A novel birnavirus identified as the causative agent of summer atrophy of pearl oyster (Pinctada fucata (Gould)).

The Akoya pearl oyster (Pinctada fucata (Gould)) is the most important species for pearl cultivation in Japan. Mass mortality of 0-year-old juvenile oysters and anomalies in adults, known as summer atrophy, have been observed in major pearl farming areas during the season when seawater temperatures exceed about 20 °C since 2019. In this study, we identified a novel birnavirus as the pathogen of summer atrophy and named it Pinctada birnavirus (PiBV). PiBV was first presumed to be the causative agent when it was detected specifically and frequently in the infected oysters in a comparative metatranscriptomics of experimentally infected and healthy pearl oysters. Subsequently, the symptoms of summer atrophy were reproduced by infection tests using purified PiBV. Infection of juvenile oysters with PiBV resulted in an increase in the PiBV genome followed by the atrophy of soft body and subsequent mortality. Immunostaining with a mouse antiserum against a recombinant PiBV protein showed that the virus antigen was localized mainly in the epithelial cells on the outer surface of the mantle. Although the phylogenetic analysis using maximum likelihood method placed PiBV at the root of the genus Entomobirnavirus, the identity of the bi-segmented, genomic RNA to that of known birnaviruses at the full-length amino acid level was low, suggesting that PiBV forms a new genus. The discovery of PiBV will be the basis for research to control this emerging disease.

Retrospective and prospective studies of transmissible viral proventriculitis in broiler chickens in Brazil.

We investigated the occurrence and pathologic findings of transmissible viral proventriculitis (TVP) associated with the chicken proventricular necrosis virus (CPNV) in commercial broiler chickens in southeastern Brazil. Seventy-three broilers, 25-36 d old, with a history of reduced growth, were referred to our veterinary pathology services from 2013 to 2017. Broilers were clinically examined, weighed, and euthanized for postmortem examination. Broilers of different ages with proventricular histologic lesions were positive for CPNV by RT-PCR; however, the intensity of histologic lesions was higher among 33-d-old animals, and viral RNA detection was more frequent among those that were 28 d old. In the proventriculi of 35 of 73 (48%) broilers, lesions were characterized by glandular epithelial necrosis, lymphoplasmacytic and histiocytic infiltrates, and metaplasia of glandular epithelium to ductal epithelium. In 24 of 73 (36%) broilers with histologic TVP-compatible lesions, CPNV was detected by RT-PCR for the viral protein 1 (VP1) gene. Broilers with histologic lesions were lighter than expected compared to the Cobb 500 standard weight. TVP has not been reported previously in broiler chickens in Brazil, to our knowledge.

A Unique Relative of Rotifer Birnavirus Isolated from Australian Mosquitoes.

The family Birnaviridae are a group of non-enveloped double-stranded RNA viruses which infect poultry, aquatic animals and insects. This family includes agriculturally important pathogens of poultry and fish. Recently, next-generation sequencing technologies have identified closely related birnaviruses in Culex, Aedes and Anopheles mosquitoes. Using a broad-spectrum system based on detection of long double-stranded RNA, we have discovered and isolated a birnavirus from Aedes notoscriptus mosquitoes collected in northern New South Wales, Australia. Phylogenetic analysis of Aedes birnavirus (ABV) showed that it is related to Rotifer birnavirus, a pathogen of microscopic aquatic animals. In vitro cell infection assays revealed that while ABV can replicate in Aedes-derived cell lines, the virus does not replicate in vertebrate cells and displays only limited replication in Culex- and Anopheles-derived cells. A combination of SDS-PAGE and mass spectrometry analysis suggested that the ABV capsid precursor protein (pVP2) is larger than that of other birnaviruses and is partially resistant to trypsin digestion. Reactivity patterns of ABV-specific polyclonal and monoclonal antibodies indicate that the neutralizing epitopes of ABV are SDS sensitive. Our characterization shows that ABV displays a number of properties making it a unique member of the Birnaviridae and represents the first birnavirus to be isolated from Australian mosquitoes.

