Recombinant fiber-1 protein of fowl adenovirus serotype 4 induces high levels of neutralizing antibodies in immunized chickens.

Virulent fowl adenovirus serotype 4 (FAdV-4) causes hydropericardium syndrome (HPS) with high mortality in chickens, leading to significant economic losses to the poultry industry. The development of an effective vaccine is essential for successful disease control. Here, we produced recombinant fiber-1 protein of FAdV-4, isolated from a Japanese HPS outbreak strain, JP/LVP-1/96, using a baculovirus expression system and evaluated its immunogenicity and protective efficacy. Recombinant fiber-1 protein induced high levels of neutralizing antibodies in immunized chickens, which were maintained for a minimum of 10 weeks. After being challenged with the virulent FAdV-4 strain JP/LVP-1/96, the immunized chickens did not exhibit clinical signs of infection or histopathological changes, there was a significant reduction in the viral load in various organs and total serum proteins, and albumin levels did not decline. These results suggest that the recombinant fiber-1 protein produced in this study can serve as a subunit vaccine to control HPS in chickens.

Genomic characterization of a fowl adenovirus serotype 4 strain isolated from a chicken with hydropericardium syndrome in Japan.

Here, we report the genomic characterization of a fowl adenovirus serotype 4 strain isolated from a chicken with hydropericardium syndrome in Japan. The viral genome of FAdV-4 strain JP/LVP-1/96 was found to be 45,688 bp long. Amino acid substitutions at position 219 (G to D) in the fiber-2 protein and at position 188 (I to R) in the hexon protein, which are commonly found in virulent FAdV-4 strains, were also found in the JP/LVP-1/96 strain. Additional specific amino acid substitutions commonly found in virulent FAdV-4 strains were found in ORFs 4 and 43, which are present only in members of the species Fowl adenovirus C. Phylogenetic analysis based on complete hexon protein gene sequences showed that strain JP/LVP-1/96 belongs to a different genetic cluster from the strains circulating in neighboring countries.

Immunity against a Japanese local strain of porcine reproductive and respiratory syndrome virus decreases viremia and symptoms of a highly pathogenic strain.

BACKGROUND: The type 2 highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) has spread throughout countries of southeast Asia, where it has caused severe economic losses. Even countries presently free of PRRSV are at high risk for infection and spread of this virus. Some of these countries, including Japan, have broad epidemics of the local type 2 PRRSV, creating chronic pathogenicity in the domestic pig population. The present study aimed to evaluate the protective efficacy of immunity by infection with a Japanese field isolate, EDRD1, against heterologous challenge with a Vietnamese HP-PRRSV field strain. To this end, four groups of PRRSV-negative crossbreed piglets were used for a challenge study. Groups 1 and 2 were inoculated with EDRD1 via the intranasal route. After 26 days, Groups 2 and 3 were inoculated with HP-PRRSV via the same route. Group 4 served as an uninfected control. Blood and oral fluid samples were taken every 3-4 days after HP-PRRSV challenge; on day 16 post-challenge, all pigs were euthanized, and examined pathologically. RESULTS: The nucleotide sequence analysis of nonstructural protein 2 gene of EDRD1 and comparison with Vietnamese HP-PRRSV showed that the 39 amino acid deletion sites of EDRD1 was nearly in the same region as the 29 amino acid deletion sites of HP-PRRSV. Immunity conferred by inoculation with EDRD1 dramatically reduced viral load in the sera and tissues besides viral shedding (Group 2) compared with those in pigs infected only with HP-PRRSV (Group 3). The clinical signs and rectal temperature were significantly reduced, and the average daily weight gain was significantly improved in the EDRD1-inoculated pigs (Group 2) compared with the Group 3 pigs. Notably, no viral RNA was detected in various organs of the Group 2 pigs 16 days post-infection with HP-PRRSV, except in one pig. Therefore, the immunity induced by EDRD1 and its genetically close field isolates may play a role in reducing viremia caused by HP-PRRSV. CONCLUSIONS: The results of the present study demonstrate that pigs are highly protected against heterologous Vietnamese HP-PRRSV challenge by immunity against a Japanese local strain, EDRD1.

Production of immunogenic recombinant L1 protein of bovine papillomavirus type 9 causing teat papillomatosis.

