and domestic avian species between February and December 2006. An RT-PCR technique was used to test for avian influenza (AI) and highly pathogenic AI subtype viruses. Among 2125 samples, 16 were type A positive; of which, 12 were later confirmed to be H5N1. Fifteen of these 16 type A positive samples were inoculated into the chorioallantoic cavity of 11-day-old embryonated hens' eggs for virus isolation. Eight produced virus with hemagglutination titres from 1/64 to 1/512. The 4/16 M-RT-PCR positive samples, which were H5N1 negative, were shown to be H7 subtype negative. The diagnostic efficiency of the laboratory for the surveillance of H5N1 in Ivory Coast was demonstrated. The positive cases of H5N1 were from a sparrowhawk (Accipter nisus); live market poultry and in free-range poultry, where the mortality rate was approximately 20% (2/10) and 96.7% (29/30) respectively. Currently, investigations into intensive poultry farms have proved negative for H5N1. No human cases have been reported this time.     

Molecular and phylogenetic analysis of the H5N1 avian influenza virus caused the first highly pathogenic avian influenza outbreak in poultry in the Czech Republic in 2007

On 19th July 2007 re-occurrence of the H5N1 highly pathogenic avian influenza (HPAI) virus was noticed in Europe. The index strain of this novel H5N1 lineage was identified in the Czech Republic where it caused historically the first HPAI outbreak in commercial poultry. In the present study we performed molecular and phylogenetic analysis of the index strain of the re-emerging H5N1 virus lineage along with the Czech and the Slovak H5N1 strains collected in 2006 and established the evolutionary relationships to additional viruses circulated in Europe in 2005-2006. Our analysis revealed that the Czech and the Slovak H5N1 viruses collected during 2006 were separated into two sub-clades 2.2.1 and 2.2.2, which predominated in Europe during 2005-2006. On the contrary the newly emerged H5N1 viruses belonged to a clearly distinguishable sub-clade 2.2.3. Within the sub-clade 2.2.3 the Czech H5N1 strains showed the closest relationships to the simultaneously circulated viruses from Germany, Romania and Russia (Krasnodar) in 2007 and were further clustered with the viruses from Afghanistan and Mongolia circulated in 2006. The origin of the Czech 2007 H5N1 HPAI strains was also discussed.     

Isolation and characterization of influenza A virus (subtype H5N1) that caused the first highly pathogenic avian influenza outbreak in chicken in Bhutan

We characterized Influenza A/H5N1 virus that caused the first outbreak of highly pathogenic avian influenza (HPAI) in chickens in Bhutan in 2010. The virus was highly virulent to chicken, killing them within two days of the experimental inoculation with an intravenous pathogenicity index (IVPI) of 2.88. For genetic and phylogenetic analyses, complete genome sequencing of 4 viral isolates was carried out. The isolates revealed multiple basic amino acids at their hemagglutinin (HA) cleavage site, similar to other "Qinghai-like" H5N1 isolates. The receptor-binding site of HA molecule contained avian-like amino acids ((222)Q and (224)G). The isolates also contained amino acid residue K at position 627 of the PB2 protein, and other markers in NS 1 and PB1 proteins, highlighting the risk to mammals. However, the isolates were sensitive to influenza drugs presently available in the market. The sequence analysis indicated that the Bhutan viruses shared 99.1-100% nucleotide homology in all the eight genes among themselves and 2010 chicken isolate from Bangladesh (A/chicken/Bangladesh/1151-11/2010) indicating common progenitor virus. The phylogenetic analysis indicated that the Bhutan isolates belonged to sub-clade 2.2.3 (EMA 3) and shared common progenitor virus with the 2010 Bangladesh virus. Based on the evidence of phylogeny and molecular markers, it could be concluded that the outbreaks in Bhutan and Bangladesh in 2010 were due to independent introductions of the virus probably through migratory birds.     