A Retrospective Study of Transmissible Viral Proventriculitis in Broiler Chickens in California: 2000-18.

Transmissible viral proventriculitis (TVP) is a disease of chickens, mostly in broilers of 2-8 wk of age. Chicken proventricular necrosis virus (CPNV), a birnavirus, is the etiologic agent. Characteristic gross lesions are enlargement, atony, and pallor of the proventriculus. Cases diagnosed in California between 2000 and 2018 (n = 477), originating from 93 different farms representing all major companies in the region, were analyzed. Frequency of cases varied widely between years, with no recognizable seasonality. The flocks were between 6 and 61 days of age; the average age was 34.0 days, and the median age was 35 days. In 166 cases, between 6.3% and 100% of the submitted birds had gross lesions in the proventriculus. The most common findings were enlarged or dilated proventriculi, thickened walls, and pale or mottled serosal appearance. Histopathologically, inflammation of the glands was the most frequent finding. Other lesions included necrosis, hyperplasia, or both conditions of the glandular epithelium; dilated glands; and occasionally fibrin deposition, fibrosis, and hemorrhages. Twenty-three proventriculi from six cases were tested by immunohistochemistry for the presence of CPNV antigen; 21 stained positive. In 209 cases, birds also had lesions in the bursa fabricii attributed to infectious bursal disease, but with no significant difference in the mean percentage of birds with gross lesions in the proventriculus between cases with or without lesions in the bursa fabricii. The results show that TVP is a common disease of broiler flocks in California and confirms that CPNV is the likely causative agent.

Retrospective study on transmissible viral proventriculitis and chicken proventricular necrosis virus (CPNV) in the UK.

Chicken proventricular necrosis virus (CPNV) is a recently described birnavirus, which has been proposed to be the cause of transmissible viral proventriculitis (TVP). The understanding of the epidemiology of both the virus and the disease is very limited. A retrospective investigation on TVP and CPNV in broiler chicken submissions from the UK from between 1994 and 2015 was performed with the aims of assessing the longitudinal temporal evolution of TVP and CPNV, and to review the histological proventricular lesions in the studied chickens. Ninety-nine of the 135 included submissions (73.3%) fulfilled the TVP-diagnostic criteria, while the remaining 36 submissions (26.7%) displayed only lymphocytic proventriculitis (LP). The first detection of CPNV by PCR dated from 2009. Results showed a rise in the number of both TVP and positive CPNV RT-PCR submissions from 2009 with a peak in 2013, suggesting that they may be an emerging or re-emerging disease and pathogen, respectively. Twenty-two out of the 99 submissions displaying TVP lesions (22%) and four out of the 36 (11%) submissions with LP gave positive CPNV RT-PCR results, further supporting the association between CPNV and TVP and confirming that CPNV is present in a low proportion of proventriculi that do not fulfil the TVP-diagnostic criteria. In addition, intranuclear inclusion bodies were observed in 22 of the submissions with TVP. The vast majority of these cases (21 of 22, 96%) gave negative CPNV RT-PCR results, raising the question of whether a virus other than CPNV is responsible for some of these TVP-affected cases.RESEARCH HIGHLIGHTSTVP and CPNV have been present in British broilers since at least 1994 and 2009, respectively.TVP and CPNV seem to be an emerging and re-emerging disease and pathogen, respectively.CPNV was detected in proventriculi with both TVP and LP-lesions.Viruses other than CPNV may be responsible for some TVP-affected cases.

ICTV virus taxonomy profile: Birnaviridae.

