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1.
J Virol Methods ; 311: 114639, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36309206

RESUMO

The titer of neutralizing antibodies (NAbs) against viral hemorrhagic septicemia virus (VHSV) has been determined by conventional neutralization assay based on the observation of cytopathic effect (CPE) and plaque formation in cultured cells. However, this method requires several days for the determination and can be affected by operator bias. To develop a rapid and high-throughput neutralization assay against VHSV, we rescued a surrogate chimeric snakehead rhabdovirus, rSHRV-Gvhsv-eGFP, which has the enhanced green fluorescent protein (eGFP) gene between N and P genes and has VHSV G gene instead of SHRV G gene in the genome. The efficacy of rSHRV-Gvhsv-eGFP to determine serum neutralization activity was evaluated using various serum samples derived from New Zealand white rabbits and olive flounder (Paralichthys oliavaceus). Although neutralization titers analyzed using rSHRV-Gvhsv-eGFP were similar to the titers measured using rVHSV-A-eGFP, the time needed for the determination of neutralization titer was much shortened (24 h for rSHRV-Gvhsv-eGFP and 48 h for rVHSV-A-eGFP), proving the usefulness of rSHRV-Gvhsv-eGFP for the neutralization assay against VHSV. In addition, as the neutralization activities using rSHRV-Gvhsv-eGFP could be well-observed without adding fresh serum as a complement source, no preparation is required for the optimization of control fresh serum from naïve fish. The present results suggest that the rapid neutralization assay using rSHRV-Gvhsv-eGFP can be used to investigate neutralization activities against VHSV.


Assuntos
Doenças dos Peixes , Linguado , Septicemia Hemorrágica Viral , Novirhabdovirus , Rhabdoviridae , Animais , Coelhos , Septicemia Hemorrágica Viral/diagnóstico , Septicemia Hemorrágica Viral/prevenção & controle , Rhabdoviridae/genética , Novirhabdovirus/genética , Glicoproteínas , Doenças dos Peixes/diagnóstico , Doenças dos Peixes/prevenção & controle
2.
Ecotoxicol Environ Saf ; 248: 114331, 2022 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-36435002

RESUMO

Azoxystrobin (AZ) is one of the most widely used strobilurin fungicides in the world, and its residue has seriously endangered aquatic ecological security. Our previous data showed that AZ exposure may reduce the resistance of fish to rhabdovirus infection in aquatic environment. Here, we further reported a potential long-term adverse effect of AZ exposure on the antiviral and immunosuppressive recovery in fish, and observed that mitochondrial dynamic balance was disturbed by AZ in which excessive mitochondrial fission occurred in response to decreased ATP levels. When a recovery operation was performed in AZ-exposed cells and fish, infectivity of our model virus, spring viraemia of carp virus (SVCV), was significantly decreased in vitro (using the epithelioma papulosum cyprini [EPC] fish cell line) and in vivo (using zebrafish) in a time-dependent manner. Also, the expression of eight innate antiviral immune genes (IFNs, ISG15, MX1, RIG-I, IRF3, Nrf2 and HO-1) showed a similar change to SVCV replication between the longer exposure period and the expression recovery. Additionally, AZ facilitated horizontal transmission of SVCV in a static cohabitation challenge model, predicting the increase of the potential for the viral outbreak. Therefore, our data suggest that long-term effect of AZ on irreparable impairment in fish made AZ residue potentially greater for ecological risks.


Assuntos
Rhabdoviridae , Peixe-Zebra , Animais , Estrobilurinas , Antivirais/toxicidade
3.
Viruses ; 14(11)2022 Nov 17.
Artigo em Inglês | MEDLINE | ID: mdl-36423155

RESUMO

Aquatic animal viruses infect and transmit in aquatic environments, causing serious harm to the aquaculture industry and a variety of wild aquatic animals. How are they affected by environmental factors and do they represent potential threat to mammalian heath or not? Here, the effects of environmental factors (ultraviolet radiation (UV), temperature, pH, and drying) and their threshold on five epidemic aquatic animal viruses infecting amphibians and bony fish, including Rana grylio virus (RGV), Andrias davidianus ranavirus (ADRV), Grass carp reovirus (GCRV), Paralichthys olivaceus rhabdovirus (PORV), and Scophthalmus maximus rhabdovirus (SMRV), were measured and compared in a fish cell line. The examination of virus titers after different treatment in fish cells showed that the two iridoviruses, RGV and ADRV, had a higher tolerance to all of the environmental factors, such as they only had a decay rate of 22-36% when incubated at 37 °C for 7 days. However, the rhabdovirus SMRV was sensitive to all of the factors, with a decay rate of more than 80% in most of the treatments; even a complete inactivation (100%) can be observed after drying treatment. To address the potential threat to mammals, infectivity and limitation factors of the five viruses in Baby hamster kidney fibroblast cells (BHK-21) were tested, which showed that three of the five viruses can replicate at a low temperature, but a high temperature strongly inhibited their infection and none of them could replicate at 37 °C. This study clarified the sensitivity or tolerance of several different types of aquatic animal viruses to the main environmental factors in the aquatic environment and proved that the viruses cannot replicate in mammalian cells at normal physiological temperature.


