Type A influenza viruses in waterfowl in Ohio and implications for domestic turkeys

Because ducks are considered an important reservoir for type A influenza virus, and type A influenza viruses had not been recovered from ducks in Ohio, a 3-year virus surveillance study was conducted in Ohio waterfowl and waterfowl passing through Ohio to determine if domestic turkeys were at risk of exposure to avian influenza (AI) viruses from the waterfowl reservoir. The prevalence of AI infections in ducks during the fall migration averaged about 5.9%. The 55 waterfowl-origin type A influenza viruses recovered from ducks during fall 1986, 1987, and 1988 represented 23 different hemagglutinin-neuraminidase sub-type combinations of type A influenza viruses. Virus recovery frequencies ranged from 3.6% to 7.8% between years, from 2.0% to 8.2% between study sites, from 0.0% to 16.7% for sampling days, and from 0.0% to 14.3% among species of ducks sampled.     

Genomic analysis of influenza A virus from captive wild boars in Brazil reveals a human-like H1N2 influenza virus

Influenza is a viral disease that affects human and several animal species. In Brazil, H1N1, H3N2 and 2009 pandemic H1N1 A(H1N1)pdm09 influenza A viruses (IAV) circulate in domestic swine herds. Wild boars are also susceptible to IAV infection but in Brazil until this moment there are no reports of IAV infection in wild boars or in captive wild boars populations. Herein the occurrence of IAV in captive wild boars with the presence of lung consolidation lesions during slaughter was investigated. Lung samples were screened by RT-PCR for IAV detection. IAV positive samples were further analyzed by quantitative real-time PCR (qRRT-PCR), virus isolation, genomic sequencing, histopathology and immunohistochemistry (IHC). Eleven out of 60 lungs (18.3%) were positive for IAV by RT-PCR and seven out of the eleven were also positive for A(H1N1)pdm09 by qRRT-PCR. Chronic diffuse bronchopneumonia was observed in all samples and IHC analysis was negative for influenza A antigen. Full genes segments of H1N2 IAV were sequenced using Illumina's genome analyzer platform (MiSeq). The genomic analysis revealed that the HA and NA genes clustered with IAVs of the human lineage and the six internal genes were derived from the H1N1pdm09 IAV. This is the first report of a reassortant human-like H1N2 influenza virus infection in captive wild boars in Brazil and indicates the need to monitor IAV evolution in Suidae populations.     

Randomly primed,strand-switching,MinION-based sequencing for the detection and characterization of cultured RNA viruses

RNA viruses rapidly mutate, which can result in increased virulence, increased escape from vaccine protection, and false-negative detection results. Targeted detection methods have a limited ability to detect unknown viruses and often provide insufficient data to detect coinfections or identify antigenic variants. Random, deep sequencing is a method that can more fully detect and characterize RNA viruses and is often coupled with molecular techniques or culture methods for viral enrichment. We tested viral culture coupled with third-generation sequencing for the ability to detect and characterize RNA viruses. Cultures of bovine viral diarrhea virus, canine distemper virus (CDV), epizootic hemorrhagic disease virus, infectious bronchitis virus, 2 influenza A viruses, and porcine respiratory and reproductive syndrome virus were sequenced on the MinION platform using a random, reverse primer in a strand-switching reaction, coupled with PCR-based barcoding. Reads were taxonomically classified and used for reference-based sequence building using a stock personal computer. This method accurately detected and identified complete coding sequence genomes with a minimum of 20× coverage depth for all 7 viruses, including a sample containing 2 viruses. Each lineage-typing region had at least 26× coverage depth for all viruses. Furthermore, analyzing the CDV sample through a pipeline devoid of CDV reference sequences modeled the ability of this protocol to detect unknown viruses. Our results show the ability of this technique to detect and characterize dsRNA, negative- and positive-sense ssRNA, and nonsegmented and segmented RNA viruses.     

Characterization of infectious laryngotracheitis viruses, antigenic comparison by kinetics of neutralization and immunization studies.

