Highly pathogenic avian influenza virus subtype H5N1 in ducks in the Northern part of Cameroon

Highly pathogenic avian influenza (HPAI) virus was first detected in Cameroon in February 2006. Analysis of NA sequences of the virus demonstrated that it is closely related to the H5N1 isolates from Northern Nigeria, Sudan and Ivory Coast, suggesting a common virus ancestor.     

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.     

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.     

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.     

禽流感病毒H5N1变异株灭活疫苗(Re-4株)对鸡、鸭和鹅的免疫效果研究

为系统评估禽流感病毒(AIV)H5N1变异株灭活疫苗(Re-4株)对家禽的免疫效果,本研究将Re-4株油乳剂灭活疫苗免疫SPF鸡和商品蛋鸡、商品鸭及商品鹅。免疫后每周采集血清测定HI抗体,绘制抗体消长曲线,免疫SPF鸡在免疫后2周、3周和50周时以105EID50剂量的强毒株(CK/SX/2/06)进行攻毒。研究结果显示,该疫苗对蛋鸡、鸭、鹅均具有良好的免疫效果,而且SPF免疫鸡血清HI抗体在4log2以上时能够完全抵抗CK/SX/2/06强毒的攻击。因此,根据实验结果推荐该油乳剂灭活疫苗的对上述禽类的免疫程序:商品蛋鸡10日龄颈部皮下注射0.3mL,60日龄和110日龄(开产前)时依次胸肌注射0.5mL和1.0mL进行免疫;商品鸭、鹅在2周龄均以0.5mL首免,5周龄和4月龄左右时以1mL的剂量肌肉注射方式进行加强免疫。     

Assessing the risk of highly pathogenic avian influenza H5N1 transmission through poultry movements in Bali,Indonesia

Indonesia continues to report the highest number of human and poultry cases of highly pathogenic avian influenza H5N1. The disease is considered to be endemic on the island of Bali. Live bird markets are integral in the poultry supply chain on Bali and are important, nutritionally and culturally, for the rural and urban human populations. Due to the lack of biosecurity practiced along the supply chain from producer to live bird markets, there is a need to understand the risks associated with the spread of H5N1 through live bird movements for effective control. Resources to control H5N1 in Indonesia are very limited and cost effective strategies are needed. We assessed the probability a live bird market is infected through live poultry movements and assessed the effects of implementing two simple and low cost control measures on this risk. Results suggest there is a high risk a live bird market is infected (0.78), and risk mitigation strategies such as detecting and removing infected poultry from markets reduce this risk somewhat (range 0.67–0.76). The study demonstrates the key role live poultry movements play in transmitting H5N1 and the need to implement a variety of control measures to reduce disease spread.     

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.     

雉鸡自然感染高致病性禽流感的病理组织学观察

本研究对已经确诊为自然感染发生高致病性禽流感的病死雉鸡的各组织器官进行病理学观察。结果发现,该养殖场的病、死雉鸡出现明显和广泛的出血和组织损伤,腹部、腿部皮下肌肉、肺、脾出血,心肌条索状坏死,胰腺、脾、肾、肝脏等器官灶状坏死。组织病理学变化以典型的病毒性心肌炎、淋巴细胞性脑炎、间质性肺炎、间质性肾炎,急性出血性肠炎,以及胰腺、肝脏、脾脏的变质性炎症等为主要特征。     

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.     

