Effect of intestinal microbiota on duck short-beak and dwarf syndrome caused by novel goose parvovirus.

Short-beak and dwarf syndrome (SBDS) is caused by infection with novel goose parvovirus (NGPV), which leads to intestinal dysbiosis, developmental delay, short beak, lameness, and paralysis in ducks and is the cause of skeletal health problems. NGPV infection can cause intestinal microbial disturbances, but it is still unclear whether the intestinal microbiota affects the pathogenicity of NGPV. Here, the effects of intestinal microbiota on NGPV-induced SBDS in Cherry Valley ducks were assessed by establishing a duck model for gut microflora depletion/reestablishment through antibiotics (ABX) treatment/fecal microbiota transplanted (FMT). By measuring body weight, beak length, beak width and tarsal length, we found that SBDS clinical symptoms were alleviated in ducks treated with ABX, but not in FMT ducks. Next, we conducted a comprehensive analysis of bone metabolism, gut barrier integrity, and inflammation levels using quantitative real-time PCR (qPCR), enzyme linked immunosorbent assay (ELISA), biochemical analysis and histological analysis. The results showed that ABX treatment improved bone quality reduced bone resorption, mitigated tissue lesions, protected intestinal barrier integrity, and inhibited systemic inflammation in NGPV-infected ducks. Moreover, cecal microflora composition and short-chain fatty acids (SCFAs) production were examined by bacterial 16S rRNA sequencing and gas chromatography. The results revealed that ABX treatment mitigated the decreased abundance of Firmicutes and Bacteroidota in NGPV-infected ducks, as well as increased SCFAs production. Furthermore, ABX treatment reduced the mucosa-associated lymphoid tissue lymphoma translocation protein 1 (Malt1) and nuclear factor κB (NF-κB) expression, which are correlated with systemic inflammation in SBDS ducks. These findings suggested that intestinal microflora depletion alleviated NGPV-induced SBDS by maintaining intestinal homeostasis, inhibiting inflammatory response and alleviating bone resorption. These results provide evidence for the pivotal role of intestinal microbiota in the process of SBDS and contribute a theoretical basis for the feasibility of microecological preparation as a method to control SBDS.

Virome of a Feline Outbreak of Diarrhea and Vomiting Includes Bocaviruses and a Novel Chapparvovirus.

An unexplained outbreak of feline diarrhea and vomiting, negative for common enteric viral and bacterial pathogens, was subjected to viral metagenomics and PCR. We characterized from fecal samples the genome of a novel chapparvovirus we named fechavirus that was shed by 8/17 affected cats and identified three different feline bocaviruses shed by 9/17 cats. Also detected were nucleic acids from attenuated vaccine viruses, members of the normal feline virome, viruses found in only one or two cases, and viruses likely derived from ingested food products. Epidemiological investigation of disease signs, time of onset, and transfers of affected cats between three facilities support a possible role for this new chapparvovirus in a highly contagious feline diarrhea and vomiting disease.

Murine and related chapparvoviruses are nephro-tropic and produce novel accessory proteins in infected kidneys.

Mouse kidney parvovirus (MKPV) is a member of the provisional genus Chapparvovirus that causes renal disease in immune-compromised mice, with a disease course reminiscent of polyomavirus-associated nephropathy in immune-suppressed kidney transplant patients. Here we map four major MKPV transcripts, created by alternative splicing, to a common initiator region, and use mass spectrometry to identify "p10" and "p15" as novel chapparvovirus accessory proteins produced in MKPV-infected kidneys. p15 and the splicing-dependent putative accessory protein NS2 are conserved in all near-complete amniote chapparvovirus genomes currently available (from mammals, birds and a reptile). In contrast, p10 may be encoded only by viruses with >60% amino acid identity to MKPV. We show that MKPV is kidney-tropic and that the bat chapparvovirus DrPV-1 and a non-human primate chapparvovirus, CKPV, are also found in the kidneys of their hosts. We propose, therefore, that many mammal chapparvoviruses are likely to be nephrotropic.