Assuntos
Ranavirus , Reoviridae , Rhabdoviridae , Animais , Raios Ultravioleta , Ranavirus/fisiologia , Urodelos , Mamíferos
4.
Viruses ; 14(11)2022 Nov 06.
Artigo em Inglês | MEDLINE | ID: mdl-36366554

RESUMO

A virome screen was performed on a new breeding line, KB1, of blackcurrant. Rhabdovirus-like particles were observed by electron microscopy in ultrathin sections of flower stalks, and the complete genome sequence of a novel virus, provisionally named blackcurrant rhabdovirus 2 (BCRV2), was determined and verified using high-throughput sequencing. The genomic organization of BCRV2 was characteristic of cytorhabdoviruses (family Rhabdoviridae) and included seven genes: 3 ́- N-P´-P-P3-M-G-L -5 ́. BLASTP analysis revealed that the putative L protein had the highest amino acid sequence identity (75 %) with strawberry virus 2. BCRV2 was detected in Cryptomyzusgaleopsidis, but efficient transmission by this aphid was not confirmed. Of note, we observed coinfection of the KB1 line with blackcurrant-associated rhabdovirus (BCaRV) by RT-PCR. This is likely the first evidence of the presence of a cyto- and a nucleorhabdovirus in a single host.


Assuntos
Coinfecção , Rhabdoviridae , Ribes , Coinfecção/genética , Genoma Viral , Fases de Leitura Aberta , Doenças das Plantas , Filogenia , Melhoramento Vegetal , Rhabdoviridae/genética
5.
J Gen Virol ; 103(11)2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36399122

RESUMO

To counteract RNA interference-mediated antiviral defence, virus genomes evolved to express proteins that inhibit this plant defence mechanism. Using six independent biological approaches, we show that orchid fleck dichorhavirus citrus strain (OFV-citrus) movement protein (MP) may act as a viral suppressor of RNA silencing (VSR). By using the alfalfa mosaic virus (AMV) RNA 3 expression vector, it was observed that the MP triggered necrosis response in transgenic tobacco leaves and increased the viral RNA (vRNA) accumulation. The use of the potato virus X (PVX) expression system revealed that the cis expression of MP increased both the severity of the PVX infection and the accumulation of PVX RNAs, further supporting that MP could act as an RNA silencing suppressor (RSS). From the analysis of the RSS-defective turnip crinkle virus (TCV), we do not find local RSS activity for MP, suggesting a link between MP suppressor activity and the prevention of systemic silencing. In the analysis of local suppressive activity using the GFP-based agroinfiltration assay in Nicotiana benthamiana (16 c line), we do not identify local RSS activity for the five OFV RNA1-encoded proteins. However, when evaluating the small interfering RNA (siRNA) accumulation, we find that the expression of MP significantly reduces the accumulation of GFP-derived siRNA. Finally, we examine whether the MP can prevent systemic silencing in 16c plants. Our findings show that MP inhibits the long-distance spread of RNA silencing, but does not affect the short-distance spread. Together, our findings indicate that MP is part of OFV's counter-defence mechanism, acting mainly in the prevention of systemic long-distance silencing. This work presents the first report of a VSR for a member of the genus Dichorhavirus.


Assuntos
Doenças das Plantas , Rhabdoviridae , Interferência de RNA , RNA Interferente Pequeno , RNA de Cadeia Dupla
6.
Zool Res ; 43(6): 966-976, 2022 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-36257828

