Pathogenicity for chickens of avian influenza viruses isolated from whistling swans and a black-tailed gull in Japan

We isolated 24 Hav1 Neq1 and 18 Hav6 Nav3 influenza viruses from such free-living wild waterfowl as whistling swans, black-tailed gulls, and tufted ducks in western Japan in 1980. Two Hav1 Neq1 viruses isolated from a whistling swan and a black-tailed gull and a Hav6 Nav3 virus from a whistling swan were examined for their pathogenicity for chickens. Five-week-old specific-pathogen-free chickens were inoculated with the viruses intratracheally or intraperitoneally. Virus was recovered successfully from all the organs, including the brain, despite the absence of signs of disease. The intracerebral pathogenicity index scores obtained for the Hav1 Neq1 viruses were 0.43 and 0.87; the score for the Hav6 Nav3 virus was 0.43. No virus produced plaques in cultivated chick embryo fibroblast cells in the absence of trypsin.     

Pathology of specific-pathogen-free chickens inoculated with H5N1 avian influenza viruses isolated in Japan in 2004

Specific-pathogen-free chickens inoculated with H5N1 highly pathogenic avian influenza (HPAI) viruses isolated in Japan in 2004 were investigated pathologically. The chickens inoculated intravenously with the viruses died within 26 hr after inoculation. Macroscopically, minimal necrosis of the tip of the comb, and hemorrhages of the palpebral conjunctiva, liver, cerebellum, and muscles were rarely observed. Histologically, dead chickens had minimal focal necrosis of hepatocytes with fibrinous thrombi in sinusoids, mild necrosis of splenic ellipsoids with fibrinous exudation, minimal necrosis of the brain, mild necrosis of epidermal cells of the comb with congestion of the lamina propria, and hemorrhages and edema of the lamina propria of the conjunctiva. Virus antigens were seen in the sinusoidal endothelial cells and hepatocytes in the liver, the capillary endothelial cells of the spleen, the capillary endothelial cells and cardiac myocytes in the heart, the capillary endothelial cells and necrotic nerve cells in the brain, the capillary endothelial cells in the lamina propria of the comb, the renal tubular epithelial cells, and the pancreatic acinar cells. The chickens inoculated by natural infectious routes died within 1-4 days after inoculation. Macroscopically, some chickens had hemorrhages in the conjunctiva, edematous swelling of the face and wattles, hydropericardium, hemorrhages of the proventriculus and bursa of Fabricius, increased secretion of tracheal mucus, and congestion and edema of lungs. Histologic lesions by natural infectious routes were similar to those by intravenous inoculation, except for the pancreatic necrosis. This study suggests H5N1 HPAI viruses isolated in Japan in 2004 cause pathologic conditions similar to natural cases.     

Molecular characterization of low pathogenicity H7N3 avian influenza viruses isolated in Italy

The complete coding regions of the surface glycoproteins, nucleoprotein (NP), polymerase 2 (PB2), and matrix (M) of A/turkey/214845/02 and A/turkey/220158/99 (H7N3) low pathogenicity avian influenza (LPAI) viruses isolated in October 2002 in Italy were amplified and sequenced to determine the epidemiologic relationships with an A/turkey/Italy/4603/99 (H7N1/4603/99) LPAI virus isolated during the 1999-2001 epizootic in Italy. The hemagglutinin (HA) of H7N3 viruses showed 97.8% nucleotide similarity with A/turkey/Italy/4603/99 (H7N1), and NP, M, and PB2 gene similarities were 93.6%, 98.2%, and 96.2%, respectively. Phylogenetic analyses of HA, PB2, and M genes showed that H7N3 and H7N1 viruses were closely related. Sequence analysis revealed a 23 amino acid deletion in the stalk of the neuraminidase of H7N3 viruses and a unique deletion of amino acid glycine in position 17 in the NP gene of H7N1 virus.     