Five isolates of infectious laryngotracheitis virus were compared by pock formation on the chorioallantoic membrane of embryonated eggs, plaque size in chicken embryo kidney tissue culture, and antigenic relationship using reciprocal kinetics of neutralization. The A4557-5 strain of infectious laryngotracheitis virus, which causes mild respiratory disease, produced pocks with a zone of edema on the chorioallantoic membrane. A virulent virus (Virus 1), isolated from an outbreak of severe disease characterized by a diphtheritic laryngotracheitis, produced the largest plaques in chicken embryo kidney cell culture. Other virulent viruses (Viruses 2, 3 and V154) did not have unique growth characteristics when grown on the chorioallantoic membrane or in chicken embryo kidney cell culture. All viruses were closely related antigenically as shown by kinetics of neutralization but viruses 2 and 3 were not homogeneous with the other three viruses when neutralized by anti-V154 chicken serum. Following aerosol infection, chickens infected with the A4557-5 virus were immune to challenge with virulent V154 virus. However, in comparison to SA-2 virus, this virus was a less effective immunizing agent when administered by the vent or drinking water methods.     

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.     

Experimental transmission of bovine viral diseases by insemination with contaminated semen or during embryo transfer

Three experimental approaches were used to study transmission of blue tongue (BT), infectious bovine rhinotracheitis (IBR) and bovine virus diarrhoea (BVD) viruses. These were insemination with contaminated semen, experimental infection of embryo donor cows, or transfer of embryos experimentally exposed to virus in vitro to normal recipients. Parameters assessed included number and quality of embryos produced, virus detection (isolation and electron microscopy), serology and histopathology. All superovulated sesceptible cows inseminated with semen containing blue tongue virus (BTV) (n = 2) or infectious bovine rhinotracheitis virus (IBRV) (n = 2) became infected. One cow inseminated with semen containing BTV produced seven virus-free seven-day-old embryos; the second cow failed to produce any embryos. One of two cows inseminated with semen containing IBRV produced two underdeveloped, virus-free embryos while no embryos were produced by the second cow. One of two cows inseminated with semen containing bovine viral diarrhoea virus (BVDV) became infected. Two poorly developed, virus-free seven-day-old embryos were recovered from one of these cows. Superovulated susceptible cows inoculated either intramuscularly with BTV (n = 3) or intranasally with IBR virus (n = 2) became infected. Virus was isolated from some tissues of two BTV-infected cows, neither of which produced embryos. A third BTV-infected cow produced two virus-free embryos collected at necropsy five days after inoculation. One of two cows experimentally infected with IBR virus, produced three embryos but virus was not detected either by electron microscopy (1 embryo) or in cell culture by cytopathic alterations (1 embryo).(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of influenza A/equine/H3N8 virus isolate variation on the measurement of equine antibody responses.

This study has tested the effect of using homologous or heterologous equine influenza A virus isolates to evaluate serum antibody levels to influenza A virus in vaccinated and naturally-infected horses. In addition, the potential effect of antigenic selection of virus variants in egg versus tissue culture propagation systems was studied. Serum antibody levels in samples from horses recently infected with a local influenza A virus isolate (A/equine 2/Saskatoon/1/90) or recently vaccinated with a prototype isolate (A/equine 2/Miami/1/63) were assessed by hemagglutination inhibition and by single radial hemolysis using cell or egg-propagated A/equine 2/Saskatoon/1/90, A/equine 2/Miami/1/63 or A/equine 2/Fontainebleau/1/79. There were no significant differences in hemagglutination inhibition or single radial hemolysis antibody levels obtained with homologous or heterologous isolates or between viruses propagated in either eggs or cell culture. However there was a trend to higher titers in the hemagglutination inhibition assay when cell-propagated virus was used. These results suggest that antigenic variation in equine influenza A virus isolates and host-cell selection of antigenic variants during virus propagation may not be of sufficient magnitude to influence serological evaluation of antibody responses by hemagglutination inhibition or single radial hemolysis.     