The fowl adenovirus (Fadv-11) outbreak in Iranian broiler chicken farms: The first full genome characterization and phylogenetic analysis

Fowl adenoviruses D and E (FAdV-D and E) can cause inclusion body hepatitis (IBH) in commercial chicken flocks. Recently, IBH outbreaks have been increasingly reported in different regions of Iran, particularly in broiler farms. The present study was conducted to perform, for the first time, a complete genome characterization of a FAdV isolate from an IBH outbreak in Iran. Briefly, liver samples were collected from affected broiler flocks and following viral DNA extraction and confirming by PCR technique; one positive sample was selected from an affected flock to conduct a complete genome sequencing. The current FAdV, named "Fowl_Adenovirus_D_isolate_iran/UT-Kiaee_2018", was placed into FAdV-11 serotype (D species). According to the complete genome sequence analysis, UT-Kiaee had high homology with Chinese and Canadian FAdV. The partial sequence of the hexon gene revealed that UT-Kiaee shared 100% identity with previous Iranian FAdVs. The present study was the first to report full genome FAdV in Iran and complete the puzzle of molecular epidemiology of FAdV in Iran through determining the possible origin of Iranian FAdvs, which are the causative agents of recent IBH outbreaks in Iran.     

Novel reassortant H5N6 highly pathogenic influenza A viruses in Vietnamese quail outbreaks

Avian influenza A H5N6 virus is a highly contagious infectious agent that affects domestic poultry and humans in South Asian countries. Vietnam may be an evolutionary hotspot for influenza viruses and therefore could serve as a source of pandemic strains. In 2015, two novel reassortant H5N6 influenza viruses designated as A/quail/Vietnam/CVVI01/2015 and A/quail/Vietnam/CVVI03/2015 were isolated from dead quails during avian influenza outbreaks in central Vietnam, and the whole genome sequences were analyzed. The genetic analysis indicated that hemagglutinin, neuraminidase, and polymerase basic protein 2 genes of the two H5N6 viruses are most closely related to an H5N2 virus (A/chicken/Zhejiang/727079/2014) and H10N6 virus (A/chicken/Jiangxi/12782/2014) from China and an H6N6 virus (A/duck/Yamagata/061004/2014) from Japan. The HA gene of the isolates belongs to clade 2.3.4.4, which caused human fatalities in China during 2014–2016. The five other internal genes showed high identity to an H5N2 virus (A/chicken/Heilongjiang/S7/2014) from China. A whole-genome phylogenetic analysis revealed that these two outbreak strains are novel H6N6-like PB2 gene reassortants that are most closely related to influenza virus strain A/environment/Guangdong/ZS558/2015, which was detected in a live poultry market in China. This report describes the first detection of novel H5N6 reassortants in poultry during an outbreak as well as genetic characterization of these strains to better understand the antigenic evolution of influenza viruses.     

H10亚型和N8亚型禽流感病毒三重RT-PCR检测方法的建立

根据基因库中H10亚型禽流感病毒(AIV)HA基因、N8亚型AIV NA基因和所有亚型AIV M基因序列,分别设计筛选出3对特异性引物,优化引物之间的浓度,建立了H10亚型和N8亚型AIV三重RT-PCR检测方法。该法对含有H10和N8亚型AIV的模板可特异性扩增出267 bp(H10亚型AIV)、464 bp(N8亚型AIV)和693 bp(AIV)目的条带,对H10亚型AIV扩增出267、693 bp目的条带,对N8亚型AIV扩增出464、693 bp目的条带,对其他亚型AIV仅扩增出693 bp目的条带,对常见禽病病原体均未扩增出任何条带。该法对H10亚型和N8亚型AIV检测下限为10~3拷贝/μL。120份临床样品检测结果与病毒分离鉴定一致。研究建立的H10亚型和N8亚型AIV三重RT-PCR检测方法特异性强、灵敏度高,为同时快速鉴别检测H10亚型和N8亚型AIV提供一种简便、快速和有效的方法。     

