Infection of zebrafish larvae with human norovirus and evaluation of the in vivo efficacy of small-molecule inhibitors.

We have recently established that human norovirus (HuNoV) replicates efficiently in zebrafish larvae after inoculation of a clinical sample into the yolk, providing a simple and robust in vivo system in which to study HuNoV. In this Protocol Extension, we present a detailed description of virus inoculation by microinjection, subsequent daily monitoring and harvesting of larvae, followed by viral RNA quantification. This protocol can be used to study viral replication of genogroup (G)I and GII HuNoVs in vivo within 3-4 d. Additionally, we describe how to evaluate the in vivo antiviral effect and toxicity of small molecules using HuNoV-infected zebrafish larvae, in multi-well plates and without the need for specific formulations. This constitutes a great advantage for drug discovery efforts, as no specific antivirals or vaccines currently exist to treat or prevent norovirus gastroenteritis.

Tephritid fruit flies have a large diversity of co-occurring RNA viruses.

Tephritid fruit flies are amongst the most devastating pests of horticulture, and Sterile Insect Technique (SIT) programs have been developed for their control. Their interactions with viruses are still mostly unexplored, yet, viruses may negatively affect tephritid health and performance in SIT programs, and, conversely, constitute potential biological control agents. Here we analysed ten transcriptome libraries obtained from laboratory populations of nine tephritid species from Australia (six species of Bactrocera, and Zeugodacus cucumis), Asia (Bactrocera dorsalis) and Europe (Ceratitis capitata). We detected new viral diversity, including near-complete (>99%) and partially complete (>80%) genomes of 34 putative viruses belonging to eight RNA virus families. On average, transcriptome libraries included 3.7 viruses, ranging from 0 (Z. cucumis) to 9 (B. dorsalis). Most viruses belonged to the Picornavirales, represented by fourteen Dicistroviridae (DV), nine Iflaviridae (IV) and two picorna-like viruses. Others were a virus from Rhabdoviridae (RV), one from Xinmoviridae (both Mononegavirales), several unclassified Negev- and toti-like viruses, and one from Metaviridae (Ortervirales). Using diagnostic PCR primers for four viruses found in the transcriptome of the Bactrocera tryoni strain bent wings (BtDV1, BtDV2, BtIV1, and BtRV1), we tested nine Australian laboratory populations of five species (B. tryoni, Bactrocera neohumeralis, Bactrocera jarvisi, Bactrocera cacuminata, C. capitata), and one field population each of B. tryoni, B. cacuminata and Dirioxa pornia. Viruses were present in most laboratory and field populations yet their incidence differed for each virus. Prevalence and co-occurrence of viruses in B. tryoni and B. cacuminata were higher in laboratory than field populations. This raises concerns about the potential accumulation of viruses and their potential health effects in laboratory and mass-rearing environments which might affect flies used in research and control programs such as SIT.

Tracking Mechanisms of Viral Dissemination In Vivo.

Dissemination and replication of viruses into hosts is a multistep process where viral particles infect, navigate, and indoctrinate various cell types. Viruses can reach tissues that are distant from their infection site by subverting subcellular mechanisms in ways that are, sometimes, disruptive. Modeling these steps, at appropriate resolution and within animal models, is cumbersome. Yet, mimicking these strategies in vitro fails to recapitulate the complexity of the cellular ecosystem. Here, we will discuss relevant in vivo platforms to dissect the cellular and molecular programs governing viral dissemination and briefly discuss organoid and ex vivo alternatives. We will focus on the zebrafish model and will describe how it provides a transparent window to unravel new cellular mechanisms of viral dissemination in vivo.

Embryo Microinjection Techniques for Efficient Site-Specific Mutagenesis in Culex quinquefasciatus.

Culex quinquefasciatus is a vector of a diverse range of vector-borne diseases such as avian malaria, West Nile virus (WNV), Japanese encephalitis, Eastern equine encephalitis, lymphatic filariasis, and Saint Louis encephalitis. Notably, avian malaria has played a major role in the extinction of numerous endemic island bird species, while WNV has become an important vector-borne disease in the United States. To gain further insight into C. quinquefasciatus biology and expand their genetic control toolbox, we need to develop more efficient and affordable methods for genome engineering in this species. However, some biological traits unique to Culex mosquitoes, particularly their egg rafts, have made it difficult to perform microinjection procedures required for genome engineering. To address these challenges, we have developed an optimized embryo microinjection protocol that focuses on mitigating the technical obstacles associated with the unique characteristics of Culex mosquitoes. These procedures demonstrate optimized methods for egg collection, egg raft separation and other handling procedures essential for successful microinjection in C. quinquefasciatus. When coupled with the CRISPR/Cas9 genome editing technology, these procedures allow us to achieve site-specific, efficient and heritable germline mutations, which are required to perform advanced genome engineering and develop genetic control technologies in this important, but currently understudied, disease vector.

