Complete genome characterization of avian influenza virus subtype H9N2 from a commercial quail flock in Egypt

The suspected presence of avian influenza virus subtype H9N2 in poultry in Egypt is a major concern since this subtype is widely distributed in different countries in the Middle East, here we describe the full genetic characterization of an avian influenza A virus (Qa/Egypt/11; H9N2) of subtype H9N2 that was previously isolated from a clinically normal quail flock in Giza, Egypt in May 2011. The nucleotide sequence analysis of the hemagglutinin gene of the isolated Egyptian virus showed the highest similarity with one group of recent Israeli strains (97?%) circulating from 2006-2010. Sequence homology and phylogenetic analysis indicated that the Qa/Egypt/11 isolate belonged to the A/quail/Hong Kong/G1/1997-like lineage with new mutations identified in all viral proteins. The phylogenetic analysis for the eight genes indicated placement of the Egyptian virus within the same lineage of H9N2 viruses that circulated in the region from 2006, especially with one group of recent Israeli strains. However, phylogenetic analysis of the internal genes like PB2, NP, and PA genes identified possible reassortment events for these genes with singular Israeli strains. This study indicates progressive evolution of this subtype in the Middle East region and possible mechanism of virus adaptation in land-based poultry like in quails.     

Isolation and serological differentiation of a herpesvirus from bobwhite quail (Colinus virginianus,L. 1758)

Summary An infectious agent was isolated from the liver of bobwhite quails (Colinus virginianus, L. 1758). The agent was sensitive to chloroform and its multiplication was inhibited by 5-iodine-2-deoxy-uridine. It passed filters with a pore diameter of 220 nm and more but not 100 nm filters. Electron microscopic examination revealed numerous nucleocapsids with hollow capsomeres and few enveloped particles in the supernatant fluids of infected cultures. The nucleocapsids were calculated to have 162 capsomeres on their surface. Using the plaque reduction method for neutralization tests no serological cross reactions could be detected between the quail isolate and sera against Marek's disease virus, turkey herpesvirus (HV), duck enteritis HV, infectious laryngotracheitis HV, amazon parrot HV, great horned owl HV, eagle owl HV, snowy owl HV, falcon HV, pigeon HV, Lake Victoria Cormorant HV, and stork HV. The isolate from bobwhite quail did only cross-react with antiserum against crane HV. It is concluded that the isolated virus is a member of the avian herpesvirus group and it is proposed to tentatively term it herpesvirus colinum (fromColinus virginianus = bobwhite quail).With 1 Figure     

Reproductive capacities of control bobwhite quail (Colinus virginianus) during one-generation reproduction studies

The Bobwhite quail (Colinus virginianus) is the avian model of choice for testing the reproductive impairment potential of pesticides and environmental contaminants. While the literature contains numerous reports on the effects of pesticides on eggshell development for quail, mallards and other avian species (Haegele and Tucker, 1974; Kolaja, 1977; Jenkins et al., 1974) limited control data for reproductive evaluation of pesticides can be found. This paper summarizes the control data from three one-generation reproductive studies in Bobwhite quail. One male and two female adult quail were housed in clean pens with tap water and game bird breeder ration available ad libitum. The pre-laying cycle was ten weeks in duration. The photoperiod was seven hours of light per day for the first eight weeks and was increased to 17 hours of light per day in order to induce egg laying. Eggs were collected daily for eight weeks and incubated. Biweekly, one egg from each pen was removed and used for determination of eggshell thickness. The mean reproductive indices for these studies are: Eggs cracked/Eggs laid--8.2%, Viable 11-day embryos/Eggs set--73.6%, Viable 21-day embryos/Viable 14-day embryos--98.9%, Hatchlings/Viable 21-day embryos--82.0%, 14-day survivors/Hatchlings--76.3%. The mean eggshell thickness for 144 eggs was 0.301 mm.     