Association between outbreaks of highly pathogenic avian influenza subtype H5N1 and migratory waterfowl (family Anatidae) populations

Highly pathogenic avian influenza (HPAI) virus subtype H5N1 threatens poultry production and human health. Understanding the role that migratory waterfowl play in introducing and maintaining this infection is critical to control the outbreaks. A study was conducted to determine if the occurrence of HPAI subtype H5N1 outbreaks in village poultry in Romania, 2005-2006, was associated with proximity to populations of migratory waterfowl. Reported outbreaks--which could be grouped into three epidemic phases--and migratory waterfowl sites were mapped. The migratory waterfowl site closest to each outbreak was identified. The distances between outbreaks occurring in phase 1 and 2 of the epidemic and the closest migratory waterfowl site were significantly (P<0.001) less than in phase 3, but these distances were only useful in predicting when outbreaks occurred during phase 1 (October-December, 2005) of the epidemic. A spatial lag (rho=0.408, P=0.041) model best fit the data, using distance and [distance]*[distance] as predictors (R2=0.425). The correlation between when outbreaks were predicted to occur and when they were observed to occur was 0.55 (P=0.006). Results support the hypothesis that HPAI virus subtype H5N1 infections of village poultry in Romania during the autumn of 2005 might have occurred via exposure to migratory populations of waterfowl.     

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 10^6.83 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.     

一株鸭源H8N4亚型禽流感病毒分离株的全基因组序列分析及致病性的研究

本研究对2012年从湖南活禽市场中分离到的一株鸭源H8N4亚型禽流感病毒(AIV)A/duck/HuN/S3160/2012(H8N4)进行全基因组序列和进化分析,并对其进行SPF鸡、SPF鸭和BALB/c小鼠的致病性试验.序列分析显示:HA裂解位点序列为339pSIEPK ↓ GLF347,为典型的低致病性AIV特征.内部基因来源较复杂,HuN/160/12的PB1、NS基因分别与A/spot-billed duck/Xianghai/427/2011 (H5N2)和A/wild bird/Korea/A81/2009(H5N2)的同源性最高,其余内部基因同源性最高的病毒株来自H2、H3、H4、H7、H10等亚型分离株,呈现明显的异源性.感染性试验结果显示,病毒在SPF鸭体内可以通过呼吸道和消化道向外排毒,并且能够在气管、肾脏、盲肠扁桃体及法氏囊检测到病毒,而不能在鸡体内有效复制及排毒.对小鼠的感染性试验结果显示,仅在鼻甲和肺检测到病毒存在,其他脏器病毒滴定结果为阴性,体重呈一过性下降,表明该病毒为低致病性AIV.     

The indirect immunofluorescence assay using cardiac tissue from chickens, quails and ducks for identification of influenza A virus during an outbreak of highly pathogenic avian influenza virus (H5N1): a rapid and simple screening tool for limited resource settings

Here we describe the diagnostic utility of the indirect immunofluorescence assay (IFA) during a recent outbreak of highly pathogenic avian influenza (HPAI) subtype H5N1 virus in southern Thailand and demonstrate the usefulness of the cardiac tissue from infected chickens, quail, and ducks for diagnosis. The most reliable sample for IFA diagnosis of influenza A virus was cardiac tissue (83.0%; 44/53) which when divided by species (chicken, quail and duck cardiac tissues) gave respective positivity rates of 88% (22/25), 88.9% (16/18) and 60.0% (6/10). Cardiac tissue also gave the highest IFA intensity for the three species. We believe that the IFA method has wide applicability in developing countries or remote settings where clinically similar avian diseases with high morbidity and mortality such as Newcastle disease and fowl cholera are common and could be rapidly excluded thereby conserving valuable reference laboratory capacity for true HPAI outbreaks.     