Birnaviridae is a family of viruses with bi-segmented dsRNA genomes totalling about 6 kbp forming icosahedral, non-enveloped virions. The family includes four genera, members of three of which (Aquabirnavirus, Avibirnavirus and Blosnavirus) infect vertebrates (excluding mammals), whereas members of the fourth genus (Entomobirnavirus) infect insects. Each genus includes 1-3 species. Infectious pancreatic necrosis virus of salmonids and infectious bursal disease virus of poultry are two economically important birnaviruses. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of Birnaviridae, which is available at www.ictv.global/report/birnaviridae.

Detection, infection dynamics and small RNA response against Culex Y virus in mosquito-derived cells.

Many insect cell lines are persistently infected with insect-specific viruses (ISV) often unrecognized by the scientific community. Considering recent findings showing the possibility of interference between arbovirus and ISV infections, it is important to pay attention to ISV-infected cell lines. One example is the Entomobirnavirus, Culex Y virus (CYV). Here we describe the detection of CYV using a combination of small RNA sequencing, electron microscopy and PCR in mosquito cell lines Aag2, U4.4 and C7-10. We found CYV-specific small RNAs in all three cell lines. Interestingly, the magnitude of the detected viral RNA genome is variable among cell passages and leads to irregular detection via electron microscopy. Gaining insights into the presence of persistent ISV infection in commonly used mosquito cells and their interactions with the host immune system is beneficial for evaluating the outcome of co-infections with arboviruses of public health concern.

The complete genomic sequence of a novel botybirnavirus isolated from a phytopathogenic Bipolaris maydis.

Bipolaris maydis is the causal agent of corn southern leaf blight. Here, we report a novel double-stranded RNA (dsRNA) mycovirus designated Bipolaris maydis botybirnavirus 1 (BmBRV1) from B. maydis strain JZ11 in Jingzhou, Hubei province of China. BmBRV1 has a genome consisting of two dsRNAs (dsRNA1 and dsRNA2) with a size of 6435 and 5987 bp, respectively, each of which contains a single open reading frame (ORF). The two polyproteins encoded by dsRNA1 and dsRNA2 share the highest amino acid identities of 81.8 and 75.3%, respectively, with the RdRp and coat protein of Sclerotinia sclerotiorum botybirnavirus 1 (SsBRV1), a tentative species of the genus Botybirnavirus. Phylogenetic analysis based on the amino acid sequences of RdRp indicated that BmBRV1 belongs to a distinct species of the newly proposed family Botybirnaviridae.

Viruses infecting marine molluscs.

Although a wide range of viruses have been reported in marine molluscs, most of these reports rely on ultrastructural examination and few of these viruses have been fully characterized. The lack of marine mollusc cell lines restricts virus isolation capacities and subsequent characterization works. Our current knowledge is mostly restricted to viruses affecting farmed species such as oysters Crassostrea gigas, abalone Haliotis diversicolor supertexta or the scallop Chlamys farreri. Molecular approaches which are needed to identify virus affiliation have been carried out for a small number of viruses, most of them belonging to the Herpesviridae and birnaviridae families. These last years, the use of New Generation Sequencing approach has allowed increasing the number of sequenced viral genomes and has improved our capacity to investigate the diversity of viruses infecting marine molluscs. This new information has in turn allowed designing more efficient diagnostic tools. Moreover, the development of experimental infection protocols has answered some questions regarding the pathogenesis of these viruses and their interactions with their hosts. Control and management of viral diseases in molluscs mostly involve active surveillance, implementation of effective bio security measures and development of breeding programs. However factors triggering pathogen development and the life cycle and status of the viruses outside their mollusc hosts still need further investigations.

Detection of transmissible viral proventriculitis and chicken proventricular necrosis virus in the UK.