The 164 K, 165 K, and 167 K residues of VP1 are vital for goose parvovirus proliferation in GEFs based on PCR-based reverse genetics system.

BACKGROUND: Goose parvovirus (GPV) is the etiological agent of Derzsy's disease and is fatal for gosling. Research on the molecular basis of GPV pathogenicity has been hampered by the lack of a reliable reverse genetics system. At present, the GPV infectious clone has been rescued by transfection in the goose embryo, but the growth character of it is unclear in vitro. METHODS: In this study, we identified the full-length genome of GPV RC16 from the clinical sample, which was cloned into the pACYC177, generating the pIRC16. The recombinant virus (rGPV RC16) was rescued by the transfection of pIRC16 into goose embryo fibroblasts (GEFs). The rescued virus was characterized by whole genome sequencing, indirect immunofluorescence assays (IFA) and western blot (WB) using rabbit anti-GPV Rep polyclonal antibody as the primary antibody. Previously, we found the 164 K, 165 K, and 167 K residues in the 160YPVVKKPKLTEE171 are required for the nuclear import of VP1 (Chen S, Liu P, He Y, et al. Virology 519:17-22). According to that, the GPV infectious clones with mutated K164A, K165A, or K167A in VP1 were constructed, rescued and passaged. RESULTS: The rGPV RC16 has been successfully rescued by transfection of pIRC16 into the GEFs and can proliferate in vitro. Furthermore, the progeny virus produced by pIRC16 transfected cells was infectious in GEFs. Moreover, mutagenesis experiments showed that the rGPV RC16 with mutated 164 K, 165 K and 167 K in VP1 could not proliferate in GEFs based on the data of IFA and WB in parental virus and progeny virus. CONCLUSIONS: The rGPV RC16 containing genetic maker and the progeny virus are infectious in GEFs. The 164 K, 165 K, and 167 K of VP1 are vital for the proliferation of rGPV RC16 in vitro.

Biodistribution of adeno-associated virus type 2 with mutations in the capsid that contribute to heparan sulfate proteoglycan binding.

We compared the phenotypes of three mutant AAV2 viruses containing mutations in arginine amino acids (R585, R588 and R484) previously shown to be involved in AAV2 heparan sulfate binding. The transduction efficiencies of wild type and mutant viruses were determined in the eye, the brain and peripheral organs following subretinal, striatal and intravenous injection, respectively, in mice and rats. We found that each of the three mutants (the single mutant R585A; the double mutant R585, 588A; and the triple mutant R585, 588, 484A) had a unique phenotype compared to wt and each other. R585A was completely defective for transducing peripheral organs via intravenous injection, suggesting that R585A may be useful for targeting peripheral organs by substitution of peptide ligands in the capsid surface. In the brain, all three mutants displayed widespread transduction, with the double mutant R585, 588A displaying the greatest spread and the greatest number of transduced neurons. The double mutant was also extremely efficient for retrograde transport, while the triple mutant was almost completely defective for retrograde transport. This suggested that R484 may be directly involved in interaction with the transport machinery. Finally, the double mutant also displayed improved transduction of the eye compared to wild type and the other mutants.

Growth characteristics of the novel goose parvovirus SD15 strain in vitro.

