Pathogenicity of avian influenza viruses isolated from wild mallard ducks and domestic turkeys

Groups of turkeys were exposed to different isolates of avian influenza virus from wild mallard ducks and domestic turkeys by the intracerebral, intravenous, intratracheal, and intra-airsac routes, and pathogenicity indices were calculated. For the intracerebral pathogenicity study, body weight was also measured. For intravenous, intratracheal, and intra-airsac pathogenicity studies, necropsy lesions were scored and serological responses were recorded. Only the intracerebral pathogenicity index and body weight gain post intracerebral infection demonstrated any differences between isolates. The other procedures failed to demonstrate any pathogenicity whatsoever. There was a correlation (R = 0.73) between intracerebral pathogenicity index and reduced weight gain postinfection. These studies suggest that growth suppression may be an objective measure of pathogenic potential of influenza viruses found to be nonpathogenic by other methods.     

The pathogenicity of four avian influenza viruses for fowls, turkeys and ducks

Groups of 10 two-week-old chicks, turkey poults and ducklings were each infected by the intranasal route with one of four avian influenza viruses: a/fowl/Germany/34 (Hav 1N))--Rostock, A/FPV/Dutch/27 (Hav 1 Neq 1)--Dutch, A/fowl/Victoria/75 (Hav 1 Neq 1)--Australian, and A/parrot/Ulster/73 (Hav 1 N1)--Ulster. Eight hours after infection 10 birds of the same age and species were placed in contact with each group and allowed to mix. The clinical signs of disease and onset of sickness and death were recorded. Ulster virus was completely avirulent for all birds. Rostock, Dutch and Australian viruses were virulent for fowls and turkeys causing death in all birds with the exception of 3/10 in contact fowls from the Rostock virus group and 2/10 in contact fowls from the Australian virus group. Only Rostock virus caused sicked sickness or death in ducks, 9/10 intranasally infected and 6/7 in contact birds showed clinical signs and 2/10 intranasally infected and 3/7 in contact ducks died. Intranasal and in contact pathogenicity indices were calculated for each virus in each bird species and indicated quantitatively the differences in virulence of the four virus strains. Virus isolation and immune response studies indicated that surviving in contact fowls in the Rostock virus group had never been infected but that surviving Australian virus in contact fowls had recovered from infection. Infection was not established in Ulster virus in contact fowls and Australian virus intranasally infected and in contact ducks. The birds in all other groups showed positive virus isolations and a high incidence of positive immune response. The last virus isolation was made at 22 days after intranasal infection of ducks with Ulster virus.     

两株鸭源H5N1亚型禽流感病毒的序列分析及致病性研究

本研究对2010年在湖北活禽市场监测分离到的两株鸭源H5N1亚型禽流感病毒(AIV) (HuB/495/10和HuB/513/10)进行了序列分析和致病性试验研究,以了解湖北地区H5N1亚型AIV的生物学特性差异.序列分析显示:2株病毒全基因组核苷酸同源性在97.3 ％～98.6％,2株病毒的8个节段基因均与青海和香港分离的野鸟源病毒A/great crested-grebe/Qinghai/1/2009 (H5N1)和A/black-crowned night heron/Hong Kong/659/2008 (H5N1)的核苷酸高度同源,HA蛋白裂解位点序列基序为341RRRKR345,呈现典型的高致病力分子特征.以105 EID50/100 μL病毒剂量感染4周龄SPF鸭发现:HuB/495/10和HuB/513/10对鸭的致死率分别为100％和20％,病毒在鸭体内呈全身性复制并可通过呼吸道和消化道向外排毒;不同滴度的病毒感染6周龄BALB/c小鼠,HuB/495/10和HuB/513/10的MLD50分别为1.38 log10 EID50和1.68 log10 EID50,对小鼠表现为高致病力,均在肺脏中高拷贝复制.     