Comparative pathobiology of low and high pathogenicity H7N3 Chilean avian influenza viruses in chickens

Chickens were intranasally inoculated with Chilean H7N3 avian influenza (AI) viruses of low pathogenicity (LP) (H7N3/LP), high pathogenicity (HP) (H7N3/HP), and a laboratory derivative (02-AI-15-#9) (H7N3/14D) from the LPAI virus to determine pathobiologic effects. All chickens inoculated with H7N3/HP AI virus became infected and abruptly died 2 or 3 days postinoculation, but a few showed moderate depression before death. The H7N3/HP AI virus produced focal hemorrhages of the comb, petechial hemorrhage at the esophageal-proventricular junction and proventricular mucosa, edema and congestion of the lung, petechiation of the spleen, and generalized decrease in body fat. Histologically, severe necrosis, hemorrhage, and inflammation were primarily identified in lungs and the lymphoid tissues. All tissues sampled from the H7N3/HP AI group were positive for the AI viral antigen, predominantly in endothelium of blood vessels throughout most tissues and less frequently in histiocytes and cellular debris of lymphoid tissues. Even less consistently, cardiac myocytes, hepatocytes, Kupffer cells, glandular epithelial cells, microglial cells, and neurons became infected. These studies suggest the Chilean H7N3/LP AI virus was poorly infectious for chickens and may have been recently introduced from a nongalliform host. By contrast, the H7N3/HP AI virus was highly infectious and lethal for chickens. The H7N3/HP AI virus had a strong tropism for the cardiovascular system, principally vascular endothelium, which is similar to the viral tropism demonstrated previously with other H5 and H7 HPAI viruses. Interestingly, the H7N3/LP AI virus on intravenous inoculation replicated in cardiac myocytes, a feature of HPAI and not LPAI viruses, which further supports the theory that the H7N3/LP AI virus was in transition from LP to HP.     

Kidney lesions associated with mortality in chickens inoculated with waterfowl influenza viruses

Seventy-six type A influenza viruses recovered from waterfowl in Wisconsin, California, South Dakota, Florida, Texas, Alabama, and Nebraska were tested for virulence in chickens. The challenge to chickens was intravenous inoculation of first-, second-, or third-egg-passage virus. Each of the virus strains was tested separately in three or four chickens. Eighteen of the 76 viruses caused the death of one or more chickens following inoculation. Postmortem lesions were similar in all dead birds. In decreasing order of frequency, gross lesions included: swollen kidneys evident as accentuated lobular patterns, urates in the pericardial sac, and urates on the surface of the liver. Microscopic lesions present in kidneys were consistent with visceral gout. Mortality was associated with inoculations having higher concentrations of infectious virus. These results indicate that the influenza A viruses circulating in duck populations may include strains potentially pathogenic for chickens.     

The pathogenicity of an avian influenza virus isolated in Victoria

An influenza virus (H7N7) isolated from an outbreak of disease in chickens in Victoria, was examined for its ability to cause disease in inoculated chickens, turkeys and ducks. The virus was highly pathogenic in chickens and turkeys but produced no clinical disease in ducks. Transmission of infection occurred from inoculated chickens to those in direct contact but other chickens separated by a distance of 3m directly downwind developed neither clinical disease nor antibody to the virus.     

Experimental assessment of the pathogenicity of H5N1 influenza A viruses isolated in Japan

We examined the pathogenicity for chickens of two H5N1 avian influenza viruses isolated in Japan, A/chicken/ Yamaguchi/7/2004 (Ck/Yamaguchi/7/04) isolated from outbreaks in commercial layer chickens, and A/duck/Yokohama/aq10/ 2003 (Dk/Yokohama/aq10/03) isolated from duck meat imported from China. All chickens inoculated intranasally with either strain died, and the viruses were reisolated from all organs examined. However, both the mean time of onset of clinical signs and the mean death time of Ck/Yamaguchi/7/04 were shorter than those of Dk/Yokohama/aq10/03.     