RESUMO

Spring viremia of carp virus (SVCV) is globally widespread and poses a serious threat to aquatic ecology and aquaculture due to its broad host range. To develop effective agents to control SVCV infection, we selected 16 naturally active small molecules to assess their anti-SVCV activity. Notably, dihydroartemisinin (DHA) (100 µmol/L) and (S, S)-(+)-tetrandrine (TET) (16 µmol/L) exhibited high antiviral effects in epithelioma papulosum cyprinid (EPC) cells, with inhibitory rates of 70.11% and 73.54%, respectively. The possible antiviral mechanisms were determined as follows: 1. Pre-incubation with DHA and TET decreased viral particle infectivity in fish cells, suggesting that horizontal transmission of SVCV in the aquatic environment was disrupted; 2. Although neither had an effect on viral adhesion, TET (but not DHA) interfered with SVCV entry into host cells (>80%), suggesting that TET may have an antiviral function in early viral replication. For in vivo study, both agents enhanced the survival rate of SVCV-infected zebrafish by 53.3%, significantly decreased viral load, and modulated the expression of antiviral-related genes, indicating that DHA and TET may stimulate the host innate immune response to prevent viral infection. Overall, our findings indicated that DHA and TET had positive effects on suppressing SVCV infection by affecting early-stage viral replication, thus holding great potential as immunostimulants to reduce the risk of aquatic rhabdovirus disease outbreaks.


Assuntos
Carpas , Doenças dos Peixes , Infecções por Rhabdoviridae , Rhabdoviridae , Animais , Infecções por Rhabdoviridae/veterinária , Infecções por Rhabdoviridae/tratamento farmacológico , Antivirais/farmacologia , Peixe-Zebra , Replicação Viral , Viremia/veterinária , Adjuvantes Imunológicos/farmacologia , Adjuvantes Imunológicos/uso terapêutico
7.
J Virol ; 96(22): e0131422, 2022 Nov 23.
Artigo em Inglês | MEDLINE | ID: mdl-36314827

RESUMO

IFN regulatory factor (IRF) 2 belongs to the IRF1 subfamily, and its functions are not yet fully understood. In this study, we showed that IRF2a was a negative regulator of the interferon (IFN) response induced by spring viremia of carp virus (SVCV). Irf2a-/- knockout zebrafish were less susceptible to SVCV than wild-type fish. Transcriptomic analysis reveals that differentially expressed genes (DEGs) in the irf2a-/- and irf2a+/+ cells derived caudal fins were mainly involved in cytokine-cytokine receptor interaction, mitogen-activated protein kinase (MAPK) signaling pathway, and transforming growth factor-beta (TGF-beta) signaling pathway. Interestingly, the basal expression levels of interferon stimulating genes (ISGs), including pkz, mx, apol, and stat1 were higher in the irf2a-/- cells than irf2a+/+ cells, suggesting that they may contribute to the increased viral resistance of the irf2a-/- cells. Overexpression of IRF2a inhibited the activation of ifnφ1 and ifnφ3 induced by SVCV and poly(I:C) in the epithelioma papulosum cyprini (EPC) cells. Further, it was found that SVCV phosphoprotein (SVCV-P) could interact with IRF2a to promote IRF2a nuclear translocation and protein stability via suppressing K48-linked ubiquitination of IRF2a. Both IRF2a and SVCV-P not only destabilized STAT1a but reduced its translocation into the nucleus. Our work demonstrates that IRF2a cooperates with SVCV-P to suppress host antiviral response against viral infection in zebrafish. IMPORTANCE Interferon regulatory factors (IRFs) are central in the regulation of interferon-mediated antiviral immunity. Here, we reported that IRF2a suppressed interferon response and promoted virus replication in zebrafish. The suppressive effects were enhanced by the phosphoprotein of the spring viremia of carp virus (SVCV) via inhibition of K48-linked ubiquitination of IRF2a. IRF2a and SVCV phosphoprotein cooperated to degrade STAT1 and block its nuclear translocation. Our work demonstrated that IRFs and STATs were targeted by the virus through posttranslational modifications to repress interferon-mediated antiviral response in lower vertebrates.


Assuntos
Doenças dos Peixes , Fator Regulador 2 de Interferon , Fosfoproteínas , Infecções por Rhabdoviridae , Rhabdoviridae , Animais , Doenças dos Peixes/virologia , Interferons/imunologia , Fosfoproteínas/metabolismo , Rhabdoviridae/fisiologia , Infecções por Rhabdoviridae/imunologia , Infecções por Rhabdoviridae/veterinária , Viremia , Peixe-Zebra/virologia , Fator Regulador 2 de Interferon/metabolismo , Técnicas de Inativação de Genes , Processamento de Proteína Pós-Traducional , Fator de Transcrição STAT1 , Replicação Viral
8.
Front Immunol ; 13: 973422, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36275642