Prenatally elevated physiological arousal interferes with perceptual learning in bobwhite quail (Colinus virginianus) embryos

Neonatal studies suggest elevated arousal can negatively influence perceptual and cognitive processes during early development. The authors explored this issue during the prenatal period by pharmacologically elevating physiological arousal in bobwhite quail (Colinus virginianus) embryos during exposure to a maternal call, then assessing preference for the familiar call following hatching. Embryos receiving norepinephrine showed a prenatal elevation in heart rate and failed to demonstrate a preference for the familiar call following hatching. Embryos not receiving norepinephrine showed no elevation in heart rate and demonstrated a preference for the familiar call. These results indicate elevated arousal can interfere with perceptual learning during the prenatal period and provide additional evidence for an optimal window of arousal necessary to foster species-typical perceptual functioning during early development.     

Prenatal visual experience influences the development of turning bias in bobwhite quail chicks (Colinus virginianus)

This study examined the effects of prenatal sensory experience on the development of turning bias in a precocial avian species (bobwhite quail). Control tests with naive bobwhite quail chicks revealed a left-side turning bias in 85% of subjects. Such large population biases are considered unusual in nonhuman species. Experiments 1, 2, and 3 demonstrated that prenatal visual experience is a significant contributor to this population level left-side turning bias in bobwhite quail chicks. In contrast, prenatal auditory experience did not appear to significantly influence the development of postnatal turning bias. The findings of this study are discussed in terms of an epigenetic theory for the development of hemispheric specialization and behavioral asymmetry. © 1998 John Wiley & Sons, Inc. Dev Psychobiol 32: 327–338, 1998     

Effects of prenatal visual stimulation on growth and heart rate in bobwhite quail (Colinus virginianus)

This study examined the effects of prenatal visual stimulation on bobwhite quail embryos' growth and heart rate. No differences in growth rate were found between embryos exposed to visual stimulation during the late prenatal period and control embryos. Embryos exposed to visual stimulation throughout incubation maintained lower heart rates in response to visual stimulation than did naïve embryos. In a subsequent experiment, naïve embryos that underwent an egg‐opening procedure exhibited heart rates that were lower than embryos measured in intact eggshells. Embryos in opened eggs maintained lower heart rates than comparison embryos across time; however, a less invasive egg‐opening procedure led to a quicker heart rate recovery than did a more invasive egg‐opening procedure. These findings indicate that prenatal heart rate responses may be mediated by multiple features of the organism's developmental context, including intensity and duration of sensory stimulation. © 2006 Wiley Periodicals, Inc. Dev Psyshobiol 48: 315–324, 2006.     

Experimental infection of non-human primates with avian influenza virus (H9N2)

Several cases of humans infected with the H9N2 avian influenza virus (AIV) have been described since 1999; however, the infectivity and pathogenicity of H9N2 in humans is not well defined. A non-human primate model in rhesus macaques was developed to study H9N2 virus infections as a means of better understanding the pathogenesis and virulence of this virus, in addition to testing antiviral drugs. Rhesus macaques inoculated with H9N2 AIV presented with biphasic fever and viral pneumonia. H9N2 was recovered from nasal washes and pharyngeal samples up to days 7-9 postinfection, followed by an increase in HI (hemagglutination inhibition) antibody titers. Tissue tropism and immunohistochemistry indicated that H9N2 AIV replicated in the upper respiratory tract (turbinate, trachea, and bronchus) and in all lobes of the lung. Our data suggest that rhesus macaques are a suitable animal model to study H9N2 influenza virus infections, particularly in the context of viral evolution and pathogenicity.     

禽H9N2亚型流感病毒能感染人的发现

目的了解禽（Ｈ９Ｎ２）亚型流感病毒是否能感染人。方法对人、鸡和猪进行Ｈ９亚型毒株血清流行病学调查。对流感样患者和鸡咽喉部采样,用常规鸡胚双腔法分离流感病毒并进行毒株鉴定。对分离出Ｈ９Ｎ２亚型毒株的患者进行个案调查。结果约１９％的人含有对Ｈ９Ｎ２毒株的抗体,其ＨＩ滴度为≥２０,从流感样患者中分离到５株Ｈ９Ｎ２病毒。结论Ｈ９Ｎ２亚型毒株能自然感染人。     