BACKGROUND: Short beak and dwarfism syndrome (SBDS) was caused by novel goose parvovirus (NGPV)--a variant of goose parvovirus (GPV). Ducks infected with NGPV shows clinical signs including growth retardation and protrusion of the tongue from an atrophied beak. SBDS outbreak was first reported at the northern coastal provinces of China during 2015 and it was again reported in Sichuan, an inland province of China in 2016. The disease caused a huge economic loss in Chinese duck feeding industry. RESULTS: The SD15 strain of NGPV was isolated from liver and intestinal tract tissue samples of infected ducks. Real-time quantitative PCR (qPCR) was used to estimate viral load in embryonated eggs and cells infected with adapted virus. The data showed that duck embryo fibroblasts (DEFs) were permissive to NGPV, while goose embryo fibroblasts (GEFs) cells were not, and the copy numbers of SD15 in the allantoic fluid of infected eggs remained at 105.0-106.5 copies/ml. The adaption procession of the virus was determined via qPCR, and viral proliferation was detected through indirect fluorescent antibody assay (IFA) in DEFs. It was further determined that viral copy numbers peaked at 96 h post-inoculation (hpi), which is the best time to harvest the virus in DEFs. Cytotoxic effects and cell death were observed at 72 hpi in SD15 infected DEFs, yet SD15 did not induce apoptosis. CONCLUSIONS: The growth characteristics of SD15 strain of NGPV determined would be beneficial for further molecular characterization of these viruses and develop potential vaccines if required.

Methylation Status of the Adeno-Associated Virus Type 2 (AAV2).

To analyze the methylation status of wild-type adeno-associated virus type 2 (AAV2), bisulfite PCR sequencing (BPS) of the packaged viral genome and its integrated form was performed and 262 of the total 266 CG dinucleotides (CpG) were mapped. In virion-packaged DNA, the ratio of the methylated cytosines ranged between 0⁻1.7%. In contrast, the chromosomally integrated AAV2 genome was hypermethylated with an average of 76% methylation per CpG site. The methylation level showed local minimums around the four known AAV2 promoters. To study the effect of methylation on viral rescue and replication, the replication initiation capability of CpG methylated and non-CpG methylated AAV DNA was compared. The in vitro hypermethylation of the viral genome does not inhibit its rescue and replication from a plasmid transfected into cells. This insensitivity of the viral replicative machinery to methylation may permit the rescue of the integrated heavily methylated AAV genome from the host's chromosomes.

Minute Virus of Canines NP1 Protein Interacts with the Cellular Factor CPSF6 To Regulate Viral Alternative RNA Processing.

The NP1 protein of minute virus of canines (MVC) governs production of the viral capsid proteins via its role in pre-mRNA processing. NP1 suppresses polyadenylation and cleavage at its internal site, termed the proximal polyadenylation (pA)p site, to allow accumulation of RNAs that extend into the capsid gene, and it enhances splicing of the upstream adjacent third intron, which is necessary to properly enter the capsid protein open reading frame. We find the (pA)p region to be complex. It contains redundant classical cis-acting signals necessary for the cleavage and polyadenylation reaction and splicing of the adjacent upstream third intron, as well as regions outside the classical motifs that are necessary for responding to NP1. NP1, but not processing mutants of NP1, bound to MVC RNA directly. The cellular RNA processing factor CPSF6 interacted with NP1 in transfected cells and participated with NP1 to modulate its effects. These experiments further characterize the role of NP1 in parvovirus gene expression.IMPORTANCE The Parvovirinae are small nonenveloped icosahedral viruses that are important pathogens in many animal species, including humans. Unlike other parvoviruses, the bocavirus genus controls expression of its capsid proteins via alternative RNA processing, by both suppressing polyadenylation at an internal site, termed the proximal polyadenylation (pA)p site, and by facilitating splicing of an upstream adjacent intron. This regulation is mediated by a small genus-specific protein, NP1. Understanding the cis-acting targets of NP1, as well as the cellular factors with which it interacts, is necessary to more clearly understand this unique mode of parvovirus gene expression.

Precision Fluorescent Labeling of an Adeno-Associated Virus Vector to Monitor the Viral Infection Pathway.

