Antiviral effect of miR-206 in rainbow trout (Oncorhynchus mykiss) against infectious hematopoietic necrosis virus infection.

Infectious hematopoietic necrosis (IHN), caused by IHN virus, is a highly contagious and lethal disease that seriously hampers the development of rainbow trout (Oncorhynchus mykiss) aquaculture. However, the immune response mechanism of rainbow trout underlying IHNV infection remains largely unknown. MicroRNAs act as post-transcriptional regulators of gene expression and perform a crucial role in fish immune response. Herein, the regulatory mechanism and function of miR-206 in rainbow trout resistance to IHNV were investigated by overexpression and silencing. The expression analysis showed that miR-206 and its potential target receptor-interacting serine/threonine-protein kinase 2 (RIP2) exhibited significant time-dependent changes in headkidney, spleen and rainbow trout primary liver cells infected with IHNV and their expression displayed a negative correlation. In vitro, the interaction between miR-206 and RIP2 was verified by luciferase reporter assay, and miR-206 silencing in rainbow trout primary liver cells markedly increased RIP2 and interferon (IFN) expression but significantly decreased IHNV copies, and opposite results were obtained after miR-206 overexpression or RIP2 knockdown. In vivo, overexpressed miR-206 with agomiR resulted in a decrease in the expression of RIP2 and IFN in liver, headkidney and spleen. This study revealed the key role of miR-206 in anti-IHNV, which provided potential for anti-viral drug screening in rainbow trout.

Tissue factor pathway inhibitors disrupt structures of rhabdovirus/ranairidovirus and inhibit viral infection in Chinese perch, Siniperca chuatsi.

Viral diseases have caused great economic losses to the aquaculture industry. However, there are currently no specific drugs to treat these diseases. Herein, we utilized Siniperca chuatsi as an experimental model, and successfully extracted two tissue factor pathway inhibitors (TFPIs) that were highly distributed in different tissues. We then designed four novel peptides based on the TFPIs, named TS20, TS25, TS16, and TS30. Among them, TS25 and TS30 showed good biosafety and high antiviral activity. Further studies showed that TS25 and TS30 exerted their antiviral functions by preventing viruses from invading Chinese perch brain (CPB) cells and disrupting Siniperca chuatsi rhabdovirus (SCRV)/Siniperca chuatsi ranairidovirus (SCRIV) viral structures. Additionally, compared with the control group, TS25 and TS30 could significantly reduce the mortality of Siniperca chuatsi, the relative protection rates of TS25 against SCRV and SCRIV were 71.25 % and 53.85 % respectively, and the relative protection rate of TS30 against SCRIV was 69.23 %, indicating that they also had significant antiviral activity in vivo. This study provided an approach for designing peptides with biosafety and antiviral activity based on host proteins, which had potential applications in the prevention and treatment of viral diseases.

USP14 negatively regulates IFN signaling by dampening K63-linked ubiquitination of TBK1 in black carp.

USP14 regulates the immune related pathways by deubiquitinating the signaling molecules in mammals. In teleost, USP14 is also reported to inhibit the antiviral immune response through TBK1, but its regulatory mechanism remains obscure. To elucidate the role of USP14 in the RLR/IFN antiviral pathway in teleost, the homolog USP14 (bcUSP14) of black carp (Mylopharyngodon piceus) has been cloned and characterize in this paper. bcUSP14 contains 490 amino acids (aa), and the sequence is well conserved among in vertebrates. Over-expression of bcUSP14 in EPC cells attenuated SVCV-induced transcription activity of IFN promoters and enhanced SVCV replication. Knockdown of bcUSP14 in MPK cells led to the increased transcription of IFNs and decreased SVCV replication, suggesting the improved antiviral activity of the host cells. The interaction between bcUSP14 and bcTBK1 was identified by both co-immunoprecipitation and immunofluorescent staining. Co-expressed bcUSP14 obviously inhibited bcTBK1-induced IFN production and antiviral activity in EPC cells. K63-linked polyubiquitination of bcTBK1 was dampened by co-expressed bcUSP14, and bcTBK1-mediated phosphorylation and nuclear translocation of IRF3 were also inhibited by this deubiquitinase. Thus, all the data demonstrated that USP14 interacts with and inhibits TBK1 through deubiquitinating TBK1 in black carp.

Autophagy mediated degradation of MITA/TBK1/IRF3 by a hnRNP family member attenuates interferon production in fish.