广州地区禽H9N2亚型流感病毒的发现及感染人调查

目的 了解广州地区禽流感病毒在家禽中的流行及感染人的情况，防止香港H5N1禽流感在广州地区流行。方法 对广州地区的主要鸡场和农贸市场的家禽和密切接触家禽的职业人群进行病原学和血清学的检测。病毒分离同时采用MDCK细胞和鸡胚双腔接种法；采用微量血凝抑制半致敏法进行血清学检测。结果 从54份鸡咽拭液中分离到1株H9N2亚型流感病毒；鸡及职业人群血对分离的H9N2毒株的血抑抗体阳性率分别为12．8％和15．1％。结论 广州地区鸡群中有H9N2，亚型流感病毒存在，禽H9N2亚型流感病毒能感染人。     

Enriched rearing facilitates spatial exploration in northern bobwhite (Colinus virginianus) neonates

Rearing with enriched environmental complexity has been shown to enhance spatial exploration and spatial learning, among other benefits. Most studies examining enriched rearing effects have used extended periods of exposure in mammals; little is known about enrichment effects on avian species. We provided objects designed to encourage spatial exploration to socially raised northern bobwhite neonates; controls were socially raised without the enrichment devices. After 3-5 days of exposure, maze performance was assessed for 48 chicks. Chicks in the enriched group showed significantly more spatial activity in the maze than control chicks. These results extend the generality of spatial enrichment effects to birds and to a relatively short duration of enrichment exposure.     

Thermal stress influences clinical chemistry values of northern bobwhite (Colinus virginianus)

Values of serum biochemistry were determined for 120 adult male Northern bobwhite (Colinus virginianus) that were either maintained at thermoneutrality or exposed to thermal stress. Thermal stress did not affect serum aspartate aminotransferase or albumin concentrations. Lacate dehydrogenase and uric acid in serum of cold-stressed birds were greater than concentrations of serum from thermoneutral or heat-stressed birds. Serum triglyceride concentrations of both cold- and heat-stressed birds were higher than concentrations from birds maintained at thermoneutrality. Serum total protein was greater in cold-stressed birds than in heat-stressed birds.     

Coinfection of avian influenza virus (H9N2 subtype) with infectious bronchitis live vaccine

Avian influenza virus of H9N2 subtype is pathotyped as a non-highly pathogenic virus. However, frequent incidences of avian influenza of high mortality that are caused by H9N2 viruses have been observed in broiler chicken farms in Iran and some other Asian countries. Coinfections or environmental factors may be involved in such cases. Infectious microorganisms have been implicating in taking part in the cases of coinfection. We studied the effect of experimental coinfection of H9N2 avian influenza virus with infectious bronchitis live vaccine, which is used extensively in chicken farms in Iran. Clinical signs, gross lesions, viral shedding and mortality rate of the experimentally infected birds were examined. Coinfection of infectious bronchitis live vaccine and H9N2 avian influenza virus led to an extension of the shedding period of H9N2 virus, increasing the severity of clinical signs and mortality rates, causing macroscopic lesions in the embryos.     

Genetic variation of the hemagglutinin of avian influenza virus H9N2

Avian influenza virus H9N2 has become the dominant subtype of influenza which is endemic in poultry. The hemagglutinin, one of eight protein-coding genes, plays an important role during the early stage of infection. The adaptive evolution and the positively selected sites of the HA (the glycoprotein molecule) of H9N2 subtype viruses were investigated. Investigating 68 hemagglutinin H9N2 avian influenza virus isolates in China and phylogenetic analysis, it was necessary that these isolates were distributed geographically from 1994, and were all derived from the Eurasian lineage. H9N2 avian influenza virus isolates from domestic poultry in China were distinct phylogenetically from those isolated in Hong Kong, including viruses which had infected humans. Seven amino acid substitutions (2T, 3T, 14T, 165D, 197A, 233Q, 380R) were identified in the HA possibly due to positive selection pressure. Apart from the 380R site, the other positively selected sites detected were all located near the receptor-binding site of the HA1 strain. Based on epidemiological and phylogenetics analysis, the H9N2 epidemic in China was divided into three groups: the 1994-1997 group, the 1998-1999 group, and the 2000-2007 group. By investigating these three groups using the maximum likelihood estimation method, there were more positive selective sites in the 1994-1997 and 1998-1999 epidemic group than the 2000-2007 groups. This indicates that those detected selected sites are changed during different epidemic periods and the evolution of H9N2 is currently slow. The antigenic determinant or other key functional amino acid sites should be of concern because their adjacent sites have been under positive selection pressure. The results provide further evidence that the pathogenic changes in the H9N2 subtype are due mainly to re-assortment with other highly pathogenic avian influenza viruses.     

