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.

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.

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.

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.

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.

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.

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.

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.

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.

Complete Genome Sequence of Infectious Bronchitis Virus Strain JP/KH/64, Isolated in Japan.

Here, we report the complete genome sequence of infectious bronchitis virus (IBV) strain JP/KH/64, which is the reference strain for the JP-I genotype in Japan. This information should be useful for an in-depth understanding of the evolution of the JP-I genotype.

Genetic Analysis of Avian Reovirus Isolated from Chickens in Japan.

Sigma C protein-coding sequences have been used to phylogenetically classify avian reovirus (ARV) strains. However, the relationship between serotype and phylogenetic cluster classification of the five prototype serotype strains of ARV in Japan has not been established. Thus, we used sigma C protein-coding sequences to characterize avian reoviruses (ARVs) isolated from chickens with tendonitis in Japan together with the five prototype serotype strains of ARV in Japan. Phylogenetic analysis of ARVs based on the sigma C protein-coding sequences revealed that the five prototype serotype strains of ARV were each classified into different, independent clusters. Two field isolates (JP/Tottori/2016 and JP/Nagasaki/2017) that were isolated from chickens with arthritis/tenosynovitis were classified into different clusters. JP/Tottori/2016 was classified into cluster VI with the CS-108 strain, and JP/Nagasaki/2017 was classified into cluster I with strain TS-142. Serologically, JP/Tottori/2016 was well-neutralized by antisera against the CS-108 strain, whereas JP/Nagasaki/2017 cross-reacted with antisera against both the CS-108 and TS-142 strains. Embryo lethality test revealed that the two field isolates induced 80% and 67% embryo mortality, respectively, whereas the five prototype strains induced 0%-33% embryo mortality. Our findings will contribute to understanding the characteristics of ARV strains in Japan.

Nota de investigación­Análisis genético de reovirus aviares aislados de pollos en Japón. Se han utilizado secuencias de proteína Sigma C para clasificar filogenéticamente cepas de reovirus aviar (ARV). Sin embargo, no se ha establecido la relación entre el serotipo y la clasificación de grupos filogenéticos de las cinco cepas prototipo de serotipo de reovirus aviares en Japón. Por lo tanto, se utilizó la secuenciación de la proteína sigma C para caracterizar los reovirus aviares (ARV) aislados de pollos con tendinitis en Japón junto con las cinco cepas prototipos de serotipos de reovirus aviares en Japón. El análisis filogenético de los reovirus extranjeros basado en el gene sigma C reveló que las cinco cepas prototipo de serotipo de reoviruses se clasificaron cada una en grupos diferentes e independientes. Dos aislamientos de campo (JP/Tottori/2016 y JP/Nagasaki/2017) que se aislaron de pollos con artritis/tenosinovitis se clasificaron en diferentes grupos. El aislamiento JP/Tottori/2016 se clasificó en el grupo VI con la cepa CS-108, y el aislamiento JP/Nagasaki/2017 se clasificó en el grupo I con la cepa TS-142. Serológicamente, el aislamiento JP/Tottori/2016 fue completamente neutralizado por antisueros contra la cepa CS-108, mientras que el virus JP/Nagasaki/2017 reaccionó de forma cruzada con antisueros contra las cepas CS-108 y TS-142. Las pruebas de patogenicidad de embriones revelaron que los dos aislados de campo indujeron 80% y 67% de mortalidad embrionaria, respectivamente, mientras que las cinco cepas prototipo indujeron 0% -33% de mortalidad embrionaria. Estos hallazgos contribuirán a comprender las características de las cepas de reovirus aviares en Japón.

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.

Genotyping of infectious bronchitis viruses isolated in Japan during 2008-2019.

Seventeen isolates of infectious bronchitis virus (IBV) were obtained from various prefectures of Japan during 2008-2019 and genetically analyzed. The IBV isolates were classified into six genetic groups, based on phylogenetic analysis of the S1 gene. The S1 genotypes were distinguishable by a newly developed restriction fragment length polymorphism (RFLP) method using three endonucleases, Hae II, Hpa I, and Fok I. Moreover, the isolates were classified into four genetic groups, based on phylogenetic analysis of the S2 gene. However, novel genetic groups based on a combination of S1 and S2 genotypes, which were undetected previously, were confirmed in this study, indicating that various recombinant IBV strains were prevalent in poultry in Japan.

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.

Identification of specific serotypes of fowl adenoviruses isolated from diseased chickens by PCR.

We have developed a polymerase chain reaction (PCR) assay to facilitate detection of the major disease-associated serotypes of fowl adenovirus (FAdV) including serotypes 1, 2, 4, 8a and 8b; primers were designed based on serotype-specific sequences of the hexon gene. We tested field isolates from chickens diagnosed with inclusion body hepatitis, gizzard erosion and hydropericardium syndrome together with reference FAdV strains characterized in Japan. We found that the primers were serotype specific; appropriate amplification of serotype-specific hexon genes was confirmed by sequence analysis of the PCR products. This PCR assay will be useful for detection of FAdV and for differentiation between disease-associated serotypes.

