Survival of Highly Pathogenic Avian Influenza H5N1 Virus in Tissues Derived from Experimentally Infected Chickens.

Eurasian lineage highly pathogenic avian influenza (HPAI) H5N1 virus has been a severe threat to the poultry industry since its emergence in 1996. The carcass or tissues derived from infected birds may present the risk of the virus spreading to humans, animals, and the surrounding environment. In this study, we investigated the survival of the virus in feather, muscle, and liver tissues collected from six chickens (Gallus gallus) experimentally infected with HPAI H5N1 virus. The tissues were stored at +4°C or +20°C, and viral isolation was performed at different times for 360 days. The maximum periods for viral survival were observed in samples stored at +4°C in all tissue types and were 240 days in feather tissues, 160 days in muscle, and 20 days in liver. The viral infectivity at +20°C was maintained for a maximum of 30 days in the feather tissues, 20 days in muscle, and 3 days in liver. The viral inactivation rates partly overlapped in the feather and muscle tissues at the two temperatures. The virus was inactivated rapidly in the liver. Our experimental results indicate that the tissue type and temperature can greatly influence the survival of HPAI H5N1 virus in the tissues of infected chickens.IMPORTANCE Highly pathogenic avian influenza virus of the H5N1 subtype can cause massive losses of poultry, and people need to handle a large number of chicken carcasses contaminated with the virus at outbreak sites. This study evaluated how long the virus can keep its infectivity in the three types of tissues derived from chickens infected with the virus. Our experimental results indicate that the virus can survive in tissues for a specific period of time depending on the tissue type and temperature. Our results are valuable for better understanding of viral ecology in the environment and for reducing the risk of the virus spreading via bird tissues contaminated with the virus.

Evaluation of the replication and pathogenicity of a variant avian paramyxovirus serotype 6 in mice.

Avian paramyxoviruses (APMVs) have been evaluated for their potential use as vaccine vectors, sparking research efforts leading to a better understanding of APMVs' replication and pathogenicity. However, within APMV serotypes, significant genetic diversity exists, and the infectivity of variant strains in mammals has not been studied. We utilized a mouse model to evaluate the pathogenicity of a variant strain of APMV-6 (APMV-6/red-necked stint/Japan/8KS0813/2008) in comparison with the prototype APMV-6 strain (APMV-6/duck/Hong Kong/18/199/1977). Although the two viruses differ substantially, both genetically and antigenically, we found that the variant and prototype strains could similarly replicate in respiratory tissues of infected mice and induce respiratory disease, sometimes resulting in death of the mice. Both viruses induced a humoral immune response that could be clearly detected by ELISA but which was poorly recognized by the hemagglutination inhibition test.

Characterization of a genetic and antigenic variant of avian paramyxovirus 6 isolated from a migratory wild bird, the red-necked stint (Calidris ruficollis).

A hemagglutinating virus (8KS0813) was isolated from a red-necked stint. Hemagglutination inhibition and neutralization tests indicated that 8KS0813 was antigenically related to a prototype strain, APMV-6/duck/Hong Kong/18/199/77, but with an 8- and 16-fold difference, respectively, in their titers. The full genome sequence of 8KS0813 showed 98.6 % nucleotide sequence identity to that of APMV-6/duck/Italy/4524-2/07, which has been reported to belong to an APMV-6 subgroup, and showed less similarity to that of the prototype strain (70.6 % similarity). The growth of 8KS0813 and the prototype strain in four different cell cultures was greatly enhanced by adding trypsin. Interestingly, this virus induced syncytia only in Vero cells. 8KS0813 was identified as APMV-6/red-necked stint/Japan/8KS0813/08, but it is antigenically and genetically distinguishable from the prototype strain, suggesting that variant APMV-6 is circulating in migratory birds.

Pathogenesis in Eurasian tree sparrows inoculated with H5N1 highly pathogenic avian influenza virus and experimental virus transmission from tree sparrows to chickens.