Isolation and characterization of H6N1 and H9N2 avian influenza viruses from Ducks in Hanoi, Vietnam

We report the genetic characterization of low pathogenic avian influenza (LPAI) viruses isolated from domestic ducks in northern Vietnam in 2009. In total, 22 influenza A viruses consisting of 21 H6N1 subtypes and one H9N2 subtype were isolated from 1488 ducks collected in February, March, and April 2009, accounting the overall virus isolation rate for 1.5%. No H5N1 strain was isolated in this study. Phylogenetic analysis indicated that all the eight genes of the H6N1 and H9N2 subtypes analyzed in this study were similar to those isolated in Korea, southeast China and northern Japan, and wild birds which migrate along the coastal East Asian Flyway are estimated to transmit these viruses. There was no evidence that the H6N1 and H9N2 subtypes share the gene segments with H5N1 subtypes. However, it is important to monitor the prevalence and genetical backgrounds of LPAI viruses among poultry in an area where several different influenza A subtypes are in circulation.     

Genetic evolution of the H9N2 avian influenza virus in Korean poultry farms

We performed whole genome sequencing of 22 H9N2 avian influenza viruses (AIV) isolated from domestic laying hens on farms between 2005 and 2008, and compared the sequences with viruses previously reported in Asia. A previous study revealed that two antigenically distinct sublineages were established within the MS96 lineage by antigenic drift since the first H9N2 AIV outbreak in South Korea. We designated them as the 01310-like lineage and the 116/04-like lineage. Since late 2004, most identified isolates in Korea have belonged to the 116/04-like lineage, however, in this study we found that six among twenty-two isolates were belonged to 01310-like lineage, indicating that the genetic divergence is still occurring after 2004. Furthermore, it is noteworthy that five isolates among the defined 01310-like lineage had a 24 amino acid deletion in the neuraminidase stalk region, which were not in any other H9N2 isolates previously reported. The internal genes analysis demonstrated extensive reassortment events among isolates from poultry farms, live bird markets, and wild birds, and multiple new genotypes were identified. We identified several features of gene evolution in H9N2 AIV suggesting that the long-term H9N2 AIV surveillance study should be continued in South Korea.     

Isolation and characterization of an H9N2 influenza virus isolated in Argentina

As part of our ongoing efforts on animal influenza surveillance in Argentina, an H9N2 virus was isolated from a wild aquatic bird (Netta peposaca), A/rosy-billed pochard/Argentina/CIP051-559/2007 (H9N2) - herein referred to as 559/H9N2. Due to the important role that H9N2 viruses play in the ecology of influenza in nature, the 559/H9N2 isolate was characterized molecularly and biologically. Phylogenetic analysis of the HA gene revealed that the 559/H9N2 virus maintained an independent evolutionary pathway and shared a sister-group relationship with North American viruses, suggesting a common ancestor. The rest of the genome segments clustered with viruses from South America. Experimental inoculation of the 559/H9N2 in chickens and quail revealed efficient replication and transmission only in quail. Our results add to the notion of the unique evolutionary trend of avian influenza viruses in South America. Our study increases our understanding of H9N2 viruses in nature and emphasizes the importance of expanding animal influenza surveillance efforts to better define the ecology of influenza viruses at a global scale.