Autophagy induced by infectious pancreatic necrosis virus promotes its multiplication in the Chinook salmon embryo cell line CHSE-214.

Infectious pancreatic necrosis virus (IPNV) is a common pathogen that causes huge economic losses for the salmonid aquaculture industry. Autophagy plays an important regulatory role in the invasion of pathogenic microorganisms. In this study, we explored the relationship between IPNV infection and autophagy in Chinook salmon embryo (CHSE-214) cells using standard methods. Transmission electron microscopy showed that IPNV infection produced typical structures of autophagosomes in CHSE-214 cells. Transformation of microtubule-associated protein 1 light chain 3 (LC3)-I to LC3-II protein, a marker of autophagy, was observed in IPNV-infected cells using confocal fluorescence microscopy and western blot analysis. Western blotting also showed that expression of the autophagy substrate p62 was significantly decreased in IPNV-infected cells. The influence of autophagy on IPNV multiplication was further clarified with cell culture experiments using autophagy inducer rapamycin and autophagy inhibitor 3-methyladenine. Rapamycin promoted IPNV multiplication at both the nucleic acid and protein levels, which led to higher IPNV yields; 3-methyladenine treatment had the opposite effect. This study has demonstrated that IPNV can induce autophagy, and that autophagy promotes the multiplication of IPNV in CHSE-214 cells.

Effect of fetal calf serum on propagation of duck hepatitis A virus genotype 3 in duck embryo fibroblast cells.

BACKGROUND: Duck viral hepatitis (DVH) is a highly contagious viral disease affecting ducks. It can be caused by five agents, including duck hepatitis A virus genotypes 1 (DHAV-1), 2 (DHAV-2), and 3 (DHAV-3), as well as duck hepatitis virus 2 and duck hepatitis virus 3. Since 2007, DHAV-3 has been known to be the most prevalent in East and South Asia. So far, the information regarding the propagation of DHAV-3 in cultured cells is limited. In this study, we describe the comparative studies on the growth properties of DHAV-3 in primary duck embryo fibroblast (DEF) cells using two different strains: a virulent strain C-GY and an attenuated strain YDF120. The effect of fetal calf serum (FCS) and chick serum (CS) on DHAV-3 replication and the mechanism of the inhibitory effect conferred by FCS were also investigated. RESULTS: Following serial passages, both C-GY and YDF120 failed to produce cytopathic effect and plaques. The combined quantitative real-time PCR and indirect immunofluorescence staining methods showed that the two viruses could be propagated productively in DEF cells. Investigation of the viral growth kinetics revealed that the two viruses replicated in DEF cells with similar efficiencies, while the viral load of the virulent C-GY strain peaked more rapidly when compared with the attenuated YDF120 strain. Neutralization assay and time-of-drug-addition study indicated that FCS displayed inhibitory effect on DHAV-3 replication. Analysis on the mechanism of action of FCS against DHAV-3 demonstrated that the inhibitory effect was reflected at three steps of the DHAV-3 life cycle including adsorption, replication, and release. CONCLUSIONS: Both virulent and attenuated DAHV-3 strains can establish noncytocidal, productive infections in DEF cells. The virulent strain replicates more rapidly than the attenuated strain in early infection period. FCS has an inhibitory effect on DHAV-3 replication, which may be attributed to action of a non-specific inhibitory factor present in FCS directly on the virus. These findings may provide new insights into the development of potential antiviral agents.

Recovery of Muscovy duck-origin goose parvovirus from an infectious clone containing an E-box motif (CACATG) deletion within the left terminal region.