野鸟源H5N8禽流感病毒遗传变异分析与致病性评估

本试验在野鸟禽流感病毒紧急疫情检测过程中鉴定并分离到1株H5N8高致病性禽流感病毒,利用病毒全基因组测序、系统发育及关键氨基酸位点分析解析了该野鸟源H5N8禽流感病毒分离株遗传进化情况,通过体外复制动力学试验及小鼠感染试验,评价了该野鸟源H5N8禽流感病毒分离株对哺乳动物致病性。进化分析显示,该病毒株属于Clade 2.3.4.4,可以不经适应直接感染小鼠并在呼吸系统内复制,表现出有限的组织嗜性,对小鼠呈低致病性。其在体内外复制能力较低。结果表明,本试验加深了对野生鸟携带H5N8禽流感病毒的认识和理解、对野鸟源H5N8禽流感病毒生物学特性的评价,为预测野鸟源H5N8禽流感病毒遗传进化趋势及其生物安全风险评估提供借鉴和参考。     

Isolation of avian influenza virus (H9N2) from emu in China

This is the first reported isolation of avian influenza virus (AIV) from emu in China. An outbreak of AIV infection occurred at an emu farm that housed 40 four-month-old birds. Various degrees of haemorrhage were discovered in the tissues of affected emus. Cell degeneration and necrosis were observed microscopically. Electron microscopy revealed round or oval virions with a diameter of 80 nm to 120 nm, surrounded by an envelope with spikes. The virus was classified as low pathogenic AIV (LPAIV), according to OIE standards. It was named A/Emu/HeNen/14/2004(H9N2)(Emu/HN/2004). The HA gene (1683bp) was amplified by RT-PCR and it was compared with other animal H9N2 AIV sequences in GenBank, the US National Institutes of Health genetic sequence database. The results suggested that Emu/HN/2004 may have come from an avian influenza virus (H9N2) from Southern China.     

A single dose of inactivated oil-emulsion bivalent H5N8/H5N1 vaccine protects chickens against the lethal challenge of both highly pathogenic avian influenza viruses

In this study, two highly pathogenic avian influenza (HPAI) H5N8 viruses were isolated from chicken and geese in 2018 and 2019 (Chicken/ME-2018 and Geese/Egypt/MG4/2019). The hemagglutinin and neuraminidase gene analyses revealed their close relatedness to the clade-2.3.4.4b H5N8 viruses isolated from Egypt and Eurasian countries. A monovalent inactivated oil-emulsion vaccine containing a reassortant virus with HA gene of the Chicken/ME-2018/H5N8 strain and a bivalent vaccine containing same reassortant virus plus a previously generated reassortant H5N1 strain (CK/Eg/RG-173CAL/17). The safety of both vaccines was evaluated in specific-pathogen-free (SPF) chickens. To evaluate the efficacy of the prepared vaccines, 2-week-old SPF chickens were vaccinated with 0.5 mL of a vaccine formula containing 10⁸/EID₅₀ /dose from each strain via the subcutaneous route. Vaccinated birds were challenged with either wild-type HPAI-H5N8 or H5N1 viruses separately at 3 weeks post-vaccine. Results revealed that both vaccines induced protective hemagglutination-inhibiting (HI) antibody titers as early as 2 weeks PV (≥5.0 log₂). Vaccinated birds were protected clinically against both subtypes (100 % protection). HPAI-H5N1 virus shedding was significantly reduced in birds that were vaccinated with the bivalent vaccine; meanwhile, HPAI-H5N8 virus shedding was completely neutralized in both tracheal and cloacal swabs after 3 days post-infection in birds that had been vaccinated with either vaccine. In conclusion, the developed bivalent vaccine proved to be efficient in protecting chickens clinically and reduced virus shedding via the respiratory and digestive tracts. The applicability of the multivalent avian influenza vaccines further supported their value to facilitate vaccination programs in endemic countries.     