Small wild birds that routinely enter poultry farms may be possible vectors of Asian lineage H5N1 highly pathogenic avian influenza virus. In this study, we conducted experimental infections using wild-caught Eurasian tree sparrows (Passer montanus) to evaluate their possible epidemiological involvement in virus transmission. When tree sparrows were intranasally inoculated with the virus at a low or high dose, all sparrows excluding euthanatized birds died within 11 days after inoculation. Viruses were frequently isolated from the drinking water, oral swabs, and visceral organs of the sparrows. Immunohistochemical analysis revealed that the virus replicated strongly in the central nervous system, heart, and adrenal gland following primary infection in the upper respiratory tract and a probable subsequent viremic stage. In the contact infection study using virus-inoculated sparrows and untreated contact chickens, more than half of all chickens died from viral infection. In the virus transmission study in which chickens were given drinking water collected from virus-inoculated sparrows, mortality due to viral infection was observed in chickens. Our data suggest that Eurasian tree sparrows could be biological vectors of the H5N1 highly pathogenic avian influenza virus. In addition to frequent virus detection in the drinking water of sparrows, the results of the virus transmission study suggest that waterborne pathways could be important for viral transmission from tree sparrows to poultry.

Detection of fowl adenovirus DNA from formalin-fixed and paraffin-embedded sections by PCR and classification of serotypes by sequencing of PCR products.

Detection of fowl adenovirus (FAV) DNA from formalin-fixed and paraffin-embedded (FFPE) sections was attempted by PCR. Serotypes of FAV were classified by sequencing the PCR products. In trials of PCR using a positive control infected with serotype 2 FAV, the best primer set was 57F forward primer (5'-CAARTTCAGRCAGACGGT-3') and 26R reverse primer (5'-GGCTTGACGTACGCTCCGTA-3'). A second PCR with the same primer set revealed a clearer band in the electrophoresis of generated PCR products. Generated PCR products were confirmed to be derived from infected FAV. In addition, PCR and sequencing of PCR products of the liver FFPE sections, from two natural inclusion body hepatitis cases that were not examined for virologic isolation, suggested that the detected FAV was serotype 8a. The PCR of FFPE sections, and serotyping by the sequencing of PCR products, are useful for diagnosis and epidemiologic analysis of FAV infections.

Inclusion body hepatitis caused by fowl adenovirus in broiler chickens in Japan, 2009-2010.

From January 2009 to June 2010, many broiler chicks suddenly died without clinical signs. The mortality rates were from 1.2% to 17.0% in affected flocks. Inclusion body hepatitis (IBH) was detected in 13 prefectures (northern, eastern, western, and southern areas) in Japan. The livers were enlarged and pale. The bursa of Fabricius and thymus had not atrophied. Multifocal necroses of hepatocytes with basophilic intranuclear inclusions were seen in the liver. Eosinophilic intranuclear inclusion bodies in hepatocytes were rare. Focal necrosis of acinar cells with basophilic intranuclear inclusions was found in the pancreas. Basophilic intranuclear inclusion bodies were detected in intact surface epithelial cells of gizzard and epithelial cells of the small intestine. The intranuclear inclusions of liver, pancreas, gizzard, and small intestine were stained positively for immunohistochemistry of fowl adenovirus (FAV) antigen. Ultrastructurally, basophilic intranuclear inclusions consisted of viral particles approximately 70 nm in diameter and arranged in a crystalline array. FAV was isolated from the liver of chickens affected with IBH. The serotype of most isolates was 2. This study suggests that IBH produced by FAV is epidemic in broiler chicks in Japan and that the present cases occurred as the primary disease without the association of infectious bursal disease virus or chicken anemia virus.

Association of increased pathogenicity of Asian H5N1 highly pathogenic avian influenza viruses in chickens with highly efficient viral replication accompanied by early destruction of innate immune responses.