Muscovy duck-origin goose parvovirus (MDGPV) is a causative agent of MDGPV-associated Derzsy's disease. To evalute the role of the cis-acting element E-box (CACATG) deletion on MDGPV eplication, an infectious plasmid clone p-PTΔE287, having one E-box deletion at nucleotide (nt) 287 of the left inverted terminal repeat sequence (L-ITR), was constructed by overlap extension PCR deleting the 287CACATG292 motif from the plasmid pMDGPVPT containing the full-length genome of the virulent MDGPV strain PT. The p-PTΔE287 plasmid was transfected into 9-day-old non-immune Muscovy duck embryos via the yolk sac, resulting in successful rescue of the deletion mutant virus r-PTΔE287. Compared with its parental virus PT, the virulence and the replication ability of r-PTΔE287 were reduced. In addition, we examined the ability of r-PTΔE287 to manipulate cell cycle progression. The results showed that r-PTΔE287 replication results in G0/G1 phase accumulation of infected duck embryo liver mesenchymal stem cells (BMSCs) and that this accumulation is caused by the prevention of cell cycle entry from G0/G1 phase into S phase. Taken together, introducing 287CACATG292 element deletion into MDGPV PT genomic DNA that induced rescued mutant virus (r-PTΔE287) cell cycle arrest function at the G0/G1 phase, which might inhibit MDGPV replication and virus progeny production. This study laid the foundation for further understanding of the relationship between E-box deletion in the L-ITR and MDGPV virulence.

ITRAQ-Based Quantitative Proteomics Reveals the Proteome Profiles of Primary Duck Embryo Fibroblast Cells Infected with Duck Tembusu Virus.

Outbreaks of duck Tembusu virus (DTMUV) have caused substantial economic losses in the major duck-producing regions of China since 2010. To improve our understanding of the host cellular responses to virus infection and the pathogenesis of DTMUV infection, we applied isobaric tags for relative and absolute quantification (iTRAQ) labeling coupled with multidimensional liquid chromatography-tandem mass spectrometry to detect the protein changes in duck embryo fibroblast cells (DEFs) infected and mock-infected with DTMUV. In total, 434 cellular proteins were differentially expressed, among which 116, 76, and 339 proteins were differentially expressed in the DTMUV-infected DEFs at 12, 24, and 42 hours postinfection, respectively. The Gene Ontology analysis indicated that the biological processes of the differentially expressed proteins were primarily related to cellular processes, metabolic processes, biological regulation, response to stimulus, and cellular organismal processes and that the molecular functions in which the differentially expressed proteins were mainly involved were binding and catalytic activity. Some selected proteins that were found to be differentially expressed in DTMUV-infected DEFs were further confirmed by real-time PCR. The results of this study provide valuable insight into DTMUV-host interactions. This could lead to a better understanding of DTMUV infection mechanisms.

Phage therapy against Pseudomonas aeruginosa infections in a cystic fibrosis zebrafish model.

Cystic fibrosis (CF) is a hereditary disease due to mutations in the CFTR gene and causes mortality in humans mainly due to respiratory infections caused by Pseudomonas aeruginosa. In a previous work we used phage therapy, which is a treatment with a mix of phages, to actively counteract acute P. aeruginosa infections in mice and Galleria mellonella larvae. In this work we apply phage therapy to the treatment of P. aeruginosa PAO1 infections in a CF zebrafish model. The structure of the CFTR channel is evolutionary conserved between fish and mammals and cftr-loss-of-function zebrafish embryos show a phenotype that recapitulates the human disease, in particular with destruction of the pancreas. We show that phage therapy is able to decrease lethality, bacterial burden, and the pro-inflammatory response caused by PAO1 infection. In addition, phage administration relieves the constitutive inflammatory state of CF embryos. To our knowledge, this is the first time that phage therapy is used to cure P. aeruginosa infections in a CF animal model. We also find that the curative effect against PAO1 infections is improved by combining phages and antibiotic treatments, opening a useful therapeutic approach that could reduce antibiotic doses and time of administration.

RNA-Seq analysis of duck embryo fibroblast cell gene expression during the early stage of egg drop syndrome virus infection.