Experimental infection of ponies with equine influenza (H3N8) viruses by intranasal inoculation or exposure to aerosols

Infection of seronegative Welsh mountain ponies was established by intranasal instillation or exposure to nebulised aerosol of egg grown H3N8 viruses. Pyrexia and coughing were noted following intranasal instillation and high titres of virus were recovered from the nasopharynx. Exposure to aerosol resulted in more severe clinical signs characterised by high temperatures, dyspnoea, anorexia and coughing; lower levels of virus were recovered from the nasopharynx. The severity of clinical signs and the kinetics of virus shedding were dose-related with the minimal infectious dose being 10(2)EID50/ml when ponies were exposed to aerosols produced by nebulisation of 20ml allantoic fluid. Full clinical signs only developed when ponies were exposed to a dose of 10(6)EID50/ml. It was concluded that exposure to nebulised aerosols of egg grown H3N8 viruses was a more reliable method of inducing clinical influenza than intranasal inoculation and would be more suitable for challenge studies.     

Isolation from turkey breeder hens of a reassortant H1N2 influenza virus with swine, human, and avian lineage genes

Type A influenza viruses can infect a wide range of birds and mammals, but influenza in a particular species is usually considered to be species specific. However, infection of turkeys with swine H1N1 viruses has been documented on several occasions. This report documents the isolation of an H1N2 influenza virus from a turkey breeder flock with a sudden drop in egg production. Sequence analysis of the virus showed that it was a complex reassortant virus with a mix of swine-, human-, and avian-origin influenza genes. A swine influenza virus with a similar gene complement was recently reported from pigs in Indiana. Isolation and identification of the virus required the use of nonconventional diagnostic procedures. The virus was isolated in embryonated chicken eggs by the yolk sac route of inoculation rather than by the typical chorioallantoic sac route. Interpretation of hemagglutination-inhibition test results required the use of turkey rather than chicken red blood cells, and identification of the neuraminidase subtype required the use of alternative reference sera in the neuraminidase-inhibition test. This report provides additional evidence that influenza viruses can cross species and cause a disease outbreak, and diagnosticians must be aware that the variability of influenza viruses can complicate the isolation and characterization of new isolates.     

Replication of a waterfowl-origin influenza virus in the kidney and intestine of chickens

Intravenous inoculation of chickens with a waterfowl-origin type A influenza virus resulted in high titers of virus in kidney tissues and viral nucleoprotein in renal tubular epithelial cells and in intestinal mucosal epithelial cells. Virus titers in kidneys of four of eight clinically normal chickens sampled on days 3 and 5 postinoculation (PI), one dead chicken on day 3 PI, and one dead chicken on day 7 PI exceeded 10(6) mean embryo infectious dose per gram of tissue. Using immunofluorescent and immunoperoxidase staining, viral nucleoprotein was identified in the cytoplasm and nucleus of tubular epithelial cells in kidneys and in nucleus of mucosal epithelial cells lining villi in the lower small intestine. Based on the low intravenous pathogenicity index for this virus (0.3) along with the high virus titers in kidney tissues and localization of viral antigen in kidney important site for replication of avian influenza (AI) virus of low pathogenicity. Recovery of type A influenza viruses from cloacal swabs could result from viral replication in kidneys as well as in the lower intestine and/or the bursa of Fabricius.     

新城疫、禽流感病毒同胚接种的研究

根据新城疫病毒、禽流感病毒两种病毒均能在鸡胚中繁殖的特点,将两种病毒以最适稀释比例稀释后,分别接种于9—11日龄的鸡胚尿囊腔,同时进行两种病毒同胚培养,并利用血凝试验进行病毒效价的测定。结果表明,在同一鸡胚中两种病毒均能较好地增殖,病毒间干扰现象不明显。本试验在增强疫苗的免疫原性和进一步阐明病毒的增殖机理方面具有重要意义。     