Adeno-associated virus 2 (AAV2) is a common vehicle for the delivery of a variety of therapeutic genes. A better understanding of the process of infection of AAV2 will advance our knowledge of AAV2 biology and allow for the optimization of AAV2 capsids with favorable transduction profiles. However, the precise fluorescent labeling of an AAV2 vector for probing virus tracking without affecting the nature of the virus remains a challenge. In this study, a lab-synthesized azide-moieties on the viral capsid at modifiable sites is precisely displayed. Upon bioorthogonal copper-less click reaction, fluorophores are subsequently conjugated to AAV2 vectors for visualization of particles. Using this principle, the authors demonstrate that it can be used for visibly studying the cell entry, and intracellular trafficking of AAV2 particles, enabling the monitoring of the intracellular dynamics of AAV2 infection. This study provides new insights into the precision labeling of AAV2 particles with important implications for a better understanding of the molecular mechanism of therapeutic gene delivery.

Fusion of Anthopleurin-B to AAV2 increases specificity of cardiac gene transfer.

AAV-mediated gene therapy has become a promising therapeutic strategy for chronic diseases. Its clinical utilization, however, is limited by the potential risk of off-target effects. In this work we attempt to overcome this challenge, hypothesizing that cardiac ion channel-specific ligands could be fused onto the AAV capsid, and narrow its tropism to cardiac myocytes. We successfully fused the cardiac sodium channel (Nav1.5)-binding toxin Anthopleurin-B onto the AAV2 capsid without compromising virus integrity, and demonstrated increased specificity of cardiomyocyte attachment. Although virus attachment to Nav1.5 did not supersede the natural heparan-mediated virus binding, heparan-binding ablated vectors carrying Anthopleurin-B eliminated hepatic and other extracardiac gene transfer, while preserving cardiac myocyte gene transfer. Virus binding to the cardiac sodium channel transiently decreased sodium current density, but did not cause any arrhythmias. Our findings expand the knowledge of attachment, infectivity, and intracellular processing of AAV vectors, and present an alternative strategy for vector retargeting.

Genomic and pathogenic analysis of a Muscovy duck parvovirus strain causing short beak and dwarfism syndrome without tongue protrusion.

In 2008, clinical cases of short beak and dwarfism syndrome (SBDS) caused by Muscovy duck parvovirus (MDPV) infection were found in mule duck and Taiwan white duck farms in Fujian, China. A MDPV LH strain causing duck SBDS without tongue protrusion was isolated in this study. Phylogenetic analysis show that the MDPV LH strain was clustered together with other MDPV strains, but divergent from GPV isolates. Two major fragment deletions were found in the inverted terminal repeats (ITR) of MDPV LH similar to the ones in the ITR of MDPV GX5, YY and SAAS-SHNH strains. To investigate the pathogenicity of the MDPV LH strain, virus infection of young mule ducks was performed. The infected ducks showed SBDS symptoms including retard growth and shorten beaks without tongue protrusion. Atrophy of thymus, spleen and bursa of Fabricius was identified in the infected ducks. The results show that MDPV LH strain is moderately pathogenic to mule duck, leading to occurrence of SBDS. As far as we know, it is the first study showing that SBDS without tongue protrusion, and atrophy of thymus, spleen and bursa of Fabricius possibly associated with immunosuppression were found in the MDPV-infected ducks. The established duck-MDPV-SBDS system will help us to further work on the virus pathogenesis and develop efficacious vaccine against MDPV infection.

The role of nuclear localization signal in parvovirus life cycle.