Comparative study of pandemic (H1N1) 2009, swine H1N1, and avian H3N2 influenza viral infections in quails

Quail has been proposed to be an intermediate host of influenza A viruses. However, information on the susceptibility and pathogenicity of pandemic H1N1 2009 (pH1N1) and swine influenza viruses in quails is limited. In this study, the pathogenicity, virus shedding, and transmission characteristics of pH1N1, swine H1N1 (swH1N1), and avian H3N2 (dkH3N2) influenza viruses in quails was examined. Three groups of 15 quails were inoculated with each virus and evaluated for clinical signs, virus shedding and transmission, pathological changes, and serological responses. None of the 75 inoculated (n = 45), contact exposed (n = 15), or negative control (n = 15) quails developed any clinical signs. In contrast to the low virus shedding titers observed from the swH1N1-inoculated quails, birds inoculated with dkH3N2 and pH1N1 shed relatively high titers of virus predominantly from the respiratory tract until 5 and 7 DPI, respectively, that were rarely transmitted to the contact quails. Gross and histopathological lesions were observed in the respiratory and intestinal tracts of quail inoculated with either pH1N1 or dkH3N2, indicating that these viruses were more pathogenic than swH1N1. Sero-conversions were detected 7 DPI in two out of five pH1N1-inoculated quails, three out of five quails inoculated with swH1N1, and four out of five swH1N1-infected contact birds. Taken together, this study demonstrated that quails were more susceptible to infection with pH1N1 and dkH3N2 than swH1N1.     

鸭源H5N1亚型禽流感病毒分离株的致病性及表面蛋白基因序列分析

分析1株2004年从广东省分离获得的H5N1型禽流感病毒株A/Duck/GuangdongJiedong/23/2004对SPF鸡和鸭的致病性,并对其血凝素(HA)基因和神经氨酸酶(NA)基因的序列进行测定,与GenBank中收录的其他序列进行比较。结果,此分离株对SPF鸡和鸭均具有高致病性,且致死率达100%。HA基因与SCK/ST/475/04的同源率最高,而NA基因与Ck/GD/178/04同源率最高。进化分析结果表明,此毒珠与DK/Ch i-na/E319-2/03的HA、NA亲缘关系较近,推测它们来源于同一祖代毒株。推导的HA基因氨基酸裂解位点为-RRRKK-,具有典型高致病性禽流感的特征序列;NA基因颈部49~68位20个氨基酸缺失是近年来H5N1亚型优势流行株共同的遗传标志。     

H5N1亚型禽流感病毒对鸭的致病性研究

为评估H5N1亚型禽流感病毒(AIV)在实验室环境下对鸭的致病力,本研究以无特殊病原(SPF)鸭为模型,对我国近年分离的7株病毒进行了致病力分析。结果发现其中4株病毒对鸭致死率为100%,2株病毒对鸭的致死率分别为60%和80%,另外1株病毒,A/goose/Hubei/51/05(GS/HB/51/05),对鸭无致病力。本研究还发现,与高致病力毒株一样,GS/HB/51/05也可在鸭体内呈全身性复制,并且可通过喉头和泻殖腔向外排泄。我们推测GS/HB/51/05可能是中国南方出现的其他对鸭呈高致病力的H5N1病毒的祖先,对这些病毒的系统研究,可揭示H5N1亚型AIV对鸭的致病力遗传机制。     

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.     

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。     

全球高致病性禽流感风险评估

H5N1高致病性禽流感暴发的风险管理需要对不同的风险因素进行综合评估,这些因素影响不同地理区域传染病的传播和持续时间。禽流感灾害风险评估的关键是制定风险路径,然后用它来发展一种定性或定量的风险评估模型。本文建立在风险分析模型框架基础之上,利用FAO、WHO以及OIE等国际组织和有关国家政府可公开获得的数据和信息,利用ArcGIS对H5N1高致病性禽流感进行风险评估,并对野生鸟类和家禽贸易在H5N1高致病性禽流感传播中的重要性进行分析。     

禽流感病毒H5N1亚型NS1蛋白的真核表达及其在细胞中的分布

为构建禽流感病毒(AIV) H5N1亚型非结构蛋白NS1的真核表达载体,并鉴定其在哺乳动物细胞中的表达与分布,本研究采用RT-PCR技术,从甲型流感病毒的总RNA中扩增NS1全长基因,并将其克隆于pXJ40中,构建真核表达载体pXJ40-HA-NSl.将该重组质粒转染293T细胞,通过western blot方法鉴定表达的NS1蛋白;并以免疫荧光技术观察NS1在H1299细胞中的分布与定位.Western blot结果显示NS1基因编码蛋白获得表达,免疫荧光检测显示NS1蛋白主要存在于细胞核中.本研究为NS1蛋白功能和H5N1亚型AIV致病机制的研究奠定了基础.     