Egg drop syndrome virus (EDSV), a member of the family Adenoviridae and an economically important pathogen with a broad host range, leads to markedly decreased egg production. However, the molecular mechanism underlying the host-EDSV interaction remains unclear. Here, we performed high-throughput RNA sequencing (RNA-Seq) to study the dynamic changes in host gene expression at 6, 12, and 24 hours post-infection in duck embryo fibroblasts (DEFs) infected with EDSV. Atotal of 441 differentially expressed genes (DEGs) were identified after EDSV infection. Gene Ontology category and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis revealed that these DEGs were associated with multiple biological functions, including signal transduction, host immunity, virus infection, cell apoptosis, cell proliferation, and pathogenicity-related and metabolic process signaling pathways. We screened and identified 12 DEGs for further examination by using qRT-PCR. The qRT-PCR and RNA-Seq results were highly consistent. This study analyzed viral infection and host immunity induced by EDSV infection from a novel perspective, and the results provide valuable information regarding the mechanisms underlying host-EDSV interactions, which will prove useful for the future development of antiviral drugs or vaccines for poultry, thus benefiting the entire poultry industry.

Specific detection of Muscovy duck parvovirus infection by TaqMan-based real-time PCR assay.

BACKGROUND: Muscovy duck parvovirus (MDPV) causes high mortality and morbidity in Muscovy ducks, with the pathogenesis of the virus still unknown in many respects. Specific MDPV detection is often rife with false positive results because of high identity at the genomic nucleotide level and antigenic similarity with goose parvovirus (GPV). The objective of this study was to develop a sensitive, highly specific, and repeatable TaqMan-based real-time PCR (qPCR) assay for facilitating the molecular detection of MDPV. RESULTS: The specific primers and probe were designed based on the conserved regions within MDPVs, but there was a variation in GPVs of the nonstructural (NS) genes after genetic comparison. After the optimization of qPCR conditions, the detection limit of this qPCR assay was 29.7 copies/µl. The assay was highly specific for the detection of MDPV, and no cross-reactivity was observed with other non-targeted duck-derived pathogens. Intra- and inter-assay variability was less than 2.21%, means a high degree of repeatability. The diagnostic applicability of the qPCR assay was proven that MDPV-positive can be found in cloacal swabs samples, Muscovy duck embryos and newly hatched Muscovy ducklings. CONCLUSIONS: Our data provided incidents that MDPV could be possible vertically transmitted from breeder Muscovy ducks to Muscovy ducklings. The developed qPCR assay in the study could be a reliable and specific tool for epidemiological surveillance and pathogenesis studies of MDPV.

Pathogenicity of a variant goose parvovirus, from short beak and dwarfism syndrome of Pekin ducks, in goose embryos and goslings.

The pathogenicity of a variant goose parvovirus (GPV), isolated from short beak and dwarfism syndrome of Pekin ducks (strain Cherry Valley), was investigated in embryonating goose eggs and goslings. The virus was easily grown in GPV antibody-free goose embryos and caused high mortality and severe lesions of goose embryos, indicating that the variant GPV has good adaptation and high pathogenicity to embryonated goose eggs similar to the classical GPV. Like the third egg-passage virus (strain H) of a classical GPV, the third egg-passage virus (strain JS1) of the variant GPV caused Derzsy's disease in 2-day-old goslings with high mortality. The findings suggest that the variant GPV strain, which had specifically adapted to Pekin ducks, still retained high pathogenicity for its original host. The mortality (73.3-80%) caused by the first and third egg-passages of the variant GPV was somewhat lower than that (93.3%) caused by the third passage virus of the classical GPV, reflecting the higher pathogenicity of the classical GPV for its original host. These findings are likely to reinforce the importance of surveillance for parvoviruses in different waterfowl species and stimulate further study to elucidate the impact of mutations in the GPV genome on its pathogenicity to goslings and ducks.

Susceptibility of Zebrafish to Vesicular Stomatitis Virus Infection.

The zebrafish, Danio rerio, has become recognized as a valuable model for infectious diseases. Here we evaluated the susceptibility of zebrafish to be infected with the mammalian vesicular stomatitis virus (VSV). Both zebrafish cells and embryos were highly susceptible to VSV infection. Mortalities exceeded 80% in infected embryos and were preceded by the invasion of the central nervous system by VSV. Live imaging of the infection with GFP-VSV as well as virus titration from infected fish confirmed the viral replication. Immunohistochemical analysis of embryonic fish provided evidence of viral antigens as well as of the apoptosis marker caspase-3 in the brain, eye, liver, pronephros, and skeletal muscle. So far, this is the first report describing the susceptibility of zebrafish to the mammalian virus VSV.

Analysis of differentially expressed proteins in Muscovy duck embryo fibroblasts infected with virulent and attenuated Muscovy duck reovirus by two-dimensional polyacrylamide gel electrophoresis.