RESUMO

To better understand the response of largemouth bass (Micropterus salmoides) to Micropterus salmoides rhabdovirus (MSRV) infection, we investigated the intestinal bacterial flora and transcriptome profile of fish at 72 hours post-infection (hpi). Total of 1574 differentially expressed genes (DEGs) were identified in largemouth bass spleen following MSRV infection, including 573 upregulated and 1001 downregulated genes. KEGG and GO enrichment analysis revealed that upregulated genes were enriched in certain antiviral related signaling pathway, including NOD-like receptor (NLR), RIG-I like receptors (RLR) and regulation of the interferon (IFN)-γ-mediated signaling pathway, whereas some immune-related DEGs enriched in focal adhesion (FA) and ECM-receptor interaction(ECM-RI) were downregulated, as well as genes associated with metabolic processes, such as peroxisome proliferator-activated receptors (PPAR), adipocytokine signaling pathway, Glycerolipid and Retinol metabolism. Furthermore, the principal component analysis (PCA) and phylogenetic analysis revealed that MSRV infection significantly affected the microbiota of largemouth bass intestine; the LEfSe analysis showed that relative abundances of Streptococcus were significantly increased, while the content of Akkermansia, Enterococcus and Lactobacillus were remarkably decreased in the fish intestine following MSRV infection. Additionally, a high correlation was determined between the expressions of interferon-related upregulated genes and the relative abundance of Streptococcus by redundancy analysis (RDA). These results collectively illustrated that intestinal microbiota composition might be associated with the immune-related gene expression in largemouth bass in response to MSRV infection.


Assuntos
Bass , Infecções por Rhabdoviridae , Rhabdoviridae , Animais , Bass/genética , Transcriptoma , RNA Ribossômico 16S , Receptores Ativados por Proliferador de Peroxissomo , Filogenia , Vitamina A , Interferons/genética , Proteínas NLR/genética , Antivirais , Adipocinas/genética
9.
Vector Borne Zoonotic Dis ; 22(11): 545-552, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36315188

RESUMO

Background: Bat flies (Diptera: Hippoboscoidea: Nycteribiidae and Streblidae) are increasingly appreciated as hosts of "bat-associated" viruses. We studied straw-colored fruit bats (Eidolon helvum) and their nycteribiid bat flies (Cyclopodia greefi) in Nigeria to investigate the role of bat flies in vectoring or maintaining viruses. Methods: We captured bats and bat flies across northern Nigeria. We used metagenomics to identify viruses in 40 paired samples (20 flies from 20 bats). We characterized viruses using genomic and phylogenetic methods, and we compared infection frequencies in bats and their bat flies. Results: In 20 bats, we detected two individuals (10%) infected with eidolon helvum parvovirus 1 (BtPAR4) (Parvoviridae; Tetraparvovirus), previously described in Ghana, and 10 bats (50%) with a novel parvovirus in the genus Amdoparvovirus (Parvoviridae). The amdoparvoviruses include Aleutian disease virus of mink and viruses of other carnivores but have not previously been identified in bats or in Africa. In 20 paired bat flies (each fly from 1 bat) all (100%) were infected with a novel virus in the genus Sigmavirus (Rhabdoviridae). The sigmaviruses include vertically transmitted viruses of dipterans. We did not detect BtPAR4 in any bat flies, and we did not detect the novel sigmavirus in any bats. However, we did detect the novel amdoparvovirus in 3 out of 20 bat flies sampled (15%), including in 2 bat flies from bats in which we did not detect this virus. Discussion: Our results show that bats and their bat flies harbor some viruses that are specific to mammals and insects, respectively, and other viruses that may transmit between bats and arthropods. Our results also greatly expand the geographic and host range of the amdoparvoviruses and suggest that some could be transmitted by arthropods. Bat flies may serve as biological vectors, mechanical vectors, or maintenance hosts for "bat-associated" viruses.


Assuntos
Quirópteros , Dípteros , Rhabdoviridae , Animais , Quirópteros/virologia , Dípteros/virologia , Nigéria/epidemiologia , Filogenia , Rhabdoviridae/genética , Infecções por Rhabdoviridae/transmissão , Infecções por Rhabdoviridae/virologia
10.
Vopr Virusol ; 67(4): 331-340, 2022 09 12.
Artigo em Russo | MEDLINE | ID: mdl-36097714