Potential of low pathogenicity avian influenza viruses of wild bird origin to establish experimental infections in turkeys and chickens

The potential of low pathogenicity (LP) avian influenza virus (AIV) isolates of wild bird origin to establish infection in commercial turkeys and broiler chickens was studied. Isolates, representing subtypes H5N1, H7N3, H6N2, and H3N6, were recovered in 2005 and 2006 from waterfowl and shorebirds in the Delmarva Peninsula region of the east coast of the United States. The LP AIV isolates were not pathogenic for 2-wk-old meat-type turkeys and broiler chickens. No mortality, clinical signs, or gross lesions were observed following intratracheal and conjunctival sac routes of exposures with 10(6.0) EID50 (embryo infectious dose) per bird. Isolates resulting in an established infection based on virus isolation were: A/mallard/Maryland/1159/ 2006 (H5N1) in the upper respiratory tract of turkeys; A/mallard/Delaware/418/2005 (H7N3) in the upper respiratory and intestinal tracts of turkeys and chickens; and A/shorebird-environment/Delaware/251/2005 (H3N6) in the upper respiratory and intestinal tracts of chickens. Infections were also confirmed by production of AIV-specific serum antibodies detected by hemagglutination inhibition.     

Assessing pathogenicity potential of waterfowl-origin type A influenza viruses in chickens.

Intravenous pathogenicity index (IVPI) tests on 29 wild duck-origin type A influenza viruses, two turkey-origin type A influenza viruses, and one chicken-origin type A influenza virus resulted in indices ranging from 0.0 to 0.49. Most of the wild duck-origin viruses and the two turkey-origin viruses had indices of 0.0, indicating they are not pathogenic. Six of the duck-origin viruses had indices ranging from 0.25 to 0.49, and the IVPI for A/chicken/Alabama/75 (H4N8) was 0.49, indicating they had low pathogenic potential. An IVPI of 1.25 up to the maximum score of 3.0 is necessary for a type A influenza virus to be classified as highly pathogenic. Gross lesions observed in chickens dying following intravenous viral challenge included kidney swelling with more prominent lobular patterns, but visceral urate deposits were not present. The usefulness of the IVPI test in evaluating the pathogenicity potential of nonpathogenic and low-pathogenic strains of avian influenza virus may be limited.     

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.     

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.     

Influence of dietary calcium stress on lethality of avian influenza viruses for laying chickens

The effect of calcium stress was studied in an attempt to reproduce lethal infections in laying chickens with A/Chicken/Alabama/75 (H4N8) influenza virus and with two nonpathogenic H5N2 influenza viruses from the 1983-84 outbreak in the eastern United States. Hens were fed calcium-deficient or standard diets for 7 to 14 days; then the calcium-deficient feed was replaced with standard feed supplemented with ad libitum oyster shell, and both groups of hens were inoculated with virus. When hens were infected with the H4N8 virus, respective mortalities of those on the calcium-deficient and standard diets were 19% (27/141) and 5% (7/143). The H5N2 viruses did not kill hens fed either diet. In standard pathogenicity tests, Alabama H4N8 viruses reisolated from the hens that died generally were more lethal for 4-week-old chickens than the stock virus. These results argue for characterization of the Alabama H4N8 virus as pathogenic rather than nonpathogenic as originally determined.     

Sequence and phylogenetic analysis of the haemagglutinin genes of H9N2 avian influenza viruses isolated from commercial chickens in Iran