Muscovy duck reovirus (MDRV) belongs to the Orthoreovirus genus of the Reoviridae family, which is a significant poultry pathogen leading to high morbidity and mortality in ducklings. However, the pathogenesis of the virus is not well understood. In the present study, two-dimensional (2D) polyacrylamide gel electrophoresis (PAGE) combined with LC-MS-MS was used to identify differentially expressed proteins between Muscovy duck embryo fibroblasts (MDEF) infected with virulent (MV9710 strain) and attenuated (CA strain) MDRV and non-infected MDEFs. A total of 115 abundant protein spots were identified. Of these, 59 of differentially expressed proteins were detected, with functions in metabolism and utilization of carbohydrates and nucleotides, anti-stress, and regulation of immune and cellular process. GO analysis of the identified proteins showed that they belonged to the classes molecular function (141 proteins), cellular component (62 proteins), and biological process (146 proteins). The results were validated by qRT-PCR, which suggests that the analysis method of 2D PAGE combined with LC-MS-MS used in this study is reliable. This study lays a foundation for further investigation of the biology of MDRV infection in MDEF.

Transfection of embryonated Muscovy duck eggs with a recombinant plasmid is suitable for rescue of infectious Muscovy duck parvovirus.

For members of the family Parvoviridae, rescue of infectious virus from recombinant plasmid is usually done in cultured cells. In this study, the whole genome of the pathogenic Muscovy duck parvovirus (MDPV) strain YY was cloned into the pBluescript II (SK) vector, generating recombinant plasmid pYY. With the aid of a transfection reagent, pYY plasmid was inoculated into 11-day-old embryonated Muscovy duck eggs via the chorioallantoic membrane route, resulting in the successful rescue of infectious virus and death of the embryos. The rescued virus exhibited pathogenicity in Muscovy ducklings similar to that of its parental strain, as evaluated based on the mortality rate. The results demonstrate that plasmid transfection in embryonated Muscovy duck eggs is a convenient and efficacious method for rescue of infectious MDPV in comparison to transfection of primary cells, which is somewhat time-consuming and laborious.

The role of hexon in egg drop syndrome virus (EDSV) inducing apoptosis in duck embryo fibroblast cells.

Although extensive efforts have been made to understand adenovirus infection in human cells, little is known for egg drop syndrome virus (EDSV) infection in the avian-derived cells. In this study, the effects of EDSV infection as well as the possible role hexon protein, the main building block of the EDSV capsid, on apoptosis induction in duck embryo fibroblast (DEF) cells was examined. Flow cytometry analysis and TUNEL assay revealed that EDSV infection induced significant apoptosis in DEF cells compared with mock infected cells. Interestingly, the increase of the apoptosis rate detected in EDSV infected DEF cells were accompanied by an increased virus load in cells in a time-dependent manner. Furthermore, a time-dependent decrease in hexon protein expression levels in hexon transfected DEF cells in parallel with a gradual decrease in TUNEL-labeling cells was also observed in the current study. In addition, caspase activity detection and western blot analysis indicates that either EDSV infection or EDSV hexon transfection both induced apoptosis of DEF cells via activating both the exogenous and the mitochondrial pathway.

Establishment of rock bream Oplegnathus fasciatus embryo (RoBE-4) cells with cytolytic infection of red seabream iridovirus (RSIV).

Red seabream iridovirus (RSIV) is a member of genus Megalocytivirus in the family Iridoviridae. RSIV infection causes significant economic losses of marine-fishes in East Asian countries. Grunt fin (GF) cell line has been commonly used for culturing RSIV. However, it is not suitable for definite evaluation of infectivity titer of RSIV because cells infected with RSIV are not completely cytolysed. Thus, we established a new cell line, RoBE-4, from rock bream (Oplegnathus fasciatus) eyed-egg embryos in this study. Morphologically, RoBE-4 cells were fibroblastic-like. They have been stably grown over two-years with 60 passages using Leibovitz's L-15 medium containing 10% (v/v) fetal bovine serum. RoBE-4 cells infected with RSIV exhibited cytopathic effects (CPE) with cell rounding. They were cytolysed completely after ≥2 weeks of culture. Numerous RSIV particles with icosahedral morphology of approximately 122nm in diameter were observed in cytoplasmic area of infected RoBE-4 cells. The RSIV-suceptibility and amount of extracellular RSIV released by RoBE-4 cells were 100-fold higher than those by GF cells. RSIV cultured with RoBE-4 cells was highly virulent to rock bream in infection experiments. Therefore, using RoBE-4 cells instead of GF cells will enable accurate and sensitive measurement of RSIV infectivity. In addition, RoBE-4 cells might be used to produce RSIV vaccine in the future with significant reduction in cost.