RESUMO

INTRODUCTION: The main approach to the rabies prevention is the vaccination of domestic and wild carnivores. For the routine evaluation the anti-rabies vaccination effectiveness, World Organization for Animal Health (OIE) recommends various enzyme-linked immunosorbent assays (ELISA).The aim of the study was to design and validate a competitive ELISA (cELISA) test system for the detection of antibodies to the rabies virus (RABV). MATERIALS AND METHODS: The development of the cELISA was carried out following the OIE recommendations. RESULTS: The repeatability of the cELISA results within one laboratory was satisfactory (coefficient of variation 7.95-13.61%). The coefficient of determination (CD) between the results of the virus neutralization reaction (FAVN) and cELISA was 0.988, p < 0.001. The lower threshold for antibody detection was less than 0.02 IU/ml. The cELISA did not demonstrate cross-reactivity against antibodies to canine distemper virus, parainfluenza virus, parvovirus, coronavirus, and canine adenovirus (types I and II). During the study of 137 dog blood sera, diagnostic specificity (DSp) and diagnostic sensitivity (DSe) for the cELISA were 83.1% and 94.9%, respectively, and CD between the cELISA and FAVN results was 0.968, p < 0.001. DISCUSSION: Indirect ELISA test systems for determining the level of antibodies to RABV are not sensitive enough compared to reference tests, unlike cELISA. The developed test system is not inferior for its DSp and DSe to the commercial cELISA BioPro ELISA Rabies Ab (DSp 66.7%, DSe 94.4%). CONCLUSION: The developed cELISA test system can be used to detect antibodies to RABV in the blood serum of dogs for evaluating the effectiveness of mass vaccination programs.


Assuntos
Lyssavirus , Vírus da Raiva , Rhabdoviridae , Animais , Anticorpos Antivirais , Cães , Ensaio de Imunoadsorção Enzimática/métodos , Ensaio de Imunoadsorção Enzimática/veterinária
11.
Plant Dis ; 106(11): 2784-2787, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36176214

RESUMO

In 2020, a novel agent was discovered in strawberry, a rhabdovirus closely related to lettuce necrotic yellows virus. The new virus, named strawberry virus 2 (StrV-2), was discovered in an accession of the Fragaria virus collection of the National Clonal Germplasm Repository (NCGR), and for this reason, it was studied in-depth. The complete StrV-2 genome was obtained and investigated in silico. Transmission was assessed using two aphid species whereas a multiplex RT-PCR test targeting plant and virus genes was developed and used to screen the NCGR Fragaria virus collection.


Assuntos
Afídeos , Fragaria , Rhabdoviridae , Animais , Fragaria/genética , Doenças das Plantas , Rhabdoviridae/genética , Genoma Viral/genética
12.
Viruses ; 14(8)2022 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-36016264

RESUMO

Largemouth bass is an important commercially farmed fish in China, but the rapid expansion of its breeding has resulted in increased incidence of diseases caused by bacteria, viruses and parasites. In this study, moribund largemouth bass containing ulcer foci on body surfaces indicated the most likely pathogens were iridovirus and rhabdovirus members and this was confirmed using a combination of immunohistochemistry, cell culture, electron microscopy and conserved gene sequence analysis. We identified that these fish had been co-infected with these viruses. We observed bullet-shaped virions (100-140 nm long and 50-100 nm in diameter) along with hexagonal virions with 140 nm diameters in cell culture inoculated with tissue homogenates. The viruses were plaque purified and a comparison of the highly conserved regions of the genome of these viruses indicated that they are most similar to largemouth bass virus (LMBV) and hybrid snakehead rhabdovirus (HSHRV), respectively. Regression infection experiments indicated fish mortalities for LMBV-FS2021 and HSHRV-MS2021 were 86.7 and 11.1%, respectively. While co-infection resulted in 93.3% mortality that was significantly (p < 0.05) higher than the single infections even though the viral loads differed by >100-fold. Overall, we simultaneously isolated and identified LMBV and a HSHRV-like virus from diseased largemouth bass, and our results can provide novel ideas for the prevention and treatment of combined virus infection especially in largemouth bass.


Assuntos
Bass , Doenças dos Peixes , Iridovirus , Rhabdoviridae , Animais , Novirhabdovirus , Rhabdoviridae/genética
13.
Front Immunol ; 13: 968348, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35990638

RESUMO

Spring viremia of carp virus (SVCV) can cause high mortality of fish. The aim of this study was to investigate the effects of Lactobacillus rhamnosus GCC-3 exopolysaccharides (GCC-3 EPS) on zebrafish (Danio rerio) infected with SVCV and elucidate the underlying mechanisms. Zebrafish were fed with a control diet or diet supplemented with 0.5% and 1% of GCC-3 EPS for 2 weeks. The results showed that supplementation of GCC-3 EPS significantly improved the survival rate of zebrafish compared with the control group. In addition, dietary 0.5% and 1% GCC-3 EPS significantly up-regulated the expression of genes related to type I interferon (IFN) antiviral immunity. Consistent with in vivo results, GCC-3 EPS significantly inhibited SVCV replication in zebrafish embryonic fibroblast (ZF4) cells while significantly increased the expression of type I IFN signaling pathway related genes. Furthermore, knocking down TANK-binding kinase 1 significantly blocked the antiviral effect of GCC-3 EPS. Dietary GCC-3 EPS improved gut microbiota, and the culture supernatant of GCC-3 EPS-associated microbiota significantly inhibited SVCV replication in ZF4 cells compared with the control-microbiota counterpart. In conclusion, our results indicate that dietary GCC-3 EPS can improve the resistance of zebrafish against SVCV infection, and the mechanism may involve enhanced type I interferon signaling.