RT-PCR快速诊断禽流感

根据禽流感病毒ＮＰ基因的序列分析结果,设计了一对ＮＰ基因特异的引物。采用该对引物,不经病毒分离,直接从禽流感病毒感染鸡的气管、泄殖腔棉拭子和组织样品中提取核酸, ＲＴ～ＰＣＲ可以扩增出 ３２６ｂｐ的 ＮＰ基因片段。采用该技术对１４个亚型禽流感病毒标准参考株,４个亚型１２株国内分离野毒株,ＲＴ－ＰＣＲ检测的结果都呈阳性;对新城疫病毒、传染性法氏囊病毒、传染性支气管炎病毒、传染性喉气管炎病毒以及减蛋综合症病毒,ＲＴ－ＰＣＲ扩增结果都呈阴性。禽流感病毒 Ａ／Ｇｏｏｓｅ／Ｇｕａｎｇｄｏｎｇ（Ｈ５Ｎ１）和 Ａ／Ａｆｒｉｃａｎ　Ｓｔａｒｌｉｎｇ／Ｅｎｇｌａｎｄ（Ｈ７Ｎ１）实验感染鸡样品 ＲＴ－ＰＣＲ检测与鸡胚病毒分离阳性率分别为３４／４２、３２／４２; ２４／５５、２４／５５, 二者符合率大于９５％。 ＲＴ－ＰＣＲ最少可检测到１０ｐｇ的病毒核酸。对山东某地发病鸡场样品进行ＲＴ－ＰＣＲ检测,只用６个小时就可得出准确的诊断结果,证明ＲＴ－ＰＣＲ检测方法敏感特异,可用于禽流感的快速诊断。     

Serological and Virological Surveillance of Avian Influenza A Virus H9N2 Subtype in Humans and Poultry in Shanghai,China, Between 2008 and 2010

We report the serological evidence of low‐pathogenic avian influenza (LPAI) H9N2 infection in an occupational poultry‐exposed population and a general population. A serological survey of an occupational poultry‐exposed population and a general population was conducted using a haemagglutinin‐inhibiting (HI) assay in Shanghai, China, from January 2008 to December 2010. Evidence of higher anti‐H9 antibodies was found in serum samples collected from poultry workers. During this period, 239 H9N2 avian influenza viruses (AIVs) were isolated from 9297 tracheal and cloacal paired specimens collected from the poultry in live poultry markets. In addition, a total of 733 influenza viruses were isolated from 1569 nasal and throat swabs collected from patients with influenza‐like symptoms in a sentinel hospital, which include H3N2, H1N1, pandemic H1N1 and B, but no H9N2 virus was detected. These findings highlight the need for long‐term surveillance of avian influenza viruses in occupational poultry‐exposed workers.     

"Constanze": a trinational project on avian influenza in wild birds at Lake Constance

When highly pathogenic avian influenza H5N1 (HPAI H5N1) arrived at Lake Constance in February 2006, little was known about its ecology and epidemiology in wild birds. In order to prevent virus transmission from wild birds to poultry, the adjacent countries initiated the tri-national, interdisciplinary research program ?Constanze? to investigate avian influenza infections in water birds at Lake Constance. In collaboration with government agencies scientists examined the prevalence of AI virus in the region of Lake Constance for a period of 33 months, compared the effectiveness of different surveillance methods and analysed the migration behaviour of water birds. Although virus introduction from regions as far as the Ural Mountains seemed possible based on the migration behaviour of certain species, no influenza A viruses of the highly pathogenic subtype H5N1 (HPAIV) was found. However, influenza A viruses of different low pathogenic subtypes were isolated in 2.2 % of the sampled birds (swabs). Of the different surveillance methods utilised in the program the sampling of so called sentinel birds was particularly efficient.     

Nephrotropic properties demonstrated by A/chicken/Alabama/75 (H4N8) following intravenous challenge of chickens.

Tissue tropism properties of A/chicken/Alabama/75 (H4N8) were examined after intravenous inoculation of 5-week-old specific-pathogen-free chickens. From 14 clinically normal chickens euthanatized on days 1-20 postinoculation, the frequencies of virus recovery were highest for cloacal swabs (86%), bursal swabs (64%), and kidney tissues (64%) and lowest for tracheal swabs (14%), thymus tissues (14%), bone-marrow swabs (7%), and brain tissues (0%). Evidence that the high frequency of virus recovery from kidney tissues was associated with virus replication in the kidney tissues was provided by high virus titers, ranging up to 10(9.5) mean embryo infectious dose per gram of kidney tissue, and by identification of intranuclear and intracytoplasmic type A influenza nucleoprotein in kidney cells using immunohistochemistry. Virus-recovery and virus titer results from three chickens that died on days 4 and 5 postinoculation paralleled the results from the clinically normal chickens. These findings indicate that A/chicken/Alabama/75 has nephrotropic properties similar to nephrotropic properties previously reported for waterfowl-origin type A influenza viruses and provide evidence that kidney lesions could be manifestations of systemic influenza infections in commercial laying chickens.