Parvoviruses are small, non-enveloped viruses with an approximately 5.0 kb, single-stranded DNA genome. Usually, the parvovirus capsid gene contains one or more nuclear localization signals (NLSs), which are required for guiding the virus particle into the nucleus through the nuclear pore. However, several classical NLSs (cNLSs) and non-classical NLSs (ncNLSs) have been identified in non-structural genes, and the ncNLSs can also target non-structural proteins into the nucleus. In this review, we have summarized recent research findings on parvovirus NLSs. The capsid protein of the adeno-associated virus has four potential nuclear localization sequences, named basic region 1 (BR), BR2, BR3 and BR4. BR3 was identified as an NLS by fusing it with green fluorescent protein. Moreover, BR3 and BR4 are required for infectivity and virion assembly. In Protoparvovirus, the canine parvovirus has a common cNLS located in the VP1 unique region, similar to parvovirus minute virus of mice (MVM) and porcine parvovirus. Moreover, an ncNLS is found in the C-terminal region of MVM VP1/2. Parvovirus B19 also contains an ncNLS in the C-terminal region of VP1/2, which is essential for the nuclear transport of VP1/VP2. Approximately 1 or 2 cNLSs and 1 ncNLS have been reported in the non-structural protein of bocaviruses. Understanding the role of the NLS in the process of parvovirus infection and its mechanism of nuclear transport will contribute to the development of therapeutic vaccines and novel antiviral medicines.

Molecular detection of chicken parvovirus in broilers with enteric disorders presenting curving of duodenal loop, pancreatic atrophy, and mesenteritis.

Enteric disorders are an important cause of economic losses in broiler chickens worldwide. Several agents have been associated with enteric problems, such as viruses, bacteria, and parasites. In this study, broiler chickens showing signs of enteric disorders were subjected to molecular diagnosis for several viral agents and also for pathological examination for elucidating this problem. Thus, the chickens were screened for avian nephritis virus (ANV), chicken astrovirus (CAstV), avian rotavirus (ArtV), avian reovirus (AReoV), infectious bronchitis virus (IBV), fowl adenovirus group I (FAdV-1), and chicken parvovirus (ChPV). Postmortem examinations revealed a curving of the duodenal loop (J-like appearance) and intestines filled with liquid and gaseous content. Histopathological analysis of the duodenal loop showed pancreatic atrophy, acute mesenteritis, and enteritis. PCR results showed that ChPV was the sole viral agent detected in samples with lesions such as the curved duodenal loop and pancreatic atrophy. Molecular characterization of the nucleotide and deduced amino acid sequences revealed a high similarity with other strains of ChPV from Brazil, Canada, United States, Europe, and Asia. These findings suggest an association between ChPV and the development of enteritis, pancreatitis, and pancreatic atrophy, which may lead to curling of the duodenal loop. Together, these alterations may disrupt the normal functioning of the digestive system, diminishing digestion and the absorption of dietary nutrients and consequently leading to reduced weight gain, flock impairment, dwarfism, and an elevated feed conversion rate.

Evidence for Vertical Transmission of Novel Duck-Origin Goose Parvovirus-Related Parvovirus.

In 2015, novel duck-origin goose parvovirus-related parvovirus (N-GPV) infection progressively appeared in commercial Cherry Valley duck flocks in North China. Diseased ducks were observed to have beak atrophy and dwarfism syndrome (BADS). A previous study showed that a high seropositive rate for N-GPV indicated a latent infection in most breeder duck flocks. To investigate this possibility in hatching eggs collected from N-GPV-infected breeder ducks, 120 eggs were collected at various stages of embryonic development for viral DNA detection and an N-GPV-specific antibody test. N-GPV DNA was present in nine hatching eggs, eleven duck embryo and eight newly hatched ducklings. Of the newly hatched ducklings, 58.33% (21/36) were seropositive. Further, two isolates were obtained from a 12-day-old duck embryo and a newly hatched duckling. N-GPV infection did not reduce the fertilization rate and hatchability. These results indicate possible vertical transmission of N-GPV and suggest that it may be transmitted from breeder ducks to ducklings in ovo.

Immunobiological activity and antiviral regulation efforts of Chinese goose (Anser cygnoides) CD8α during NGVEV and GPV infection.