Assuntos
Carpas , Doenças dos Peixes , Interferon Tipo I , Lactobacillus rhamnosus , Infecções por Rhabdoviridae , Animais , Antivirais/uso terapêutico , Suplementos Nutricionais , Interferon Tipo I/uso terapêutico , Rhabdoviridae , Infecções por Rhabdoviridae/veterinária , Viremia , Peixe-Zebra
14.
J Gen Virol ; 103(8)2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-36018884

RESUMO

The Sf9 cell line, originally isolated from the ovarian tissue of Spodoptera frugiperda larvae, is widely used in academia and industry for the baculovirus-mediated production of recombinant proteins and virus-like particles. RNA interference (RNAi) is a conserved antiviral pathway present in eukaryotic organisms and is the primary antiviral defence mechanism in insects. Recent evidence has implicated RNAi as an antiviral response to baculovirus infection in Sf9 cells. To test this hypothesis, CRISPR/Cas9 technology was used to disable the RNAi pathway in Sf9 cells by knocking out Dicer-2, the protein responsible for cleaving viral double-stranded RNA precursors into short interfering RNAs. Infection of Dicer-2 knockout Sf9 cells with either the wild-type baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV), recombinant AcMNPV (rAcMNPV) expressing ß-galactosidase (ß-gal), or rAcMNPV expressing a wasp venom protein (Vn50) at a multiplicity of infection (m.o.i.) of 1 resulted in a modest increase in virus replication compared to control Sf9 cells under adherent culture conditions. In contrast, Dicer-2 knockout Sf9 monolayer or suspension cultures infected by the rAcMNPV expressing ß-gal at higher m.o.i.s (3.5 and 20) did not exhibit increases in either viral DNA replication or ß-gal production. Intriguingly, during long-term passaging in suspension, Dicer-2 knockout Sf9 cultures underwent transient crashes in cell proliferation and viability. It was discovered that these periods of low growth and viability coincided with a dramatic increase in the RNA levels of S. frugiperda rhabdovirus, a recently identified adventitious virus that persistently infects the Sf9 cell line, suggesting a role for Dicer-2 in managing chronic viral infections in this industrially relevant insect cell line.


Assuntos
Baculoviridae , Rhabdoviridae , Animais , Antivirais , Linhagem Celular , Replicação do DNA , DNA Viral , Nucleopoliedrovírus , Células Sf9 , Spodoptera , Replicação Viral
15.
J Virol ; 96(16): e0079122, 2022 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-35913215

RESUMO

Spring viremia of carp virus (SVCV) is a severe infectious pathogen that causes high rates of mortality in cyprinids and other fish species. Despite numerous investigations of SVCV infection, the underlying molecular mechanisms remain poorly understood. In this study, we found that the SVCV matrix protein (SVCV-M) played an inhibitory role in the host interferon (IFN) response by targeting the MAVS/TRAF3 signaling axis, thereby uncovering a previously unrecognized mechanism of SVCV escape from host innate antiviral immunity. Mechanistically, SVCV-M was located at the mitochondria independent of MAVS, which allowed SVCV-M to build an arena for competition with the MAVS platform. A microscale thermophoresis assay showed that SVCV-M had a high affinity for TRAF3, as indicated by a lower equilibrium dissociation constant (KD) value than that of MAVS with TRAF3. Therefore, the association of MAVS with TRAF3 was competitively impaired by SVCV-M in a dose-dependent manner. Accordingly, SVCV-M showed a potent ability to inhibit the K63-linked polyubiquitination of TRAF3. This inhibition was accompanied by the impairment of the IFN response, as shown by the marked decline in IFN-φ1-promoter (pro) luciferase reporter activity. By constructing truncated TRAF3 and SVCV-M proteins, the RING finger, zinc finger, and coiled-coil domains of TRAF3 and the hydrophobic-pocket-like structure formed by the α2-, α3-, and α4-helices of SVCV-M may be the major target and antagonistic modules responsible for the protein-protein interaction between the TRAF3 and SVCV-M proteins. These findings highlighted the intervention of SVCV-M in host innate immunity, thereby providing new insights into the extensive participation of viral matrix proteins in multiple biological activities. IMPORTANCE The matrix protein of SVCV (SVCV-M) is an indispensable structural element for nucleocapsid condensation and virion formation during viral morphogenesis, and it connects the core nucleocapsid particle to the outer membrane within the mature virus. Previous studies have emphasized the architectural role of SVCV-M in viral construction; however, the potential nonstructural functions of SVCV-M in viral replication and virus-host interactions remain poorly understood. In this study, we identified the inhibitory role of the SVCV-M protein in host IFN production by competitively recruiting TRAF3 from the MAVS signaling complex and impairing TRAF3 activation via inhibition of K63-linked polyubiquitination. This finding provided new insights into the regulatory role of SVCV-M in host innate immunity, which highlighted the broader functionality of rhabdovirus matrix protein apart from being a structural protein. This study also revealed a previously unrecognized mechanism underlying SVCV immune evasion by inhibiting the IFN response by targeting the MAVS/TRAF3 signaling axis.