The Asian H5N1 highly pathogenic avian influenza (HPAI) viruses have been increasing in pathogenicity in diverse avian species since 1996 and are now widespread in Asian, European, and African countries. To better understand the basis of the increased pathogenicity of recent Asian H5N1 HPAI viruses in chickens, we compared the fevers and mean death times (MDTs) of chickens infected with the Asian H5N1 A/chicken/Yamaguchi/7/04 (CkYM7) strain with those infected with the H5N1 Duck/Yokohama/aq10/03 (DkYK10) strain, using a wireless thermosensor. Asian H5N1 CkYM7 caused peracute death in chickens before fever could be induced, whereas DkYK10 virus induced high fevers and had a long MDT. Real-time PCR analyses of cytokine mRNA expressions showed that CkYM7 quickly induced antiviral and proinflammatory cytokine mRNA expressions at 24 h postinfection (hpi) that suddenly decreased at 32 hpi. In contrast, these cytokine mRNA expressions increased at 24 hpi in the DkYK10 group, but decreased from 48 hpi onward to levels similar to those resulting from infection with the low-pathogenicity H5N2 A/chicken/Ibaraki/1/2004 strain. Sequential titrations of viruses in lungs, spleens, and kidneys demonstrated that CkYM7 replicated rapidly and efficiently in infected chickens and that the viral titers were more than twofold higher than those of DkYK10. CkYM7 preferentially and efficiently replicated in macrophages and vascular endothelial cells, while DkYK10 grew moderately in macrophages. These results indicate that the increased pathogenicity in chickens of the recent Asian H5N1 HPAI viruses may be associated with extremely rapid and high replication of the virus in macrophages and vascular endothelial cells, which resulted in disruption of the thermoregulation system and innate immune responses.

Persistence of avian influenza virus (H5N1) in feathers detached from bodies of infected domestic ducks.

Asian lineage highly pathogenic avian influenza virus (H5N1) continues to cause mortality in poultry and wild bird populations at a panzootic scale. However, little is known about its persistence in contaminated tissues derived from infected birds. We investigated avian influenza virus (H5N1) persistence in feathers detached from bodies of infected ducks to evaluate their potential risk for environmental contamination. Four-week-old domestic ducks were inoculated with different clades of avian influenza virus (H5N1). Feathers, drinking water, and feces were collected on day 3 postinoculation and stored at 4 degrees C or 20 degrees C. Viral persistence in samples was investigated for 360 days by virus isolation and reverse transcription-PCR. Infectious viruses persisted for the longest period in feathers, compared with drinking water and feces, at both 4 degrees C and 20 degrees C. Viral infectivity persisted in the feathers for 160 days at 4 degrees C and for 15 days at 20 degrees C. Viral titers of 10(4.3) 50% egg infectious doses/ml or greater were detected for 120 days in feathers stored at 4 degrees C. Viral RNA in feathers was more stable than the infectivity. These results indicate that feathers detached from domestic ducks infected with highly pathogenic avian influenza virus (H5N1) can be a source of environmental contamination and may function as fomites with high viral loads in the environment.

Characterization of Fowl adenovirus serotype 4 isolated from chickens with hydropericardium syndrome based on analysis of the short fiber protein gene.

The sequences of short fiber genes of the Fowl adenovirus serotype 4 (FAdV-4), including isolates from chickens with hydropericardium syndrome (HPS), in Japan, India, and Pakistan were compared. By phylogenetic analysis based on complete nucleotide sequences of this gene, FAdV-4 strains from HPS (HPS-FAdV-4) in Japan, India, and Pakistan fell into a different cluster from FAdV-4 strains not derived from HPS. Hydropericardium syndrome-FAdV-4 isolates were differentiated from other FAdV-4 strains by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis using the enzyme the Alu I. The use of PCR-RFLP analysis of short fiber genes may be useful to distinguish among FAdV-4 strains.

Pathogenesis of venous hypertrophy associated with schistosomiasis in whooper swans (Cygnus cygnus) in Japan.

Thirteen whooper swans (Cygnus cygnus) affected with schistosomiasis were examined pathologically. Venous hypertrophy, characterized by marked nodular proliferation of medial smooth muscle fibers with frequent obliteration of the vascular lumen, was observed in eight of the 13 whooper swans. Venous hypertrophy was located in the medium-sized veins of the mesentery, the serosa, and the muscular layer of the duodenum, jejunum, ileum, and cecum. In addition, vascular lesions were seen in the capsule and parenchymal interstitia of the liver, spleen, kidney, heart, aorta, air sac, and pleura. In mild lesions, segmental proliferation of medial smooth muscles was observed in the venous medium of the mesentery and serosa. Moderate lesions had a proliferation of smooth muscles in the veins with obliteration of venous lumens. In marked lesions, more severe proliferation of veins extended into the intestinal muscular layers and depressed them. Schistosome parasites were found in the venous lumens of each of the eight whooper swans with vascular lesions. Bile pigments and hemosiderin were observed in the livers of whooper swans. In addition, adult nematodes (Sarconema sp.) were localized in the myocardium of four of the eight whooper swans. The venous hypertrophy may be caused by the proliferation of medial smooth muscle fibers induced by schistosomiasis.