Bovine papillomavirus type 9 (BPV9) is a causative agent of severe teat papillomatosis. Considering the lack of efficient BPV culture methods, recombinant proteins such as virus-like particles developed through genetic engineering may serve as a useful tool for developing effective vaccines against BPV infection. In this study, we successfully produced immunogenic particles composed of recombinant L1 protein of BPV9 (rBPV9-L1), using a baculovirus expression system. rBPV9-L1-immunized mice produced BPV9-specific IgG, which did not cross-react with BPV type 6, which is another causative agent of teat papillomatosis. Hence, immunogenic rBPV9-L1 is potentially applicable as a vaccine candidate for teat papillomatosis.

Restricted viral cDNA synthesis in cell lines that fail to support productive infection by bovine leukemia virus.

Bovine leukemia virus (BLV) is the causative agent of enzootic bovine leucosis, which results in significant economic losses on many affected farms. BLV infects a wide range of animals as well as cell lines derived from various mammalian species and organs; however, studies show that only some cell lines support sustained production of viral progeny. The differences between cells that produce viral progeny and those that do not are unclear. The aim of this study was to identify the steps of BLV replication that are associated with the capacity of a cell to support a productive infection. Eleven cell lines derived from various species were categorized into two groups, those that produce BLV progeny and those that do not, and the efficiency of viral attachment was compared. In addition, viral entry and reverse transcription were compared for two BLV-producing cell lines and three non-producing cell lines. BLV attached to and entered all of the tested cells. However, synthesis of viral DNA was inhibited in all three non-virus-producing cell lines, suggesting that BLV production was blocked either prior to or at the stage of reverse transcription. These results increase our understanding of the BLV life cycle and should enable better control over the spread of BLV.

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.

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.

Complete Genome Sequences of Infectious Bronchitis Virus Genotype JP-II (GI-7) and JP-III (GI-19) Strains Isolated in Japan.

We report the complete genome sequences of strains JP/Yamanashi/93 and JP/Shimane/98, which are classified in JP-II (GI-7) and JP-III (GI-19), respectively, the major genotypes of infectious bronchitis virus (IBV) in Japan. This information will be useful for the in-depth understanding of the evolution of IBV in Japan.

Susceptibility and Pathogenesis of Eurasian Tree Sparrows Experimentally Inoculated with Velogenic Newcastle Disease Virus.

Wild-caught Eurasian tree sparrows (Passer montanus) were experimentally inoculated with genotype VII velogenic Newcastle disease virus (NDV) APMV1/chicken/Japan/Fukuoka-1/2004 to investigate the susceptibility and pathogenesis of infected sparrows. Intranasal inoculation of two groups with high or low doses of the virus resulted in the mortality of some birds in both groups on days 7-15 postinoculation. Neurologic signs, ruffled feathers, labored breathing, emaciation, diarrhea, depression, and ataxia were observed in a few birds that eventually succumbed to death. The inoculation of the higher viral load resulted in higher mortality and hemagglutination inhibition antibody detection rates. Tree sparrows that survived the 18-day observation period after inoculation exhibited no apparent clinical signs. Histologic lesions in dead birds were observed in the nasal mucosa, orbital ganglion, and central nervous system, accompanied by NDV antigens detected by immunohistochemistry. Viral inclusion bodies were rarely observed in the cytoplasm of neurons. NDV was isolated from the oral swab and brain of dead birds but not from other organs, including the lung, heart, muscle, colon, and liver. In another experimental group, tree sparrows were intranasally inoculated with the virus and then examined 1-3 days later to examine the early pathogenesis of the disease. Inoculated birds exhibited inflammation of the nasal mucosa with viral antigens, and virus was isolated from some oral swab samples on days 2 and 3 postinoculation. The results of the present study suggest that tree sparrows are susceptible to velogenic NDV, and the infection could be fatal, although some birds can exhibit asymptomatic or mild infection. The unique pathogenesis regarding the neurologic signs and viral neurotropism of velogenic NDV was characteristic in infected tree sparrows.