HnRNP A/B belongs to the heterogeneous nuclear ribonucleoprotein (hnRNP) family and plays an important role in regulating viral protein translation and genome replication. Here, we found that overexpression of hnRNP A/B promoted spring viremia of carp virus (SVCV) and cyprinid herpesvirus 3 (CyHV3) replication. Further, hnRNP A/B was shown to act as a negative regulator of type I interferon (IFN) response. Mechanistically, hnRNP A/B interacted with MITA, TBK1 and IRF3 to initiate their degradation. In addition, hnRNP A/B bound to the kinase domain of TBK1, the C terminal domain of MITA and IAD domain of IRF3, and the RRM1 domain of hnRNP A/B bound to TBK1, RRM2 domain bound to IRF3 and MITA. Our study provides novel insights into the functions of hnRNP A/B in regulating host antiviral response.

Innate immune response of snakehead fish to Indian strain of snakehead rhabdovirus (SHRV-In) infection and the infectivity potential of the virus to other freshwater fishes.

A new virus known as snakehead rhabdovirus (SHRV-In) was discovered in South India in striped snakehead (Channa striata) that had hemorrhagic patches and cutaneous ulcerations. The virus is the most potentially harmful pathogen of snakehead because it could cause 100% mortality within 5 days. The goal of the current investigation was to evaluate the infectivity of rhabdovirus in freshwater fishes and to analyze the immune response in snakehead fish after challenge with SHRV-In. The infectivity study of SHRV-In against three freshwater fish such as tilapia, grass carp and loach showed that the virus could not induce mortality in any of them. Snakehead fish challenged with SHRV-In showed significant (p < 0.05) changes in haematological parameters such as red blood cell (RBC), haemoglobin (HGB), haematocrit (HCT), mean corpuscular haemoglobin concentration (MCHC), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), white blood cell (WBC), total platelet (PLT) counts, mean platelet volume (MPV) and immunological markers such as respiratory burst, superoxide dismutase, catalase activity and myeloperoxidase activity at 6, 12, 24 and 48 hpi. Real time PCR was executed to examine the expression profile of innate immune genes such as IRF-7, IL-8 and IL-12 in Snakehead fish at 6, 12, 24 and 48 h post SHRV-In infection. Immune gene expression of IRF-7, IL-8 and IL-12 were up-regulated in the spleen when compared to kidney at 6 and 12 hpi. However, the expression level of all the genes was down-regulated at 24 and 48 hpi. The down regulation of innate immune genes after 24 hpi in these tissues may be the result of increased multiplication of SHRV-In by interfering with the immune signaling pathway.

High-Throughput Sequencing Reveals Three Rhabdoviruses Persisting in the IRE/CTVM19 Cell Line.

Cell cultures derived from ticks have become a commonly used tool for the isolation and study of tick-borne pathogens and tick biology. The IRE/CTVM19 cell line, originating from embryos of Ixodes ricinus, is one such line. Previously, reovirus-like particles, as well as sequences with similarity to rhabdoviruses and iflaviruses, were detected in the IRE/CTVM19 cell line, suggesting the presence of multiple persisting viruses. Subsequently, the full genome of an IRE/CTVM19-associated rhabdovirus was recovered from a cell culture during the isolation of the Alongshan virus. In the current work, we used high-throughput sequencing to describe a virome of the IRE/CTVM19 cell line. In addition to the previously detected IRE/CTVM19-associated rhabdovirus, two rhabdoviruses were detected: Chimay rhabdovirus and Norway mononegavirus 1. In the follow-up experiments, we were able to detect both positive and negative RNA strands of the IRE/CTVM19-associated rhabdovirus and Norway mononegavirus 1 in the IRE/CTVM19 cells, suggesting their active replication in the cell line. Passaging attempts in cell lines of mammalian origin failed for all three discovered rhabdoviruses.

Heterologous Exchanges of Glycoprotein and Non-Virion Protein in Novirhabdoviruses: Assessment of Virulence in Yellow Perch (Perca flavescens) and Rainbow Trout (Oncorhynchus mykiss).