Increasing evidence suggests that a new birnavirus, named chicken proventricular necrosis virus (CPNV), is the aetiological agent of transmissible viral proventriculitis (TVP). The present work aimed to explore the possible presence of both TVP and CPNV in the UK. Forty-four chickens showing TVP-compatible gross lesions were classified into three groups based on the histological lesions: (i) TVP-affected chickens: lymphocytic infiltration and glandular necrosis (n = 15); (ii) lymphocytic proventriculitis (LP)-affected chickens: lymphocytic infiltration without necrosis (n = 18); and (iii) without proventriculitis (WP): no lymphocytic infiltration or necrosis (n = 11). Nine proventriculi (seven out of 15 corresponding to TVP, and two out of 11 corresponding to LP) were positive for CPNV by reverse transcriptase polymerase chain reaction (RT-PCR). These results support the previously suggested idea of CPNV as causative agent of TVP. Moreover, these data show that CPNV can also be detected in a number of cases with LP, which do not fulfil the histological TVP criteria. Phylogenetic analysis of partial sequences of gene VP1 showed that British CPNV sequences were closer to other European CPNV sequences and might constitute a different lineage from the American CPNV. TVP cases with negative CPNV PCR results may be due to chronic stages of the disease or to the reduced PCR sensitivity on formalin-fixed paraffin-embedded tissues. However, involvement of other agents in some of the cases cannot totally be ruled out. As far as the authors are aware, this is the first peer-reviewed report of TVP as well as of CPNV in the UK, and the first exploratory CPNV phylogenetic study.

Runting Stunting Syndrome Associated with Transmissible Viral Proventriculitis in Broiler Chickens.

This report describes an outbreak of transmissible viral proventriculitis (TVP) associated with runting stunting syndrome (RSS) in 25- and 28-day-old broiler chickens, in which chicken proventricular necrosis virus (CNPV) was detected. Clinical signs included poor uniformity, very small birds for their age, increased mortality, and culling of smaller birds. Almost all birds necropsied exhibited moderate to severely enlarged proventriculi with diffusely pale serosa and thickened walls. Microscopically the proventriculi had lesions of degeneration and necrosis of the epithelium of the proventricular glands, accompanied by lymphocytic inflammation and glandular hyperplasia, with occasional formation of lymphoid nodules within the glandular parenchyma. Immunohistochemistry staining for CPNV was positive. Positive staining was generally found in the cytoplasm of glandular epithelial cells in the form of finely granular brown pigment. CPNV RNA was detected in the proventriculi by reverse transcriptase-PCR (RT-PCR). Other findings included mild enteritis in a few birds and small bursa of Fabricius. Direct electron microscopy performed on the intestinal samples was negative for viral particles. RT-PCR analysis of bursae was positive for infectious bursal disease virus (IBDV). In conclusion, this report associates TVP with RSS by describing an outbreak in which TVP attributable to CPNV was the most commonly found lesionin chickens with a clinical history compatible with RSS. Therefore, TVP should be considered as a possible differential diagnosis in cases compatible with RSS.

Isolation of a novel aquatic birnavirus from rainbow trout Oncorhynchus mykiss in Australia.

In November 2010, a rainbow trout (Oncorhynchus mykiss) hatchery in Victoria reported increased mortality rates in diploid and triploid female fingerlings. Live and moribund fish were submitted for laboratory investigation. All fish showed hyperpigmentation of the cranial half of the body. Histological lesions were seen in all areas of skin examined despite the localised nature of the gross lesions. There was irregular hyperplasia and spongiosis, alternating with areas of thinning and architectural disturbance. Occasionally, particularly in superficial layers of epithelium, cells showed large, eosinophilic inclusions that obscured other cellular detail. A small number of fish had necrosis in dermis, subcutis and superficial muscles. Bacteriological culture of skin and gills was negative for all bacterial pathogens, including Flavibacterium columnare, the agent of columnaris disease. Attempts at virus isolation from the skin of affected fish resulted in the development of a cytopathic effect in RTG-2 cell cultures suggestive of the presence of a virus. Negative contrast electron microscopy of cell culture supernatant demonstrated the presence of viral particles with the typical morphology of birnaviruses. Preliminary molecular characterisation identified an aquabirnavirus that differed from both the Tasmanian aquabirnavirus (TABV) and other aquabirnaviruses exotic to Australia. Previous isolates of aquabirnaviruses in Australia and New Zealand have been from healthy fish in a marine environment. This is the first report of an aquabirnavirus isolated from young salmonids at a freshwater hatchery in Australia. The role of the virus in the mortality event on the farm is uncertain as no further deaths attributable to this virus have occurred in the 4 yr since its initial discovery. The virus has been provisionally named Victorian trout aquabirnavirus (VTAB).