一株H5N3亚型禽流感病毒全基因组序列分析及其对小鼠的感染性研究

为了解H5N3亚型流感病毒的生物学特性,本研究对2017年浙江省分离到的一株H5N3亚型禽流感病毒(AIV)[DK/ZJ/S1368/2017(H5N3)]进行了遗传演化分析及小鼠感染性实验。遗传演化分析结果显示,该株病毒的HA蛋白裂解位点处仅含一个碱性氨基酸,属于低致病性AIV。同时,该病毒的血凝素(HA)基因与H5N7亚型流感病毒亲缘关系较近;聚合酶碱性蛋白2(PB2)基因和核蛋白(NP)基因与H10N7亚型流感病毒亲缘关系较近;碱性聚合酶蛋白1(PB1)基因与H1N1亚型流感病毒亲缘关系较近;酸性聚合酶蛋白(PA)基因与H6N2亚型流感病毒亲缘关系较近;神经氨酸酶(NA)基因与H10N3亚型流感病毒亲缘关系较近;基质蛋白(M)基因与H3N8亚型流感病毒亲缘关系较近;非结构蛋白(NS)基因与H1N1亚型流感病毒亲缘关系较近。表明其基因来源复杂。小鼠感染实验结果显示,该分离株无需提前适应即可以在小鼠肺脏和鼻甲中复制,小鼠感染病毒后无明显临床症状,与对照组相比其体质量变化不明显,表明该病毒对小鼠呈低致病性。本研究通过对该H5N3亚型AIV的生物学特性的分析,发现该病毒基因来源复杂,无需提前适应就可以在小鼠体内复制,具有感染哺乳动物的潜在威胁,提示应当持续加强对H5N3亚型AIV的监测和相关生物学特性的研究工作。     

Pathogenicity and transmission of H5N1 avian influenza viruses in different birds

In this study, we selected three H5N1 highly pathogenic avian influenza viruses (HPAIVs), A/Goose/Guangdong/1/1996 (clades 0), A/Duck/Guangdong/E35/2012 (clade 2.3.2.1) and A/Chicken/Henan/B30/2012 (clade 7.2) isolated from different birds in China, to investigate the pathogenicity and transmission of the viruses in terrestrial birds and waterfowl. To observe the replication and shedding of the H5N1 HPAIVs in birds, the chickens were inoculated intranasally with 10⁶ EID₅₀ of GSGD/1/96, 10³ EID₅₀ of DkE35 and CkB30, and the ducks and geese were inoculated intranasally with 10⁶ EID₅₀ of each virus. Meanwhile, the naive contact groups were set up to detect the transmission of the viruses in tested birds. Our results showed that DkE35 was highly pathogenic to chickens and geese, but not fatal to ducks. It could be detected from all the tested organs, oropharyngeal and cloacal swabs, and could transmit to the naive contact birds. GSGD/1/96 could infect chickens, ducks and geese, but only caused death in chickens. It could transmit to the chickens and ducks, but was not transmittable to geese. CkB30 was highly pathogenic to chickens, low pathogenic to ducks and not pathogenic to geese. It could be transmitted to the naive contact chickens, but not to the ducks or geese. Our findings suggested that H5N1 HPAIVs from different birds show different host ranges and tissue tropisms. Therefore, we should enhance serological and virological surveillance of H5N1 HPAIVs, and pay more attention to the pathogenic and antigenic evolution of these viruses.     