Infectious hematopoietic necrosis virus (IHNV) and viral hemorrhagic septicemia virus (VHSV) are rhabdoviruses in two different species belonging to the Novirhabdovirus genus. IHNV has a narrow host range restricted to trout and salmon species, and viruses in the M genogroup of IHNV have high virulence in rainbow trout (Oncorhynchus mykiss). In contrast, the VHSV genotype IVb that invaded the Great Lakes in the United States has a broad host range, with high virulence in yellow perch (Perca flavescens), but not in rainbow trout. By using reverse-genetic systems of IHNV-M and VHSV-IVb strains, we generated six IHNV:VHSV chimeric viruses in which the glycoprotein (G), non-virion-protein (NV), or both G and NV genes of IHNV-M were replaced with the analogous genes from VHSV-IVb, and vice versa. These chimeric viruses were used to challenge groups of rainbow trout and yellow perch. The parental recombinants rIHNV-M and rVHSV-IVb were highly virulent in rainbow trout and yellow perch, respectively. Parental rIHNV-M was avirulent in yellow perch, and chimeric rIHNV carrying G, NV, or G and NV genes from VHSV-IVb remained low in virulence in yellow perch. Similarly, the parental rVHSV-IVb exhibited low virulence in rainbow trout, and chimeric rVHSV with substituted G, NV, or G and NV genes from IHNV-M remained avirulent in rainbow trout. Thus, the G and NV genes of either virus were not sufficient to confer high host-specific virulence when exchanged into a heterologous species genome. Some exchanges of G and/or NV genes caused a loss of host-specific virulence, providing insights into possible roles in viral virulence or fitness, and interactions between viral proteins.

STAT2 negatively regulates RIG-I in the antiviral innate immunity of black carp.

The signal transducer and activator of transcription 2 (STAT2), a downstream factor of type I interferons (IFNs), is a key component of the cellular antiviral immunity response. However, the role of STAT2 in the upstream of IFN signaling, such as the regulation of pattern recognition receptors (PRRs), remains unknown. In this study, STAT2 homologue of black carp (Mylopharyngodon piceus) has been cloned and characterized. The open reading frame (ORF) of bcSTAT2 comprises 2523 nucleotides and encodes 841 amino acids, which presents the conserved structure to that of mammalian STAT2. The dual-luciferase reporter assay and the plaque assay showed that bcSTAT2 possessed certain IFN-inducing ability and antiviral ability against both spring viremia of carp virus (SVCV) and grass carp reovirus (GCRV). Interestingly, we detected the association between bcSTAT2 and bcRIG-I through co-immunoprecipitation (co-IP) assay. Moreover, when bcSTAT2 was co-expressed with bcRIG-I, bcSTAT2 obviously suppressed bcRIG-I-induced IFN expression and antiviral activity. The subsequent co-IP assay and immunoblotting (IB) assay further demonstrated that bcSTAT2 inhibited K63-linked polyubiquitination but not K48-linked polyubiquitination of bcRIG-I, however, did not affect the oligomerization of bcRIG-I. Thus, our data conclude that black carp STAT2 negatively regulates RIG-I through attenuates its K63-linked ubiquitination, which sheds a new light on the regulation of the antiviral innate immunity cascade in vertebrates.

Black carp ATG16L1 negatively regulates STING-mediated antiviral innate immune response.

The precise control of interferon (IFN) production is indispensable for the host to eliminate invading viruses and maintain a homeostatic state. In mammals, stimulator of interferon genes (STING) is a prominent adaptor involved in antiviral immune signaling pathways. However, the regulatory mechanism of piscine STING has not been thoroughly investigated. Here, we report that autophagy related 16 like 1 (bcATG16L1) of black carp (Mylopharyngodon piceus) is a negative regulator in black carp STING (bcSTING)-mediated signaling pathway. Initially, we substantiated that knockdown of bcATG16L1 increased the transcription of IFN and ISGs and enhanced the antiviral activity of the host cells. Subsequently, we identified that bcATG16L1 inhibited the bcSTING-mediated IFN promoter activation and proved that bcATG16L1 suppressed bcSTING-mediated antiviral ability. Furthermore, we revealed that bcATG16L1 interacted with bcSTING and the two proteins shared a similar subcellular distribution. Mechanically, we found that bcATG16L1 attenuated the oligomerization of bcSTING, which was a key step for bcSTING activation. Taken together, our results indicate that bcATG16L1 interacts with bcSTING, dampens the oligomerization of bcSTING, and negatively regulates bcSTING-mediated antiviral activity.

GSTP1 is a negative regulator of MAVS in the antiviral signaling against SVCV infection.