The CD8 molecule is a cell membrane glycoprotein expressed on cytotoxic T lymphocytes, which are involved in the clearance of viruses. However, the functional characterization of goose CD8α is still unclear. The immunobiological characterization of goose CD8α in goose spleen mononuclear cells (MNCs) was examined by real-time quantitative PCR (qPCR). It was shown that CD8α mRNA levels were significantly up-regulated by in vitro treatment of MNCs with phytohemagglutinin (PHA), concanavalin A (ConA), and polyinosinic-polycytidylic acid (poly I:C) in a dose-dependent way, but lipopolysaccharides (LPSs) did not have this same effect. Moreover, the time-course effect of CD8α expression in response to mitogens (PHA, ConA, and poly I:C) was evaluated in MNCs. A significant increase in the transcriptional levels of CD8α was detected in new type gosling viral enteritis virus (NGVEV)-infected goose MNCs at 48 h postinfection (PI) and in goose parvovirus (GPV)-infected MNCs at 72 h PI. Also, the number of CD8α+ cells was significantly increased during viral infection from 72 h on. The seminal changes in mRNA profiles of antiviral cytokines (IFN-α, IFN-γ, and IL-18) were observed and were significantly increased during late phases of NGVEV and GPV infection. Accordingly, our data not only contribute to the understanding of the immune characteristics of goose CD8α, but they also provide new insight into the innate antiviral immunity of geese.

Complete genome sequence of goose parvovirus Y strain isolated from Muscovy ducks in China.

A goose parvovirus (GPV) Y strain was isolated from Muscovy ducks in Anhui Province of China. By polymerase chain reaction method, its complete genomic sequence was found to be 5,106 bp in length, consisting of 444-bp inverted terminal repeat, 1,844-bp non-structural protein and 2,199-bp capsid protein (VP) regions. Then its sequence was aligned with the sequences of GPV and Muscovy duck parvovirus published in the GenBank using the neighbor-joining method. The phylogenetic analyses based on the VP3 gene sequences revealed that the GPV Y strain along with those from Taiwan belonged to the subgroup IIb, while other GPV strains from Muscovy ducks belonged to the subgroup Ib and most of other GPV strains isolated in China mainland were clustered in the subgroup IIa. The absence of the deduced 703-705NRT glycosylation site in VP region may explain the host specificity of the GPV Y strain. The complete genomic sequence of the GPV Y strain from Muscovy ducks will help to understand the molecular and evolutionary characteristics of GPV.

Prevalence and effect of spawner-isolated mortality virus on the hatchery phases of Penaeus monodon and P. merguiensis in Australia.

Spawner-isolated mortality virus (SMV) has been associated with mortalities in broodstock of Penaeus monodon and with mid-crop mortality syndrome on grow-out farms. Epidemiological evidence suggested an association between the SMV status of broodstock and subsequent survival of their progeny, and this paper describes investigations into that association. The faeces of 909 broodstock in 9 different groups were tested by a polymerase chain reaction (PCR) for SMV and positive results were confirmed by an internal dot-blot. Seventy-seven spawners (8.5%) were positive for SMV with prevalence ranging from 0 to 24% among groups. The prevalence in spawners of P. monodon was higher (24%) than in P. merguiensis (4%). Three longitudinal studies were undertaken to compare the survival of progeny from broodstock that were positive to SMV with those that were not. Survival in hatchery tanks of progeny from SMV-positive spawners was lower than those from SMV-negative spawners with reductions of 23% (p = 0.01), 7.3% (p = 0.214) and 18.9% (p = 0.129) in the 3 studies. The conclusions were less consistent when examined during each of the later stages of growth in hatchery pools, nursery and grow-out ponds, with progeny from SMV-postive spawners sometimes having better survival rates. However, survival was better overall in progeny from SMV-negative spawners. Simple linear regression showed survival was negatively related to the proportion of postlarvae from SMV-positive spawners, with a decrease in survival of 5.6% for each 10% increase in the proportion of postlarvae coming from SMV-positive spawners (p = 0.006). Data from 38 ponds showed 6.71% of losses were due to SMV. If these losses were consistent across the entire industry, the annual loss due to SMV would have been approximately AUD 3 million in 1999/2000.