To determine the genetic relationship of Iranian viruses, the haemagglutinin (HA) genes from ten isolates of H9N2 viruses isolated from commercial chickens in Iran during 1998–2002 were amplified and sequenced. Sequence analysis and phylogenetic studies were conducted by comparing each isolate with those of the available H9N2 strains at GenBank. All these ten isolates had the same sequence –R-S-S-R/G-L– of proteolytic cleavage site of the HA. Nucleotide sequence comparisons of HA gene from Iranian isolates showed 95.2–99.1% identity within the group. Five isolates had leucine (L) at position 226 instead of glutamine (Q). Phylogenetic analysis showed that all our isolates belonged to the G1-like sublineage. Also these isolates showed some degree of homology with other H9N2 isolates e.g., 94.3–96.9% with qu/HK/G1/97, 96.1–98.6% with pa/Chiba/1/97, 95.6–98.2% with pa/Narita/92A/98, and 94.0–96.3% with HK/1073/99. On the basis of phylogenetic and molecular characterization evidence, we concluded that the H9N2 subtype influenza viruses circulating in chicken flocks in Iran since 1998–2002 had a common origin. The results of this study indicated that all Iranian viruses have the potential to emerge as highly pathogenic influenza virus, and considering the homology of these isolates with human H9N2 strains, it seems that the potential of these avian influenza isolates to infect human should not be overlooked.     

2013—2014年H7亚型禽流感病毒遗传进化分析

对2013—2014年15株不同来源的H7亚型禽流感病毒国内分离株进行了基因组测序与遗传进化分析。结果显示,15株H7亚型禽流感病毒具有遗传多样性,可分为2种亚型:13株为H7N9亚型,2株为H7N3亚型。HA蛋白裂解位点附近氨基酸分析显示所有H7亚型流感分离株均为低致病性毒株。基因组遗传进化分析显示,13株H7N9亚型禽流感病毒均与2013年人源分离株同源性较高,但具有2种遗传学特性,13株病毒的PB2、PA、HA、NP、NA、M和NS基因高度同源,而PB1基因来源于2个不同分支。2株H7N3亚型流感病毒与国内鸭源分离株同源性较高。15株H7亚型禽流感病毒PB2蛋白均未出现627K、701N等与哺乳动物适应性相关的氨基酸变异。13株H7N9亚型禽流感病毒HA裂解位点附近氨基酸(EIPKGR/GL)与人源H7N9病毒一致,且HA蛋白和M2蛋白分别具有与哺乳动物适应性和金刚烷胺耐药性相关的标志性变异,而2株H7N3亚型禽流感病毒未出现上述氨基酸变异。     

Low pathogenicity avian influenza viruses infect chicken layers by different routes of inoculation

In order to develop better control measures against avian influenza, it is necessary to understand how the virus transmits in poultry. In a previous study in which the infectivity and transmissibility of the pandemic H1N1 influenza virus was examined in different poultry species, we found that no or minimal infection occurred in chicken and turkeys intranasally (IN) inoculated with the virus. However, we demonstrated that the virus can infect laying turkey hens by the intracloacal (IC) and intraoviduct (IO) routes, possibly explaining the drops in egg production observed in turkey breeder farms affected by the virus. Such novel routes of exposure have not been previously examined in chickens and could also explain outbreaks of low pathogenicity avian influenza (LPAI) that cause a decrease in egg production in chicken layers and breeders. In the present study, 46-wk-old specific-pathogen-free chicken layers were infected by the IN, IC, or IO routes with one of two LPAI viruses: a poultry origin virus, A/chicken/CA/1255/02 (H6N2), and a live bird market isolate, A/chicken/NJ/12220/97 (H9N2). Only hens IN inoculated with the H6N2 virus presented mild clinical signs consisting of depression and anorexia. However, a decrease in number of eggs laid was observed in all virus-inoculated groups when compared to control hens. Evidence of infection was found in all chickens inoculated with the H6N2 virus by any of the three routes and the virus transmitted to contact hens. On the other hand, only one or two hens from each of the groups inoculated with the H9N2 virus shed detectable levels of virus, or seroconverted and did not transmit the virus to contacts, regardless of the route of inoculation. In conclusion, LPAI viruses can also infect chickens through other routes besides the IN route, which is considered the natural route of exposure. However, as seen with the H9N2 virus, the infectivity of the virus did not increase when given by these alternate routes.     

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.     

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.