Glutathione S-transferase P1 (GSTP1), the most ubiquitous member of the GST superfamily, plays vital roles in the detoxification, antioxidant defense, and modulation of inflammatory responses. However, limited studies have been conducted on the function of GSTP1 in antiviral innate immunity. In this study, we have cloned the homolog of GSTP1 in triploid hybrid crucian carp (3nGSTP1) and investigated its regulatory role in the interferon signaling pathway. The open reading frame of 3nGSTP1 is composed of 627 nucleotides, encoding 209 amino acids. In response to spring viremia of carp virus (SVCV) infection, the mRNA level of 3nGSTP1 was up-regulated in the liver, kidney, and caudal fin cell lines (3 nF C) of triploid fish. The knockdown of 3nGSTP1 in 3 nF C improved host cell's antiviral capacity and attenuated SVCV replication. Additionally, overexpression of 3nGSTP1 inhibited the activation of IFN promoters induced by SVCV infection, poly (I:C) stimulation, or the RLR signaling factors. The co-immunoprecipitation assays further revealed that 3nGSTP1 interacts with 3nMAVS. In addition, 3nGSTP1 dose-dependently inhibited 3nMAVS-mediated antiviral activity and reduced 3nMAVS protein level. Mechanistically, 3nGSTP1 promoted ubiquitin-proteasome degradation of MAVS by promoting its K48-linked polyubiquitination. To conclude, our results indicate that GSTP1 acts as a novel inhibitor of MAVS, which negatively regulates the IFN signaling.

Natural product honokiol exhibits antiviral effects against Micropterus salmoides rhabdovirus (MSRV) both in vitro and in vivo.

Micropterus salmoides rhabdovirus (MSRV) is a formidable pathogen, presenting a grave menace to juvenile largemouth bass. This viral infection frequently leads to epidemic outbreaks, resulting in substantial economic losses within the aquaculture industry. Unfortunately, at present, there are no commercially available vaccines or pharmaceutical treatments to combat this threat. In order to address the urgent need for therapeutic strategy to resist MSRV infection, the antiviral activity of natural product honokiol against MSRV was explored in this study. Firstly, cellular morphology was directly observed in an inverted microscope when treated with honokiol after MSRV infection. The results clarified that honokiol significantly lessened cytopathic effect (CPE) induced by MSRV and protected the integrity of GCO cells. Furthermore, the viral nucleic acid expression (G gene) was detected by reverse transcription real-time quantitative PCR (RT-qPCR) and the results indicated that honokiol significantly decreased the viral loads of MSRV in a concentration-dependent manner, and honokiol showed a high antiviral activity with IC50 of 2.92 µM. Besides, honokiol significantly decreased the viral titre and suppressed apoptosis caused by MSRV. Mechanistically, honokiol primarily inhibited the initial replication of MSRV and discharge of progeny virus to exert anti-MSRV activity. More importantly, in vivo experiments suggested that honokiol (40 mg/kg) expressed a fine antiviral activity against MSRV when administrated with intraperitoneal injection, which led to a notable 40% improvement in the survival rate among infected largemouth bass. In addition, it also resulted in significant reduction in the viral nucleic acid expression within liver, spleen and kidney at 2, 4 and 6 days following infection. What is more, 100 mg/kg honokiol with oral administration also showed certain antiviral efficacy in MSRV-infected largemouth bass via improving the survival rate by 10.0%, and decreasing significantly the viral nucleic acid expression in liver, spleen and kidney of largemouth bass on day 2. In summary, natural product honokiol is a good candidate to resist MSRV infection and has promising application prospects in aquaculture.

Separation of spring viraemia of carp virus from large-volume samples using immunomagnetic beads.

A method for separation of spring viraemia of carp virus (SVCV) from large-volume samples using immunomagnetic beads (IMBs) coated with a polyclonal antibody against SVCV was developed. The optimum amount of IMBs was 2 mg in 100 mL. After IMB treatment, the detection limit of SVCV in reverse transcription quantitative PCR (RT-qPCR) was 103 times the 50% tissue culture infectious dose per mL in 100-mL samples. The concentration of viral RNA extracted from SVCV that had been separated using IMBs was 5.18 × 103-fold higher than that of the unseparated SVCV. When fish samples were tested, the concordance rates of the IMBs/RT-qPCR and RT-qPCR were 100% and 67.5%, respectively.