Assuntos
Carpas , Infecções por Rhabdoviridae/veterinária , Rhabdoviridae/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Imunidade Inata , Interferons/metabolismo , Infecções por Rhabdoviridae/imunologia , Fator 3 Associado a Receptor de TNF/genética , Fator 3 Associado a Receptor de TNF/metabolismo , Proteínas da Matriz Viral/metabolismo , Viremia/veterinária
16.
J Immunol ; 209(6): 1165-1172, 2022 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-36002231

RESUMO

The signaling adaptor MAVS is a critical determinant in retinoic acid-inducible gene 1-like receptor signaling, and its activation is tightly controlled by multiple mechanisms in response to viral infection, including phosphorylation and ubiquitination. In this article, we demonstrate that zebrafish sirt5, one of the sirtuin family proteins, negatively regulates mavs-mediated antiviral innate immunity. Sirt5 is induced by spring viremia of carp virus (SVCV) infection and binds to mavs, resulting in attenuating phosphorylation and ubiquitination of mavs. Disruption of sirt5 in zebrafish promotes survival ratio after challenge with SVCV. Consistently, the antiviral responsive genes are enhanced, and the replication of SVCV is diminished in sirt5-dificient zebrafish. Therefore, we reveal a function of zebrafish sirt5 in the negative regulation of antiviral innate immunity by targeting mavs.


Assuntos
Sirtuínas , Peixe-Zebra , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Antivirais , Imunidade Inata , Fosforilação , Rhabdoviridae , Sirtuínas/metabolismo , Tretinoína/metabolismo , Ubiquitinação , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
17.
Arch Virol ; 167(11): 2381-2385, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-35920980

RESUMO

Through high-throughput RNA sequencing, we discovered a putative new cytorhabdovirus in the seeds of Rudbeckia sp., which we have tentatively named "rudbeckia virus 1" (RudV1). Its complete 12,502-nt genomic sequence contains five open reading frames (ORFs): ORF1 (putative nucleocapsid protein, N), ORF2 (putative phosphoprotein, P), ORF3 (putative cell-to-cell movement protein, P3), ORF4 (putative matrix protein, M), and ORF5 (putative RNA-dependent RNA polymerase, L). BLASTp searches showed that ORF1, ORF3, ORF4, and ORF5 of RudV1 are most closely related to the corresponding proteins of Tagetes erecta virus 1 (a putative member of the genus Cytorhabdovirus) with 33.87% (88% query coverage), 55.98% (89% query coverage), 35.33% (94% query coverage), and 57.75% (98% query coverage) sequence identity at the amino acid level, respectively. Phylogenetic analysis and pairwise comparisons indicated that RudV1 is a novel member of the genus Cytorhabdovirus within the family Rhabdoviridae.