Experimental infection and pathology of clade 2.2 H5N1 virus in gulls

During 2006, H5N1 HPAI caused an epizootic in wild birds, resulting in a die-off of Laridae in the Novosibirsk region at Chany Lake. In the present study, we infected common gulls (Larus canus) with a high dose of the H5N1 HPAI virus isolated from a common gull to determine if severe disease could be induced over the 28 day experimental period. Moderate clinical signs including diarrhea, conjunctivitis, respiratory distress and neurological signs were observed in virus-inoculated birds, and 50% died. The most common microscopic lesions observed were necrosis of the pancreas, mild encephalitis, mild myocarditis, liver parenchymal hemorrhages, lymphocytic hepatitis, parabronchi lumen hemorrhages and interstitial pneumonia. High viral titers were shed from the oropharyngeal route and virus was still detected in one bird at 25 days after infection. In the cloaca, the virus was detected sporadically in lower titers. The virus was transmitted to direct contact gulls. Thus, infected gulls can pose a significant risk of H5N1 HPAIV transmission to other wild migratory waterfowl and pose a risk to more susceptible poultry species. These findings have important implications regarding the mode of transmission and potential risks of H5N1 HPAI spread by gulls.     

不同H9N2亚型禽流感病毒株致病力的比较试验

参照NDV评价毒力的方法对1998—2008年间收集的25株H9N2AIV的致病性进行了比较研究。结果显示,25株H9N2AIV不同毒株间致病性有较大差异,大致分为3类：致病性偏强、致病性中等和致病性较弱。从中选出致病力有差异的8个毒株进行了1日龄雏鸡脑内接种指数（ICPI）、6周龄鸡静脉接种指数（IVPI）以及8周龄SPF鸡人工感染排毒试验,结果证明大部分毒株ICPI和IVPI基本上为0,排毒散毒的高峰期在攻毒后第5天到第6天。在25个毒株中,3#和12#表现出了比较高的致病性,不仅其EID50值最高（分别为10-8.8/0.2mL和10-8.8/0.2mL）、ELD50值最高（分别为10-7.9/0.2mL和10-8.7/0.2mL）,而且鸡胚平均死亡时间也最短（分别为66.5,69h）。3#、12#还出现了1日龄雏鸡脑内接种指数（分别为0.238,0.437）和6周鸡静脉内接种指数（分别为0.34,0.51）。在8周龄SPF鸡人工感染排毒试验中3#和12#毒株的排毒量大,排毒时间也明显长于其他毒株（第2～9天）。以上试验结果表明我国H9N2AIV不同毒株致病性有明显差异,呈多态性,致病性偏强的毒株造成鸡群较高的死亡率。     

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.     

利用假病毒技术筛选H5N1亚型禽流感病毒中和活性单克隆抗体

为筛选H5N1禽流感病毒(AIV)血凝素蛋白(HA)特异性中和抗体,本研究构建了表达HA蛋白的重组质粒,利用该重组质粒及缺失表达人免疫缺陷病病毒1型(HIV-1)囊膜蛋白基因的骨架质粒,构建了表面整合有H5亚型AIV HA蛋白的HIV假病毒。利用该假病毒系统,筛选得到一株具有中和活性的单克隆抗体(MAb)。经测定,该MAb与纯化的全病毒具有较好的反应性,其对HIV-H5HA假病毒的中和效价为64。中和试验表明该MAb能够有效阻断野生型病毒对鸡胚的感染。本研究结果为开发H5N1 AIV的被动免疫治疗方法奠定了基础,同时对亚单位疫苗的研制也具有一定的意义。