Infectious dose‐dependent accumulation of live highly pathogenic avian influenza H5N1 virus in chicken skeletal muscle—implications for public health

Highly pathogenic avian influenza viruses (HPAIV) of H5N1 subtype are a major global threat to poultry and public health. Export of poultry products, such as chicken and duck meat, is a known source for the cross‐boundary spread of HPAI H5N1 viruses. Humans get infected with HPAI H5N1 viruses either by close contact with infected poultry or through consumption of fresh/undercooked poultry meat. Skeletal muscle is the largest soft tissue in chicken that has been shown to contain virus during systemic HPAIV infection and supports productive virus infection. However, the time between infection of a chicken with H5N1 virus and presence of virus in muscle tissue is not yet known. Further, it is also not clear whether chicken infected with low doses of H5N1 virus that cause non‐fatal subclinical infections continue to accumulate virus in skeletal muscle. We investigated the amount and duration of virus detection in skeletal muscle of chicken experimentally infected with different doses (10², 10³ and 10⁴ EID₅₀) of a HPAI H5N1 virus. Influenza viral antigen could be detected as early as 6 hr after infection and live virus was recovered from 48 hr after infection. Notably, chicken infected with lower levels of HPAI H5N1 virus (i.e., 10² EID₅₀) did not die acutely, but continued to accumulate high levels of H5N1 virus in skeletal muscle until 6 days post‐infection. Our data suggest that there is a potential risk of human exposure to H5N1 virus through meat from clinically healthy chicken infected with a low dose of virus. Our results highlight the need to implement rigorous monitoring systems to screen poultry meat from H5N1 endemic countries to limit the global spread of H5N1 viruses.     

Control versus no control: options for avian influenza H5N1 in Nigeria

In January 2006, an outbreak of a highly pathogenic avian influenza (HPAI) was recorded in Nigeria for the first time. This present work describes an estimation of possible costs associated with a vaccination-based control policy added to other measures to restrict HPAI H5N1 virus infections. The evaluations used epidemiological and production data, including budgets necessary for the vaccine acquisition, distribution and administration in arriving at the final costs. Using decision tree and cost benefit analysis the economical benefits for Nigeria and countries with similar veterinary infrastructures, biosecurity and farming systems are calculated. The result indicated that a halting in the continued spread of the virus through effective control measure will be 52 times better than taking no action. This should help policy makers in deciding in favour of vaccination combined with other tools as an effective means of controlling avian influenza H5N1. * Control of HPAI H5N1 will best be understood by policy makers in financial terms. * Effective control through vaccination of poultry is much cheaper and reduces the chances of human zoonoses. * Poultry vaccination combined with other control measures will be the most effective means of control in most developing economies.     

The spread of highly pathogenic avian influenza (subtype H5N1) clades in Bangladesh, 2010 and 2011

Since the global spread of highly pathogenic avian influenza H5N1 during 2005–2006, control programs have been successfully implemented in most affected countries. HPAI H5N1 was first reported in Bangladesh in 2007, and since then 546 outbreaks have been reported to the OIE. The disease has apparently become endemic in Bangladesh. Spatio-temporal information on 177 outbreaks of HPAI H5N1 occurring between February 2010 and April 2011 in Bangladesh, and 37 of these outbreaks in which isolated H5N1 viruses were phylogenetically characterized to clade, were analyzed.     

一株鸭源H5N2禽流感病毒全基因组序列分析及对鸡的致病性研究

2012年在我国重庆市活禽交易市场进行流行病学调查时,从鸭体内分离到1株H5N2亚型禽流感病毒(AIV),DK/CQ036/12(H5N2).为了解该株H5N2亚型AIV的生物学特性,本研究对其进行了全基因组分析及对SPF鸡和BALB/c小鼠的致病性试验.序列分析显示:HA裂解位点序列为341R-----346TRGLF350,为低致病性AIV特征.内部基因来源较复杂,与KD/CQ/036/12分离株的M基因NP基因同源性最高的病毒株均来自H4、H7等亚型分离株,呈明显的异源性.分离株的感染性试验显示,该分离株在鸡体内可以通过呼吸道和消化道向外排毒,但并不能在鸡体内有效的复制.对小鼠的感染性试验结果显示,仅在鼻甲和肺能检测到病毒存在,其他脏器病毒滴定结果为阴性,表明病毒对鸡和小鼠均呈低致病性.