Fowl Adenoviruses Type 8b Isolated from Chickens with Inclusion Body Hepatitis in Japan.

Fowl adenovirus (FAdV) type 8b isolated from chickens with inclusion body hepatitis (IBH) in Japan from 2018 to 2019 were characterized serologically and genetically. Serologically, all isolates were well neutralized by antisera against the FAdV-8b strain, but they were not neutralized by antisera against the FAdV-8a strain. Phylogenetic analysis of the part of the hexon protein gene that includes the L1 region revealed that these isolates were all identical. They were also identical to foreign strains such as the SD1356 strain isolated in China and belonged to FAdV-8b. Furthermore, the 2018-19 Japanese IBH 8b isolates were genetically identical to the SD1356 strain by phylogenetic analysis of fiber genes, but they were different from previous Japanese 8b strains. These findings suggest that the 2018-19 Japanese IBH isolates might have been introduced from other countries.

Development of immunogenic chimeric virus-like particles based on bovine papillomavirus type 6.

Virus-like particles (VLPs) are considered useful tools for vaccine development because they induce an immune response and are safe. In addition, VLPs may be useful as a platform for the presentation of foreign antigens to elicit immune responses. In this study, we aimed to produce a chimeric VLP composed of L1 protein of bovine papillomavirus type 6 (BPV6-L1) that can display an entire foreign protein on its surface. Based on prediction of the conformational structure of VLP of BPV6-L1 (BPV6-VLP), candidate insertion sites for the foreign protein into BPV6-VLP were identified. Fusion proteins of BPV6-L1 and EGFP as a model foreign protein were constructed and produced. Only the fusion protein in which EGFP was inserted between amino acids 136 and 137 of BPV6-L1 self-assembled into VLPs and did not exhibit hindrance of the conformation of EGFP. Chimeric BPV6-VLP-immunized mice produced specific IgG against both BPV6 and EGFP. This is the first demonstration of the production of an immunogenic, bivalent, chimeric BPV6-VLP incorporating an entire protein for stable surface display. Thus, immunogenic chimeric BPV6-VLP may constitute a promising vaccine platform.

Inclusion body hepatitis caused by Aviadenovirus in a tropical screech owl (Megascops choliba).

In 2016, tropical screech owl (Megascops choliba) (Tso) chicks were suddenly found dead in a Japanese breeding facility. We autopsied a 9-day-old Tso and discovered white spots scattered on the liver surface. Multifocal necrosis was diffused, and macrophages had infiltrated the necrotic hepatic lesions. Hepatocytes contained numerous intranuclear inclusion bodies. Immunohistochemical staining detected Adenovirus antigen only in the liver. Next, PCR and sequencing (LC536616) identified Tso Adenovirus (TsoAd). Basic Local Alignment Search Tool (BLAST) and phylogenic analyzes suggested TsoAd is an owl Aviadenovirus. Our study contributes to an improved understanding of infectious disease among captive raptors.

Studies on heterologous protection between Japanese type 1 and type 2 porcine reproductive and respiratory syndrome virus isolates.

The objective of the present study was to evaluate the cross-protective immunity between type 1 and type 2 porcine reproductive and respiratory syndrome virus (PRRSV) isolates in growing pigs. Japanese type 1 PRRSV, first isolated from a pig with respiratory disorders in a farm in 2009, exhibits unique genetic characteristics. The pathogenicity of a Japanese standard strain of type 2 PRRSV, EDRD1, in pigs immunized by the type 1 PRRSV isolate, Jpn EU 4-37 was determined by evaluating clinical signs, viremia, antibody response, and pathological lesions. Similarly, we evaluated the pathogenicity of Jpn EU 4-37 in pigs immunized by EDRD1 and compared the cross-protective immunity between these isolates. The EDRD1 challenge after Jpn EU 4-37 inoculation reduced viral clearance and shedding in pigs, compared to those treated with the EDRD1 single infection. On the other hand, the pathogenicity of Jpn EU 4-37 after EDRD1 infection did not differ significantly compared to non-immunized pigs treated with Jpn EU 4-37. Therefore, exposure to Jpn EU 4-37 could not induce enough immunity to reduce the viremia against subsequent infection by type 2 PRRSV. However, the immunity induced by Jpn EU 4-37 infection may play a role in reducing viremia caused by type 2 PRRSV. Moreover, the immunity induced by the EDRD1 and other genetically related viruses, which are broadly distributed in Japan, may not contribute to cross-protection against Jpn EU 4-37 as an emerging virus.