Susceptibilidad y patogenia en los gorriones molineros inoculados experimentalmente con un virus velogénico de la enfermedad de Newcastle. Gorriones molineros (Passer montanus) capturados de la naturaleza se inocularon experimentalmente con un virus velogénico de la enfermedad de Newcastle (NDV) del genotipo VII APMV1/chicken/Japan/Fukuoka-1/2004 para investigar la susceptibilidad y la patogenia de los gorriones infectados. La inoculación intranasal de dos grupos con dosis altas o bajas del virus resultó en la mortalidad de algunas aves en ambos grupos en los días siete a 15 posteriores a la inoculación. Se observaron signos neurológicos, plumas erizadas, dificultad para respirar, emaciación, diarrea, depresión y ataxia en algunas aves que finalmente sucumbieron a la muerte. La inoculación de la carga viral más alta resultó en tasas más altas de detección de anticuerpos inhibidores de hemaglutinación y mortalidad. Los gorriones molineros que sobrevivieron al período de observación de 18 días después de la inoculación no mostraron signos clínicos aparentes. En las aves muerta se observaron lesiones histológicas en la mucosa nasal, ganglio orbitario y sistema nervioso central, acompañadas de antígenos virales detectados por inmunohistoquímica. Rara vez se observaron cuerpos de inclusión virales en el citoplasma de las neuronas. El virus de Newcastle se aisló del hisopo orales y del cerebro de aves muertas, pero no de otros órganos, incluidos los pulmones, el corazón, los músculos, el colon y el hígado. En otro grupo experimental, el virus se inoculó por vía intranasal a los gorriones molineros y luego se examinaron al día uno y tres para examinar la patogenia temprana de la enfermedad. Las aves inoculadas exhibieron inflamación de la mucosa nasal con antígenos virales y se aisló el virus de algunas muestras de hisopos orales en los días dos y tres posteriores a la inoculación. Los resultados del presente estudio sugieren que los gorriones molineros son susceptibles al virus de Newcastle velogénico y que la infección podría ser mortal, aunque algunas aves pueden presentar una infección asintomática o leve. La patogénesis única con respecto a los signos neurológicos y el neurotropismo viral del virus de Newcastle velogénico fue característica en los gorriones molineros infectados.

Development and application of one-step multiplex reverse transcription PCR for simultaneous detection of five diarrheal viruses in adult cattle.

A one-step multiplex reverse transcription (RT)-PCR method was developed for the simultaneous detection of five viruses causing diarrhea in adult cattle: bovine group A rotavirus (GAR), bovine group B rotavirus (GBR), bovine group C rotavirus (GCR), bovine coronavirus (BCV), and bovine torovirus (BToV). The detection limit of the one-step multiplex RT-PCR for GAR, GCR, BCV, and BToV was 10(2), 10(0), 10(1), and 10(2) TCID(50)/ml, respectively, and that for GBR was 10(6) copies/ml. The one-step multiplex RT-PCR with newly designed primers to detect GAR had higher sensitivity than a single RT-PCR with conventional primers, with no false-positive reactions observed for ten other kinds of bovine RNA viruses To assess its field applicability, 59 of 60 fecal samples containing one of these five viruses from all 25 epidemic diarrhea outbreaks in adult cattle were positive in the one-step multiplex RT-PCR assay. Furthermore, using four additional fecal samples containing two viruses (GBR and BCV or BToV), two amplified products of the expected sizes were obtained simultaneously. In contrast, all 80 fecal samples lacking the five target viruses from normal adult cattle were negative in the multiplex assay. Taken together, our results indicate that the one-step multiplex RT-PCR developed here for the detection of GAR, GBR, GCR, BCV, and BToV can be expected to be a useful tool for the rapid and cost-effective diagnosis and surveillance of viral diarrhea in adult cattle.

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.

Hemagglutinin-neuraminidase gene of genotype VII Newcastle disease virus strains isolated in Japan.

Recently, genotype VII of Newcastle disease virus (NDV) has become the most prevalent NDV genotype in Asia. Here the hemagglutinin-neuraminidase (HN) gene of genotype VII NDV strains isolated in Japan was analyzed. Notably, point amino acid substitutions in the HN protein at position 347, which is located on the major linear epitope of the HN protein, were found in two strains. However, by a hemagglutination inhibition assay, major antigenic differences did not exist between the studied strains. Additionally, chickens vaccinated with the B1 strain did not exhibit clinical effects after challenge with variants possessing the substitution at position 347 (E to K), whereas all unvaccinated chickens subjected to this challenge died within 5 days.

Genetic Analysis of the Complete S1 Gene in Japanese Infectious Bronchitis Virus Strains.

The complete nucleotide sequence of the S1 glycoprotein gene of the Japanese infectious bronchitis virus (IBV) strains was determined and genetically analyzed. A total of 61 Japanese IBV strains were classified into seven genotypes, namely GI-1, 3, 7, 13, 18, 19, and GVI-1 using the classification scheme that was proposed by Valastro et al, with three exceptions. These genotypes practically corresponded to those defined in Japan, namely Mass, Gray, JP-II, 4/91, JP-I, JP-III, and JP-IV, which have been identified through their partial nucleotide sequences containing hypervariable regions 1 and 2. In addition, three exceptive strains were considered to be derived from recombination within the S1 gene of IBV strains G1-13 and GI-19. By analyzing the amino acid polymorphism of the S1 glycoprotein among Japanese genotypes, a diversity was observed based on the genotype-specific amino acid residue, the proteolytic cleavage motif at the S1/S2 cleavage site, and the position of the potential N-glycosylation sites.