A survey of fish viruses isolated from wild marine fishes from the coastal waters of southern Korea.

A survey was conducted to investigate viral infection in 253 wild marine fishes harvested in the southern coastal area of Korea from 2010 to 2012. The fish that were captured by local anglers were randomly bought and sampled for virus examination. The samples were tested for presence of virus by virus isolation with FHM, FSP, and BF-2 cells and molecular methods (polymerase chain reaction and sequencing). Of the 253 fish sampled, 9 fish were infected with virus. Aquabirnaviruses (ABVs), Viral hemorrhagic septicemia virus (VHSV), and Red seabream iridovirus (RSIV) were detected in 7, 1, and 1 fish, respectively. Molecular phylogenies demonstrated the detected viruses (ABV, VHSV, and RSIV) were more closely related to viruses reported of the same type from Korea and Japan than from other countries, suggesting these viruses may be indigenous to Korean and Japanese coastal waters.

Viral diseases of wild and farmed European eel Anguilla anguilla with particular reference to the Netherlands.

Diseases are an important cause of losses and decreased production rates in freshwater eel farming, and have been suggested to play a contributory role in the worldwide decline in wild freshwater eel stocks. Three commonly detected pathogenic viruses of European eel Anguilla anguilla are the aquabirnavirus eel virus European (EVE), the rhabdovirus eel virus European X (EVEX), and the alloherpesvirus anguillid herpesvirus 1 (AngHV1). In general, all 3 viruses cause a nonspecific haemorrhagic disease with increased mortality rates. This review provides an overview of the current knowledge on the aetiology, prevalence, clinical signs and gross pathology of these 3 viruses. Reported experimental infections showed the temperature dependency and potential pathogenicity of these viruses for eels and other fish species. In addition to the published literature, an overview of the isolation of pathogenic viruses from wild and farmed A. anguilla in the Netherlands during the past 2 decades is given. A total of 249 wild A. anguilla, 39 batches of glass eels intended for farming purposes, and 239 batches of farmed European eels were necropsied and examined virologically. AngHV1 was isolated from wild yellow and silver A. anguilla from the Netherlands from 1998 until the present, while EVEX was only found sporadically, and EVE was never isolated. In farmed A. anguilla AngHV1 was also the most commonly isolated virus, followed by EVE and EVEX.

Transmissible viral proventriculitis identified in broiler breeder and layer hens.

Transmissible viral proventriculitis (TVP) is a recognized cause of production losses in broiler chickens, but previously it has not been reported in broiler breeder and commercial layer hens. In this study, TVP was identified in broiler breeder and commercial layer hens, 9-20 wk of age, based on histopathologic detection of characteristic microscopic lesions. Microscopic lesions in proventriculi of affected hens consisted of glandular epithelial necrosis, ductal epithelial hyperplasia, replacement of glandular epithelium with ductal epithelium, and diffuse interstitial lymphoid infiltration. Additionally, chicken proventricular necrosis virus (CPNV), a virus previously identified as the etiology of TVP in broiler chickens, was detected in proventriculi of TVP-affected hens using a reverse transcriptase-polymerase chain reaction procedure. The findings identify TVP as a potential cause of production losses in broiler breeder and commercial layer hens and provide additional evidence for etiologic involvement in TVP by CPNV.

Espirito Santo virus: a new birnavirus that replicates in insect cells.