Assuntos
Rhabdoviridae , Rudbeckia , Aminoácidos/genética , Genoma Viral , Sequenciamento de Nucleotídeos em Larga Escala , Proteínas do Nucleocapsídeo/genética , Fases de Leitura Aberta , Fosfoproteínas/genética , Filogenia , RNA Viral/genética , RNA Polimerase Dependente de RNA , Rudbeckia/genética
18.
J Fish Dis ; 45(11): 1757-1765, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-35944110

RESUMO

The disease caused by Micropterus salmoides rhabdovirus (MSRV) has brought substantial economic losses to the largemouth bass aquaculture industry in China. Vaccination was considered as a potential way to prevent and control this disease. As a kind of sustained and controlled release system, alginate and chitosan microspheres (SA-CS) are widely used in the development of oral vaccination for fish. Here, we prepared a king of alginate-chitosan composite microsphere to encapsulate the second segment of MSRV glycoprotein (G2 protein) and then evaluated the immune effect of the microsphere vaccine on largemouth bass. Largemouth bass were vaccinated via intragastric immunization by different treatments (PBS, SA-CS, G2 and SA-CS-G2). The results showed that a stronger immune response including serum antibody levels, immune-related physiological indexes (acid phosphatase, alkaline phosphatase, superoxide dismutase and total antioxidant capacity) and the expression of immune-related gene (IgM、IL-8、IL-1ß、CD4、TGF-ß、TNF-α) can be induced obviously with SA-CS-G2 groups compared with G2 groups when fish were vaccinated. Furthermore, fish were injected with a lethal dose of MSRV after immunization for 28 days, and the highest relative percentage survival (54.8%) was observed in SA-CS-G2 group (40 µg per fish), which is significantly higher than that of G2 group (25.8%). This study showed that alginate-chitosan microspheres as the vaccine carrier can effectively improve the immune effect of oral vaccination and induce better immune protection effect against MSRV infection.


Assuntos
Bass , Quitosana , Doenças dos Peixes , Rhabdoviridae , Fosfatase Ácida , Alginatos , Fosfatase Alcalina , Animais , Antioxidantes , Preparações de Ação Retardada , Imunoglobulina M , Interleucina-8 , Microesferas , Superóxido Dismutase , Fator de Crescimento Transformador beta , Fator de Necrose Tumoral alfa , Vacinas de Subunidades , Vacinas Sintéticas
19.
J Fish Dis ; 45(10): 1429-1437, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-35930453

RESUMO

Micropterus salmoides rhabdovirus (MSRV) is one of the common pathogens in the largemouth bass industry, which can cause lethal diseases in juvenile fish and enormous economic losses. To establish effective means to prevent MSRV infection, the pcDNA3.1-G plasmid containing the MSRV glycoprotein gene was successfully constructed and intramuscularly injected into the largemouth bass to evaluate the immune responses and protective effects in our study. As the results showed, the serum antibody levels of the fish vaccinated with different doses of pcDNA3.1-G were significantly higher compared with the control groups (PBS and pcDNA3.1). Meanwhile, the immune parameters (acid phosphatase and alkaline phosphatase) were also significantly up-regulated. Several immune-related genes (IgM, IL-8, IL-12p40 and CD40) were expressed in the pcDNA3.1-G groups at higher levels than in the control groups, which indicated that strong immune responses were induced. Besides, the survival percentages of fish in the control groups (PBS and pcDNA3.1) and pcDNA3.1-G groups (2.5, 5, 10 and 20 µg/fish) at 14 days after challenge experiment with MSRV were 0%, 0%, 6.1%, 15.2%, 29.0% and 48.5% respectively. This study indicated that pcDNA3.1-G was a prospective DNA vaccine candidate against MSRV-induced mortality.


Assuntos
Bass , Doenças dos Peixes , Rhabdoviridae , Vacinas de DNA , Animais , Estudos Prospectivos , Rhabdoviridae/genética
20.
J Fish Dis ; 45(12): 1831-1837, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-35962585

RESUMO

Variants of perch rhabdovirus (PRV) circulate across European percid farms via the fish trade. To trace their circulation, they are usually isolated by cell culture and subsequently identified genetically by sequencing partial or complete genes. Here, a newly developed nested PCR-based method was used to amplify and sequence the complete N and P genes directly from clinical samples obtained during an outbreak on a farm as well as from four batches of fish sampled from two other farms in another country. In an attempt to trace the origin of the five detected viruses, their N and P sequences were concatenated and compared with related viruses. One virus found in pike-perch was highly related to a virus isolated in 2016 in Belgium. Two other viruses detected on a single farm were distinct from one another, with one being almost identical to another virus isolated in 2016 in Belgium and the other being more closely related to a subgroup with different origins, France and Belgium. Two other viruses found in perch from a third farm were identical and were more related to a subgroup of viruses isolated in France. Identifying variants by a direct PCR approach will help to prevent further dissemination in farms.


Assuntos
Doenças dos Peixes , Percas , Infecções por Rhabdoviridae , Rhabdoviridae , Animais , Infecções por Rhabdoviridae/epidemiologia , Infecções por Rhabdoviridae/veterinária , Doenças dos Peixes/epidemiologia , Filogenia , Rhabdoviridae/genética
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