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。     

Public health risk from avian influenza viruses

Since 1997, avian influenza (AI) virus infections in poultry have taken on new significance, with increasing numbers of cases involving bird-to-human transmission and the resulting production of clinically severe and fatal human infections. Such human infections have been sporadic and are caused by H7N7 and H5N1 high-pathogenicity (HP) and H9N2 low-pathogenicity (LP) AI viruses in Europe and Asia. These infections have raised the level of concern by human health agencies for the potential reassortment of influenza virus genes and generation of the next human pandemic influenza A virus. The presence of endemic infections by H5N1 HPAI viruses in poultry in several Asian countries indicates that these viruses will continue to contaminate the environment and be an exposure risk with human transmission and infection. Furthermore, the reports of mammalian infections with H5N1 AI viruses and, in particular, mammal-to-mammal transmission in humans and tigers are unprecedented. However, the subsequent risk for generating a pandemic human strain is unknown. More international funding from both human and animal health agencies for diagnosis or detection and control of AI in Asia is needed. Additional funding for research is needed to understand why and how these AI viruses infect humans and what pandemic risks they pose.     

Pathogenicity in quails and mice of H5N1 highly pathogenic avian influenza viruses isolated from ducks

In our study, the pathogenicity of H5N1 influenza A viruses circulating in waterfowls in Southern China was investigated. Three H5N1 highly pathogenic avian influenza (HPAI) viruses isolated from ducks, A/Duck/Guangdong/383/2008(DK383), A/Duck/Guangdong/378/2008(DK378) and A/Duck/Guangdong/212/2004(DK212) were inoculated at 10(6) fifty-percent egg infectious doses (EID(50)) into ducks, quails and mice and showed varying levels of pathogenicity. In ducks, the mortality rates ranged from 0 to 60% and the mean death time (MDT) was 0-6.7 days post-inoculation (DPI). While the viruses were highly pathogenic in quails, resulting in 83.3-100% mortality and the MDT of 2.3-3 DPI, they were completely lethal in mice (100% mortality). The viruses replicated in many organs of ducks and quails and were found in the brain, and kidney, lung and spleen of the mice. Phylogenetic analysis revealed that DK383 and DK378 viruses of clade 2.3.2 belonged to genotype 11, while DK212 virus of clade 9 was genotype 3. Our study illustrated H5N1 influenza viruses within Clade 2.3.2 and 9 from duck in Southern China had very highly pathogenicity to Japanese quails and BALB/c mice, but viruses within Clade 2.3.2 had more highly lethality than those of clade 9 to Muscovy ducks. Therefore, they had posed a continued challenge for disease control and public health.     

Avian influenza viruses from migratory and resident ducks of coastal Louisiana

Cloacal and tracheal swabs were collected from 1389 hunter-killed ducks in Cameron Parish, Louisiana, during the 1986 and 1987 waterfowl seasons. Twenty-eight avian influenza viruses (AIVs) were isolated from 605 blue-winged teal (Anas discors), 75 mottled ducks (A. fulvigula), 375 gadwalls (A. stepera) and 334 green-winged teal (A. crecca). Prevalence estimates of AIV in ducks sampled during September, November, and December through January were 3.1%, 2.0%, and 0.4%, respectively. Differences in prevalence were detected by season (P = 0.044) and age class (P = 0.036). Two isolations from resident mottled ducks document transmission of AIV on these wintering areas. Much subtype diversity was present, with nine of 13 hemagglutinin (HA) and nine of nine neuraminidase (NA) subtypes recovered. Predominant HA and NA subtypes were typical of AIVs commonly associated with waterfowl. Results indicate that AIVs are transmitted in the wintering areas, and, although prevalence is low, these viruses continue to circulate within these duck populations during winter.     

Evolution of avian influenza viruses

Although influenza viruses can infect a wide variety of birds and mammals, the natural host of the virus is wild waterfowl, shorebirds, and gulls. When other species of animals, including chickens, turkeys, swine, horses, and humans, are infected with influenza viruses, they are considered aberrant hosts. The distinction between the normal and aberrant host is important when describing virus evolution in the different host groups. The evolutionary rate of influenza virus in the natural host reservoirs is believed to be slow, while in mammals the rate is much higher. The higher rate of evolution in mammals is thought to be a result of selective pressure on the virus to adapt to an aberrant host species. Chickens and turkey influenza virus isolates have previously and incorrectly been lumped together with wild waterfowl, gull, and shorebird influenza viruses when determining rates of evolutionary change. To determine mutational and evolutionary rates of a virus in any host species, two primary assumptions must be met: first, all isolates included in the analysis must have descended from a single introduction of the virus, and second, the outbreak must continue long enough to determine a trend. For poultry, three recent outbreaks of avian influenza meet these criteria, and the sequences of the hemagglutinin and nonstructural genes were compared. Sequences from all three outbreaks were compared to an avian influenza virus consensus sequence, which at the amino acid level is highly conserved for all the internal viral proteins. The consensus sequence also provides a common point of origin to compare all influenza viruses. The evolutionary rates determined for all three outbreaks were similar to what is observed in mammals, providing strong evidence of adaptation of influenza to the new host species, chickens and turkeys.     