Complete Genome Sequences of Two JP-I (GI-18) Genotype Infectious Bronchitis Virus Strains Isolated from Chickens with Nephritis in Japan.

We report the complete genome sequences of two strains of JP-1 genotype (GI-18) infectious bronchitis virus (IBV) isolated from the kidneys of dead chickens in Japan in 2000 and 2017. This information will help researchers better understand the evolution and epidemiology of IBV in Japan.

Correction: Mase et al. Genetic Analysis of the Complete S1 Gene in Japanese Infectious Bronchitis Virus Strains. Viruses 2022, 14, 716.

In the original publication [...].

Detection of Gyrovirus galga 1 in Cryopreserved Organs from Two Commercial Broiler Flocks in Japan.

Gyrovirus galga 1 (GyVg1, previously recognized as avian gyrovirus 2), which was first reported in chicken in 2011, is a new member of the genus Gyrovirus. The presence of GyVg1 has also been confirmed in different regions of Europe, South America, Africa, and Asia, indicating its global distribution. However, because there are no reports of examining the distribution of GyVg1 in animals in Japan, the epidemiology of this virus is unknown. In this study, we attempted to retrospectively detect GyVg1 in cryopreserved chicken materials derived from different two commercial broiler flocks in 1997. The GyVg1 genome was detected in organ materials derived from both flocks by PCR. GyVg1 detected in both flocks was classified into four genetic groups by analyzing the nucleotide sequences of the detected PCR products. These results suggest that diverse GyVg1 strains were present in commercial chicken flocks as early as 1997 in Japan.

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.

Analysis of the fusion protein gene of Newcastle disease viruses isolated in Japan.

The complete nucleotide sequences of the fusion (F) protein gene of Newcastle disease viruses (NDV) isolated in Japan from 1930 to 2007 (45 strains total) were determined and genetically analyzed. In the deduced amino acid sequences of fusion protein, the 5 potential asparagine-linked glycosylation sites and 10 cysteine residues were all conserved in the NDV examined in this study. The major epitopes involved in virus neutralization are conserved in most of the NDV strains isolated in Japan except a few strains. By virus neutralization test, no major antigenic differences were observed among representative strains of each genotype in Japan. All chickens vaccinated with the B1 strain survived without clinical signs after challenge with 2 NDV strains isolated in Japan (velogenic strains, JP/Ibaraki/2000 and JP/Kagoshima/91), which possess amino acids substitutions involved in virus neutralization in the F protein gene.

A potential system for the isolation and propagation of porcine deltacoronavirus using embryonated chicken eggs.

Porcine deltacoronavirus (PDCoV) is a novel swine enteropathogenic coronavirus that leads to acute diarrhea/vomiting, dehydration, and mortality in seronegative neonatal piglets. As widely known, attempts to culture porcine enteropathogenic coronaviruses, such as PDCoV and porcine epidemic diarrhea virus, in cells have been proven to be difficult. This study aimed to establish an efficient and cost-effective culture system for PDCoV using embryonated chicken eggs (ECEs) to enable future vaccine production and efficient virus isolation from infected animals. The inoculation of samples into the allantoic cavity of 3- to 7-day-old ECEs yielded efficient virus propagation even from porcine fecal samples. Virus propagation in 2- and 8-day-old ECEs were confirmed in 30.0 % and 11.1 % of the samples, respectively. This indicates that susceptible cells rapidly develop in 2-day-old ECEs and differentiate to mature cells that are nonsusceptible to PDCoV in 8-day-old layer chicken ECEs. Furthermore, our study demonstrated that PDCoV can be passaged in 6-day-old ECEs with high viral replicative efficiency. This technique for propagating PDCoV using ECEs is a powerful tool that could be utilized for PDCoV vaccine development and virus isolation from poultry, livestock, and wild animals.

Complete Genome Sequence of a Fowl Adenovirus D Strain Isolated from Chickens with Inclusion Body Hepatitis in Japan.

We report the complete genome sequence of fowl adenovirus D (FAdV-D) strain JP/Tokushima/2010IBH, which was isolated from chickens with inclusion body hepatitis in Japan. This FAdV-D isolate was genetically highly similar to recent isolates from China, suggesting a common origin.