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.

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.

Immunohistochemistry and molecular epidemiology of avian paramyxovirus 1 from formalin-fixed and paraffin-embedded sections of Japanese doves (Columba livia) affected with neurological signs.

Four doves (Nos. 1-4 birds) affected with neurological signs (ataxia, circling and torticollis) were investigated pathologically and microbiologically. Viral isolation was tried from the tracheal and cloacal swabs of all 4 birds and from liver, spleen, kidney, heart, lung and brain of Nos. 1 and 2 birds. No viruses were isolated from 4 birds, but they had high serum antibody titers against avian paramyxovirus 1 (APMV-1). Histologically, they had the characteristic histological changes of pigeon APMV-1 infection; nonpurulent encephalitis and interstitial nephritis. Immununohistochemically, APMV-1 antigens were detected in the necrotic renal tubular epithelial cells of 1 bird of them (No. 3 bird). Detection of APMV-1 ribonucleic acid (RNA) from formalin-fixed and paraffin-embedded (FFPE) sections was attempted by reverse transcription-polymerase chain reaction (RT-PCR). Sequencing the RT-PCR product showed the virus RNA belonged to the same APMV-1 genotype (VI) as the strains isolated from the world previous cases of pigeon APMV-1 infection. The RT-PCR of FFPE sections and sequencing of RT-PCR products are useful for molecular epidemiology of the virus when viral isolation from fresh samples is unsuccessful.

Phylogenetic analysis of fowl adenoviruses isolated from chickens with gizzard erosion in Japan.

Thirty-four fowl adenoviruses (FAdVs) isolated from chickens with gizzard erosion (GE) from 1999 to 2010 were characterized phylogenetically together with foreign isolates. The phylogenetic analysis based on part of the hexon gene classified these 34 FAdV isolates into 3 groups: FAdV-1, -8a and 8b, thereby suggesting that FAdVs associated with GEs in chickens are diverse. All 30 FAdV-1 isolates were genetically identical, and they were also identical with FAdV-1 isolates from GEs in chickens in European countries (Germany, Poland, Austria, Hungary and Italy). Thus, the same type of FAdV-1 has been associated with outbreaks of GE in Japanese chickens for the past 10 years, which may have spread from a common ancestor, although the epidemiological relationship is unknown.

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 of Newcastle disease in vaccinated chickens: pathogenicity of isolated virus and vaccine effect on challenge of its virus.

The pathogenicity of Newcastle disease (ND) virus, isolated from ND outbreak in vaccinated chickens, was evaluated through experiments. The pathogenicity indexes (mean death time (MDT); 58 hr, intracerebral pathogenicity index (ICPI); 1.7 and intravenous pathogenicity index (IVPI); 2.51) indicated that the ND virus was velogenic. The ND virus caused lymphocytic necrosis in the spleen with fibrinous exudation and proliferation of macrophages, sinusoidal fibrin exudation in the liver, proliferation of macrophages in the lung, lymphocytic necrosis and depletion in the bursa of Fabricius, cecal tonsils and thymus, necrosis of bone marrow, tracheitis, conjunctivitis and necrosis of feather epithelial cells in specific-pathogen-free chickens. Immunohistochemically, ND virus antigens were seen in the lesions mentioned above. The ND virus could not induce the encephalitis and pancreatitis that were observed in the natural case of ND in vaccinated chickens. There was no clinical disease in vaccinated chickens after the challenge of the ND virus. In diluted ND vaccine experiments, chickens vaccinated with a high dilution of vaccine and then challenged with the ND virus showed clinical sign and mortality with pancreatic focal necrosis. Vaccine diluted with fresh tap water had no effect on protection against the challenge of the ND virus. This study suggests that improper vaccination may be involved in outbreaks of ND in vaccinated chickens.

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.

Fowl adenoviruses isolated from chickens with inclusion body hepatitis in Japan, 2009-2010.

Nine fowl adenoviruses (FAdVs) isolated from chickens with inclusion body hepatitis (IBH) in Japan from 2009 to 2010 were characterized serologically and genetically. These isolates were all neutralized by antisera against the SR-48 strain (FAdV-2). Phylogenetic analysis based on the part of the hexon gene that included the L1 region revealed that all isolates were almost identical except one isolate in 2009. This suggests a common ancestor for the FAdVs obtained from chickens with IBH in Japan in 2010.

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.

Inactivation of high and low pathogenic avian influenza virus H5 subtypes by copper ions incorporated in zeolite-textile materials.