Espirito Santo virus (ESV) is a newly discovered virus recovered as contamination in a sample of a virulent strain of dengue-2 virus (strain 44/2), which was recovered from a patient in the state of Espirito Santo, Brazil, and amplified in insect cells. ESV was found to be dependent upon coinfection with a virulent strain of dengue-2 virus and to replicate in C6/36 insect cells but not in mammalian Vero cells. A sequence of the genome has been produced by de novo assembly and was not found to match to any known viral sequence. An incomplete match to the nucleotide sequence of the RNA-dependent RNA polymerase from Drosophila X virus (DXV), another birnavirus, could be detected. Mass spectrometry analysis of ESV proteins found no matches in the protein data banks. However, peptides recovered by mass spectrometry corresponded to the de novo-assembled sequence by BLAST analysis. The composition and three-dimensional structure of ESV are presented, and its sequence is compared to those of other members of the birnavirus family. Although the virus was found to belong to the family Birnaviridae, biochemical and sequence information for ESV differed from that of DXV, the representative species of the genus Entomobirnavirus. Thus, significant differences underscore the uniqueness of this infectious agent, and its relationship to the coinfecting virus is discussed.

BAFF enhances B-cell-mediated immune response and vaccine-protection against a very virulent IBDV in chickens.

Infectious bursal disease (IBD) is an acute, highly infectious and immunosuppressive disease caused by IBDV, which specifically targets destruction of B cells in the bursa of Fabricius. B-cell activating factor belonging to the TNF family (BAFF, also called BLyS, TALL-1, THANK, or zTNF4) is an important factor for B-cell proliferation and survival. Here we demonstrate that human soluble BAFF (hsBAFF) may enhance humoral immune response by elevating B lymphocyte activity of secretion of immunoglobulin (Ig) such as IgA, IgM and IgG in chickens immunized or unimmunized with an inactivated IBDV vaccine from a very virulent strain. Of importance, we found that hsBAFF, as a co-immunostimulant for vaccination, may play a vital role in amplifying the specific protective immune response, thereby potently preventing very virulent IBDV challenge. This is supported by serological evidence that hsBAFF may effectively enhance higher specific IgG activity and titre in serum of immunized chickens. The findings strongly suggest that BAFF may be exploited in combination with specific vaccination for prevention of IBD.

Electron microscopic identification of viruses associated with poult enteritis in turkeys grown in California 1993-2003.

Poult enteritis (PE) is one of the most common diseases seen in young turkey flocks. Since 1993, more than 1800 cases of suspected PE have been submitted for examination by negative stain electron microscopy; this has involved more than 2400 individual results, because in many cases more than one virus was identified; at least 1500 individual results were positive for viruses. Viruses have been identified in poults as young as 3 days and up to 9 wk of age. The most commonly found viruses are rotavirus-like viruses and small round viruses ranging from 15 nm to 30 nm, either alone or in combination. Reovirus, birnavirus, and adenovirus have also been detected. There has been no evidence to suggest the presence of coronaviruses. This report summarizes our findings.

Isolation, characterization and genome sequence of a birnavirus strain from flounder Paralichthys olivaceus in China.

A birnavirus strain, Paralichthys olivaceus birnavirus (POBV), was isolated and characterized from cultured flounder in China, and its complete genomic sequence was subsequently determined. The virus could induce cytopathic effects (CPE) in four of seven fish cell lines and was resistant to chloroform, 5-iodo-2'-deoxyuridine, acid and alkaline pH, and heat treatment. Purified virus particles had a typical icosahedral shape, with a diameter of approximately 55-60 nm. The genomic segments A and B of POBV were 3,091 and 2,780 bp in length and shared many of the features of the members of the family Birnaviridae. Segment A contained two partially overlapping ORFs encoding a polyprotein, pVP2-VP4-VP3, and a nonstructural protein, VP5, while segment B had only one ORF encoding for the VP1, a viral RNA-dependent RNA polymerase (RdRp). This is the first report about a birnavirus strain from a new non-salmonid host in China and its complete genome sequence.