Pathology and microbiology of dermal squamous cell carcinoma in young brown chickens reared on reused litter.

Dermal squamous cell carcinoma (DSCC) was found in young brown chicken flocks reared on reused litter in Japan. DSCC was often detected at slaughter from April 2007 to March 2009, especially in June and July 2007. No DSCC was observed in the broiler chickens on the farms. Twelve 11-wk-old brown chickens with DSCC were investigated pathologically and microbiologically. Various degrees of crater-like skin lesions were found on the back, waist, neck, legs, abdomen, and wings of the carcasses. The feather follicles were enlarged. The feather follicular epithelial cells proliferated, and the squamous cells proliferated neoplastically in association with collagen fibers and fibroblasts in the dermis under the feather follicular epithelium. "Keratin pearl" structures were often seen in the dermis. Immunohistochemically, the keratin antigen was positive in the neoplastically proliferated squamous cells in the dermis. Avian leukosis virus antigens could not be found in the neoplastic squamous cells in the dermis. Ultrastructurally, no viral agents could be detected in the skin with DSCC. Virologically, reverse transcription-polymerase chain reactions of the skin with DSCC for fowlpox virus and avian leukosis virus were negative. No viruses could be isolated from the skin with DSCC. This study suggests that the chicken breed, reused litter, and season may be associated with the incidence of DSCC in brown chickens.

Zoonotic risk for influenza A (H5N1) infection in wild swan feathers.

We examined whooper swans naturally infected with avian influenza virus (H5N1) to evaluate the possible zoonotic risk of swan feathers. Viruses were isolated from feather calami. Immunohistochemical testing revealed that virus antigens were present in the feather epidermis and feather follicle wall epidermis of some feathers. RT-PCR and genetic sequencing using paraffin sections of swan feathers confirmed the presence of avian influenza virus (H5N1) in the feather tissue. These results indicate that the feathers could have the risk for zoonotic infection from infected wild swans.

Pathology of whooper swans (Cygnus cygnus) infected with H5N1 avian influenza virus in Akita, Japan, in 2008.

Two (1 adult and 1 young bird) of 4 H5N1-highly-pathogenic-avian-influenza (HPAI)-virus-infected whooper swans in Akita, Japan, in 2008 were investigated pathologically. Macroscopically, white spots with hemorrhages were scattered in the pancreas in the adult bird. Histologically, the adult bird had severe necrotizing pancreatitis and mild nonpurulent encephalitis. The young bird had severe nonpurulent encephalitis and nonpurulent enteric ganglionitis, and intestinal venous wall thickening. Virus antigens were detected in the lesions of pancreatitis in the adult bird and of encephalitis in adult and young birds. These findings suggest that the swans died or became moribund due to neurological disorders and necrotizing pancreatitis caused by H5N1 HPAI virus infection.

Genetic characterization of fowl adenoviruses isolated from chickens with hydropericardium syndrome in Japan.

We genetically characterized fowl adenoviruses (serotype 4 FAdV, FAdV-4) isolated from chickens with hydropericardium syndrome (HPS) in Japan by the polymerase chain reaction (PCR) method coupled with direct sequencing. Phylogenetic analysis based on the part of the hexon gene that included the L1 region revealed that all FAdV-4 isolates from chickens with HPS in Japan were identical and were distinguished completely from the cluster including FAdV strains from chickens with HPS in India and Pakistan. This suggested that FAdV-4 from the HPS chickens in India and Pakistan was derived from a common ancestor, but the origin of the FAdV-4 from the HPS chickens in Japan was completely different.

Highly pathogenic avian influenza virus (H5N1) isolated from whooper swans, Japan.

On April 21, 2008, four whooper swans were found dead at Lake Towada, Akita prefecture, Japan. Highly pathogenic avian influenza virus of the H5N1 subtype was isolated from specimens of the affected birds. The hemagglutinin (HA) gene of the isolate belongs to clade 2.3.2 in the HA phylogenetic tree.

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.

Immunohistochemical distribution of viral antigens in pigs naturally infected with porcine teschovirus.