PPM1A silences cytosolic RNA sensing and antiviral defense through direct dephosphorylation of MAVS and TBK1.

Cytosolic RNA sensing is a prerequisite for initiation of innate immune response against RNA viral pathogens. Signaling through RIG-I (retinoic acid-inducible gene I)-like receptors (RLRs) to TBK1 (Tank-binding kinase 1)/IKKε (IκB kinase ε) kinases is transduced by mitochondria-associated MAVS (mitochondrial antiviral signaling protein). However, the precise mechanism of how MAVS-mediated TBK1/IKKε activation is strictly controlled still remains obscure. We reported that protein phosphatase magnesium-dependent 1A (PPM1A; also known as PP2Cα), depending on its catalytic ability, dampened the RLR-IRF3 (interferon regulatory factor 3) axis to silence cytosolic RNA sensing signaling. We demonstrated that PPM1A was an inherent partner of the TBK1/IKKε complex, targeted both MAVS and TBK1/IKKε for dephosphorylation, and thus disrupted MAVS-driven formation of signaling complex. Conversely, a high level of MAVS can dissociate the TBK1/PPM1A complex to override PPM1A-mediated inhibition. Loss of PPM1A through gene ablation in human embryonic kidney 293 cells and mouse primary macrophages enabled robustly enhanced antiviral responses. Consequently, Ppm1a(-/-) mice resisted to RNA virus attack, and transgenic zebrafish expressing PPM1A displayed profoundly increased RNA virus vulnerability. These findings identify PPM1A as the first known phosphatase of MAVS and elucidate the physiological function of PPM1A in antiviral immunity on whole animals.

[THE PROOF OF VERTICAL TRANSMISSION OF THE NUCLEOPOLYHEDROVIRUS IN MANY GENERATIONS OF THE GYPSY MOTH LYMANTRIA DISPAR L].

INTRODUCTION: Insect viruses can play an important role in population dynamics of their hosts. That is why the problem of permanent viral infection support among virus-positive insects is associated with one of the intriguing problems of general biology and virology. MATERIALS AND METHODS: Under laboratory conditions, the modeling of the vertical transmission of the nucleopolyhedrovirus (NPV) gypsy moth was implemented at relatively high level of mortality among insects of parental generation (60%). The diagnostics of the occult virus was executed by the PCR method among insects before their infection under laboratory conditions, as well as among insects that survived after inoculation. RESULTS: The NPV-caused mortality among insects that survived after infection in generations F1, F2, and F3 was 14 ± 4%, 10 ± 4%, and 5 ± 0.5%, respectively. In the following three generations NPV-induced mortality was not noticed. DISCUSSION: The level of the virus-positive individuals among the gypsy moth embryos in all occasions was higher than the NPV-induced mortality of insects. Thus, the given results show that the presence of virus among insect does not mean inevitable mortality of their hosts. Perhaps, the viral DNA can completely or partly lose its infectivity but may exist in the analyzed insect samples. CONCLUSIONS: The viral infection can be formed among progeny surviving after inoculation of insects. It can be actuated during three generations of the gypsy moth. The level of the virus-positive individuals among the gypsy moth embryos determined by the PCR method in daughter generations was higher than the NPV- induced mortality of insects.

Novel duck parvovirus identified in Cherry Valley ducks (Anas platyrhynchos domesticus), China.

An unknown infectious disease in Cherry Valley ducks (Anas platyrhynchos domesticus) characterized by short beak and strong growth retardation occurred in China during 2015. The causative agent of this disease, tentatively named duck short beak and dwarfism syndrome (DSBDS), as well as the evolutionary relationships between this causative agent and all currently known avian-origin parvoviruses were clarified by virus isolation, transmission electron microscope (TEM) observation, analysis of nuclear acid type, (RT-)PCR identification, whole genome sequencing, and NS1 protein sequences-based phylogenetic analyses. The results indicated that the causative agent of DSBDS is closely related with the goose parvovirus-like virus, which is divergent from all currently known avian-origin parvoviruses and should be a novel duck parvovirus (NDPV).