Quantitative transmission characteristics of different H5 low pathogenic avian influenza viruses in Muscovy ducks

EU annual serosurveillance programs show that domestic duck flocks have the highest seroprevalence of H5 antibodies, demonstrating the circulation of notifiable avian influenza virus (AIV) according to OIE, likely low pathogenic (LP). Therefore, transmission characteristics of LPAIV within these flocks can help to understand virus circulation and possible risk of propagation. This study aimed at estimating transmission parameters of four H5 LPAIV (three field strains from French poultry and decoy ducks, and one clonal reverse-genetics strain derived from one of the former), using a SIR model to analyze data from experimental infections in SPF Muscovy ducks. The design was set up to accommodate rearing on wood shavings with a low density of 1.6 ducks/m²: 10 inoculated ducks were housed together with 15 contact-exposed ducks. Infection was monitored by RNA detection on oropharyngeal and cloacal swabs using real-time RT-PCR with a cutoff corresponding to 2–7 EID50. Depending on the strain, the basic reproduction number (R₀) varied from 5.5 to 42.7, confirming LPAIV could easily be transmitted to susceptible Muscovy ducks. The lowest R₀ estimate was obtained for a H5N3 field strain, due to lower values of transmission rate and duration of infectious period, whereas reverse-genetics derived H5N1 strain had the highest R₀. Frequency and intensity of clinical signs were also variable between strains, but apparently not associated with longer infectious periods. Further comparisons of quantitative transmission parameters may help to identify relevant viral genetic markers for early detection of potentially more virulent strains during surveillance of LPAIV.     

Genetic relationship of H3 subtype avian influenza viruses isolated from domestic ducks and wild birds in Korea and their pathogenic potential in chickens and ducks

The H3 subtype avian influenza virus (AIV) is one of the most frequently isolated subtypes in domestic ducks, live poultry markets, and wild birds in Korea. In 2002-2009, a total of 45 H3 subtype AIVs were isolated from the feces of clinically normal domestic ducks (n=28) and wild birds (n=17). The most prevalent subtypes in domestic ducks were H3N2 (35.7%), H3N6 (35.7%), H3N8 (25.0%), and H3N1 (3.6%, novel subtype in domestic duck in Korea). In contrast, H3N8 (70.6%) is the most prevalent subtype in wild birds in Korea. In the phylogenetic analysis, HA genes of the Korean H3 AIVs were divided into 3 groups (Korean duck, wild bird 1, and wild bird 2) and all viruses of duck origin except one were clustered in a single group. However, other genes showed extensive diversity and at least 17 genotypes were circulating in domestic ducks in Korea. When the analysis expanded to viruses of wild bird origin, the genetic diversity of Korean H3 AIVs became more complicated. Extensive reassortments may have occurred in H3 subtype influenza viruses in Korea. When we inoculated chickens and ducks with six selected viruses, some of the viruses replicated efficiently without pre-adaptation and shed a significant amount of viruses through oropharyngeal and cloacal routes. This raised concerns that H3 subtype AIV could be a new subtype in chickens in Korea. Continuous surveillance is needed to prepare the advent of a novel subtype AIV in Korea.     