The effect of cotton textiles containing Cu(2+) held by zeolites (CuZeo-textile) on the inactivation of H5 subtype viruses was examined. Allantoic fluid (AF) containing a virus (AF virus) (0.1 ml) was applied to the textile (3×3-cm), and incubated for a specific period at ambient temperature. After each incubation, 0.9 ml of culture medium was added followed by squeezing to recover the virus into the medium. The recovered virus was titrated using Madin-Darby canine kidney (MDCK) cells or 10-day-old embryonated chicken eggs. The highly pathogenic H5N1 and the low pathogenic H5N3 viruses were inactivated on the CuZeo-textile, even after short incubation. The titer of A/chicken/Yamaguchi/7/04 (H5N1) in MDCK cells and in eggs declined by >5.0 log(10) and 5.0 log(10), respectively, in 30 s. The titer of A/whooper swan/Hokkaido/1/08 (H5N1) in MDCK cells declined by 2.3 and 3.5 in 1 and 5 min, respectively. When A/whistling swan/Shimane/499/83 (H5N3) was treated on the CuZeo-textile for 10 min, the titer declined by >5.0 log(10) in MDCK cells and by >3.5 log(10) in eggs. In contrast, no decrease in the titers was observed on cotton textiles containing zeolites alone (Zeo-textile). Neither cytopathic effects nor NP antigens were detected in MDCK cells inoculated with the H5N1 virus treated on the CuZeo-textile. The viral genes (H5, N1, M, and NP) were amplified from the virus treated on the CuZeo-textile by RT-PCR. The hemagglutinating activity of the CuZeo-textile treated virus was unaffected, indicating that virus-receptor interactions were maintained. Electron microscopic analysis revealed a small number of particles with morphological abnormalities in the H5N3 virus samples recovered immediately from the CuZeo-textile, while no particles were detectable in the 10-min treated sample, suggesting the rapid destruction of virions by the Cu(2+) in the CuZeo-textile. The loss of infectivity of H5 viruses could, therefore, be due to the destruction of virions by Cu(2+). Interestingly, CuCl(2) treatment (500 and 5000 µM) did not have an antiviral effect on the AF viruses (H5N1 and H5N3) even after 48 h of incubation, although the titer of the purified H5N3 virus treated with CuCl(2) declined greatly. The antiviral effect was inhibited by adding the AF to the purified H5N3 virus prior to the CuCl(2) treatment. The known antibacterial/antifungal activities of copper suggest that the CuZeo-textile can be applied at a high level of hygiene in both animals and humans.

H4N8 subtype avian influenza virus isolated from shorebirds contains a unique PB1 gene and causes severe respiratory disease in mice.

H4N8 subtype avian influenza viruses were isolated from shorebirds in eastern Hokkaido. All the isolates shared >99.7% nucleotide homology, and all the viral genes except for PB1 were highly related to those of A/red-necked stint/Australia/1/04. Thus, the isolates were regarded as PB1 reassortants. The most similar PB1 gene was identified in A/mallard/New Zealand/1615-17/04 (H4N6) with nucleotide homology of 90.9%. BALB/c mice intranasally inoculated with the H4N8 isolates developed severe respiratory disease, which eventually led to death in some mice. The virus was isolated from the lungs, and viral antigen was detected in the lungs with pneumonia. Other H4 subtype viruses tested did not cause any symptoms in mice, although these viruses were also isolated from the lungs. The PB2 gene of the H4N8 isolates contains K482R, but not the E627K or D701N substitutions. The PB1-F2 gene of the isolates consists of a 101-amino acid unique sequence, but lacks the N66S mutation.

Limited susceptibility of pigeons experimentally inoculated with H5N1 highly pathogenic avian influenza viruses.

An experimental infection study was performed using pigeons reared for racing or meat production in Japan and clade 2.2 and 2.3.2 isolates of H5N1 highly pathogenic avian influenza virus to evaluate the possible role of pigeons in virus transmission to poultry. In experiment 1, when 20 pigeons were intranasally inoculated with high or low viral doses, no inoculated pigeon exhibited clinical signs for 14 days. Drinking water and almost all swab samples were negative for virus isolation. Virus isolation was positive in 3 oral swab samples from 2 pigeons from day 2 through 4 postinoculation, but viral titers of positive samples were extremely low. Immunohistochemical analysis for virus detection was negative in all tissue samples. Along with seroconversion in a limited number of pigeons postinoculation, these results suggest that pigeons have limited susceptibility to the virus used for experimental infection. In experiment 2, when uninoculated chickens were housed with virus-inoculated pigeons, all pigeons and contact chickens survived for 14 days without exhibiting any clinical signs. According to serological analysis, the chickens did not exhibit seroconversion after close contact with inoculated pigeons. Our data suggest that the risk posed by pigeons with respect to the transmission of the H5N1 highly pathogenic avian influenza virus to poultry would be less than that for other susceptible avian species.

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.

Genetic characterization and susceptibility on poultry and mammal of H7N6 subtype avian influenza virus isolated in Japan in 2009.

From February to March 2009, six strains of H7N6 subtype avian influenza virus were isolated from quails in three farms in Aichi prefecture in Japan. The isolates were shown to be low pathogenic for chicken by the examination performed using the "Manual of Standards for Diagnostic Tests and Vaccines" by World organisation for Animal Health (OIE). The deduced amino acid sequence at the cleavage site was PE (I/Q/L) PKRR (nucleotide sequences were cct gaa (a/c) (t/a) a cc (a/g) aaa aga aga), suggesting persistence in domestic poultry for some time. The direct putative ancestor strain could not be elucidated by phylogenetic analysis of all genome segments of the quail isolates. Diverged date from a putative common ancestor in a non-rooted phylogenetic tree among quail viruses was estimated between March 2002 and July 2004. Three putative N-linked glycosylation sites resided in the vicinity of the receptor binding pocket of HA1 region. They are considered to decrease the reactivity of neutralizing antibody against the virus. Experiments for the infectivity and pathogenicity of a quail strain to poultry indicated that the quail isolate had higher infectivity to quails than chickens and ducks. Direct and dust-borne and/or droplet-borne transmissions among quail were proven in quails with and without direct contact with experimentally infected quails. The virus is seldom transmitted among chickens either directly or indirectly, and indirect transmission from infected quails to chickens was not observed. The pathogenicity of the quail strain for mammalian, pig and mouse was low, although it could replicate in those animals.

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.

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.

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.

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.

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.

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.