A distribution of porcine teschovirus (PTV) antigens in pigs naturally infected with PTV is presented using the method of immunohistochemical examination. In the nervous system, PTV antigens were found in the cytoplasm of neuronal cells and glial cells distributed in the spinal ventral horn and brain stem, and also in the cytoplasm of ganglion cells in the spinal ganglion. No antigens were seen in the cerebral hemisphere. In the nervous system, the distribution of PTV antigens was consistent with lesions characteristic of nonsuppurative encephalomyelitis. In the other examined organ, PTV antigens were observed in bronchiolar epithelial cells in the lung, hepatocytes in the liver, epithelial cells in the tonsils and the myenteric nerve plexus in the small and large intestine.

Low pathogenicity H5N2 avian influenza outbreak in Japan during the 2005-2006.

At the end of May 2005, a low-pathogenicity avian influenza (LPAI) virus of subtype H5N2 was isolated for the first time from chickens in Japan. Through active and epidemiological surveillance, 5.78 million chickens on 41 farms were found to be affected and 16 H5N2 viruses were isolated. Antigenic analysis revealed antigenic similarity of these isolates. Phylogenetic analysis showed that they originated from a common ancestor and clustered with the H5N2 strains prevalent in Central America that have been circulating since 1994. Experimental infection of chickens with the index isolate (A/chicken/Ibaraki/1/05) demonstrated that this virus replicated efficiently in the respiratory tract without clinical signs, and dust-borne and/or droplet-borne transmission was considered as a possible mode of transmission. These results suggested that the H5N2 LPAI viruses isolated in Japan were highly adapted to chickens.

Severe nonpurulent encephalitis with mortality and feather lesions in call ducks (Anas platyrhyncha var. domestica) inoculated intravenously with H5N1 highly pathogenic avian influenza virus.

One-day-old, 2-wk-old, and 4-wk-old call ducks (Anas platyrhyncha var. domestica) inoculated intravenously with the H5N1 highly pathogenic avian influenza virus A/chicken/Yamaguchi/7/2004 isolate (Ck/Yama/7/04) were examined clinically, pathologically, and virologically. Clinically, the birds exhibited mild-to-severe neurologic signs and corneal opacity. All birds in the 1-day-old group and one bird in the 4-wk-old group died within 4 days after the virus inoculation. Histologic changes were characterized by severe nonpurulent encephalitis and necrotic lesions of feather epithelium on day 3 postinoculation (PI) or later. Focal necrosis of myocardial cells, pancreatic acinar cells, skeletal myocytes, and corneal epithelial cells was observed. Viral antigens were detected in association with necrotic changes. Viruses were isolated from all examined organs including the skin with many feathers. Serum antibody against the virus was detected in all surviving birds on day 10 PI by hemagglutination-inhibition tests. These results suggest that Ck/Yama/7/04 has a pathogenicity that causes neurologic sign, nonpurulent encephalitis with mortality, and feather lesions for call ducks. Feather lesions with viral antigens and the virus isolation from the skin suggest that Ck/Yama/ 7/04 has a predilection for feathers in call ducks.

Pathogenesis in call ducks inoculated intranasally with H5N1 highly pathogenic avian influenza virus and transmission by oral inoculation of infective feathers from an infected call duck.

To evaluate the possibility of virus transmission through feathers of call ducks, we performed two experiments, intranasal infection study and transmission study, using the Japanese H5N1 highly pathogenic avian influenza virus (HPAIV) A/chicken/Yamaguchi/7/2004 (Ck/Yama/7/04). In Experiment 1, 1-day-old, 2-wk-old, and 4-wk-old birds were inoculated intranasally with Ck/Yama/7/04. Birds in all age groups exhibited necrosis and/or viral antigens in the feather epithelium. Nonpurulent encephalitis and focal necrosis of the pancreas and heart also were common to inoculated birds. In Experiment 2, nine 2-wk-old birds that were orally inoculated with feathers of an infected call duck exhibited the nonpurulent encephalitis, necrosis of the feather epithelium, and focal necrosis of the pancreas and heart, accompanied by viral antigens. These lesions were similar to those in intranasal infection. Some birds were positive for the virus isolation from cloacal swabs and hemagglutination inhibition antibody. The infection was confirmed in seven of nine birds. This study confirmed that the Japanese HPAIV can replicate in the feather epithelium, causing necrosis in call ducks through the natural infection route. It also suggests that feathers of call ducks infected with Ck/Yama/7/04 can be a potential source of infection for unaffected birds in nature.