H5亚型鸭源流感病毒HA基因序列生物信息学分析

A型流感病毒（AIV）引起的禽类禽流行性感冒（avian influenza,AI）或相关疾病,被国际兽疫局定为甲类传染病。AIV中最容易突变的基因是血凝素（HA）基因,具有亚型和株的特异性,是区分病毒亚型的依据之一。本研究从GenBank中下载了所有209条鸭源H5禽流感病毒HA基因的全序列,利用生物信息学软件构建进化树研究序列的聚类特点;比较不同年份毒株的HA基因的受体结合位点氨基酸,找出受体结合位点氨基酸的变异规律;比较不同年份分离的鸭源H5N1序列的HA1和HA2蛋白的剪切位点,找出各年份毒株的剪切位点的特点;比较不同年份分离序列的潜在糖基化位点,统计各年份潜在糖基化位点的数量以及序列变化。期望找到H5亚型鸭源流感病毒的变异规律和变异方向,为鸭源流感的防控起到积极的作用。     

Persistence of avian influenza viruses in water

Persistence of five avian influenza viruses (AIVs) derived from four waterfowl species in Louisiana and representing five hemagglutinin and neuraminidase subtypes was determined in distilled water at 17 C and 28 C. Infectivity was determined over 60 days by microtiter endpoint titration. One AIV was tested over 91 days at 4 C. Linear regression models for these viruses predicted that an initial concentration of 1 x 10(6) TCID50/ml water could remain infective for up to 207 days at 17 C and up to 102 days at 28 C. Significant differences in slopes for AIV persistence models were detected between treatment temperatures and among viruses. Results suggest that these viruses are adapted to transmission on waterfowl wintering habitats. Results also suggest a potential risk associated with waterfowl and domestic poultry sharing a common water source.     

胶体金方法检测H3和H7亚型禽流感病毒

用微波法制备金溶胶,对禽流感病毒(AIV)H3和H7亚型单克隆抗体(H3-1D6和H7-1F7)用亲和层析法进行纯化,优化单抗与金溶胶的最佳结合条件后,喷涂于玻璃纤维上制成金标垫.将纯化的马抗H3和H7亚型AIV多克隆抗体和山羊抗鼠二抗分别包被于硝酸纤维素(NC)膜上作为检测线和质控线,制备胶体金检测试纸条.用制备的试纸条对H3和H7亚型AIV标准抗原、毕赤酵母真核表达蛋白和已知样品进行检测,结果与血凝试验、血凝抑制试验、AC-ELISA和RT-PCR方法相符.同时用该方法对H5和H9亚型AIV标准抗原、病料以及传染性支气管炎、新城疫等抗原进行检测,结果均为阴性.该方法操作简单,肉眼于10 min内可判定结果,且达到了血凝试验和血凝抑制试验的敏感性.     

Intracerebral pathogenicity for chickens of avian influenza viruses isolated from free-living waterfowl in Japan

The pathogenicity for chickens of 91 strains of avian influenza A virus isolated from such free-living waterfowl as whistling swan, pintail, tufted duck, mallard and black-tailed gull in Japan was tested. The majority of the virus strains infected and were pathogenic for the chickens. The virulence of these viruses seemed not to be as high as that of fowl plague virus. There were no significant differences in the intracerebral index score among the viruses belonging to the same subtype, irrespective of year of isolation or host.     

Antigenic and genetic analyses of H5 influenza viruses isolated from ducks in Asia.

The hemagglutinin (HA) of six H5 influenza virus strains isolated from ducks in Japan and China in 1976 to 1996 were analyzed antigenically and genetically. Antigenic analysis using a panel of monoclonal antibodies revealed that the HA of H5 influenza viruses isolated from ducks are antigenically closely related to each other. Phylogenetic analysis indicates that the isolates from ducks in Hokkaido were derived from an ancestor common with the highly pathogenic isolates from chickens and humans in Hong Kong in 1997.     

Pathological lesions in the lungs of ducks infected with influenza A viruses

To determine histopathological damage in the respiratory tract, ducks were inoculated with five different influenza A viruses, including viruses virulent for other avian hosts. Lungs were collected for detection of virus and histopathological examination. Small amounts of infectious virus were recovered from lungs, and viral antigens were demonstrated by immunoperoxidase staining with monoclonal antibodies to the viral nucleoprotein. Although clinical signs were not detected, lungs of ducks infected with both virulent and avirulent viruses had mild pneumonia characterized by infiltrates of lymphocytes and macrophages. These findings show that although clinical signs are not evident, ducks may have damage to the respiratory tract during influenza.