Characterization and pathogenicity analysis of a newly isolated strain of infectious hematopoietic necrosis virus.

Rainbow trout is one of the fastest-growing aquaculture species and infectious hematopoietic necrosis virus (IHNV) is endemic throughout almost all rainbow trout farms in China nowadays. In this study, IHNV GS21 was identified as the causative pathogen, which resulted in massive mortality of rainbow trout occurring in northwest China. GS21 isolate was propagated in Chinook salmon embryonic cell line (CHSE-214) and induced apparent cytopathic effects (CPE) at 3 days post-infection (dpi). Phylogenetic analysis revealed that GS21 isolate was clustered with other reported Chinese isolates within the J genogroup. Moreover, the complete cDNA sequence of GS21 isolate was obtained and it possesses more than 98 % of ANI values and 89 % of DDH values with other Chinese IHNV isolates. The detailed sequence analysis of G gene revealed the distinct amino acid substitutions of G230, G252, G270, and I277 in GS21 isolate. Furthermore, the artificially infected rainbow trout exhibited similar clinical disease symptoms as natural infection did. The cumulative mortality infected by GS21 isolate of 104 PFU/mL reached 93 % at approximately 13.5 °C. Additionally, viral loads in tissues increased first and declined then as well as the expression of immune-associated genes. Collectively, our results characterized a novel IHNV GS21 isolate that can lead to massive mortality in juvenile rainbow trout and provided a basis to define the pathogenic characteristics and evolutionary relationship of IHNV and host immune response against IHNV infection.

Eukaryotic translation elongation factor 1 alpha (eEF1A) inhibits Siniperca chuatsi rhabdovirus (SCRV) infection through two distinct mechanisms.

IMPORTANCE: Although a virus can regulate many cellular responses to facilitate its replication by interacting with host proteins, the host can also restrict virus infection through these interactions. In the present study, we showed that the host eukaryotic translation elongation factor 1 alpha (eEF1A), an essential protein in the translation machinery, interacted with two proteins of a fish rhabdovirus, Siniperca chuatsi rhabdovirus (SCRV), and inhibited virus infection via two different mechanisms: (i) inhibiting the formation of crucial viral protein complexes required for virus transcription and replication and (ii) promoting the ubiquitin-proteasome degradation of viral protein. We also revealed the functional regions of eEF1A that are involved in the two processes. Such a host protein inhibiting a rhabdovirus infection in two ways is rarely reported. These findings provided new information for the interactions between host and fish rhabdovirus.

Evaluation of Taxonomic Characteristics of Matlo and Phala Bat Rabies-Related Lyssaviruses Identified in South Africa.

We report the genetic characterization of two potentially novel rabies-related lyssaviruses identified from bats in Limpopo province, South Africa. Matlo bat lyssavirus (MBLV) was identified in two Miniopterus natalensis (Natal long-fingered) bats in 2015 and 2016, and Phala bat lyssavirus (PBLV) was identified in a Nycticeinops schlieffeni (Schlieffen's) bat in 2021. The distribution of both of these bat species is largely confined to parts of Africa, with limited reports from the Arabian Peninsula. MBLV and PBLV were demonstrated to group with the unassigned and phylogroup I lyssaviruses, respectively. MBLV was most closely related to Lyssavirus caucasicus (WCBV), whereas PBLV was most closely related to Lyssavirus formosa (TWBLV-1) and Taiwan bat lyssavirus 2 (TWBLV-2), based on analysis of the N and G genes, the concatenated N + P + M + G + L coding sequence, and the complete genome sequence. Based on our analysis, MBLV and WCBV appeared to constitute a phylogroup separate from Lyssavirus lleida (LLEBV) and Lyssavirus ikoma (IKOV). Analysis of the antigenic sites suggests that PBLV will likely be serologically distinguishable from established lyssaviruses in virus-neutralization tests, whereas MBLV appeared to be antigenically highly similar to WCBV. Taken together, the findings suggested that, while PBLV is likely a new lyssavirus species, MBLV is likely related to WCBV.

Generation of Recombinant Snakehead Rhabdovirus (SHRV) Expressing Artificial MicroRNA Targeting Spring Viremia of Carp Virus (SVCV) P Gene and In Vivo Therapeutic Use Against SVCV Infection.

Spring viremia of carp virus (SVCV) is a highly lethal virus in common carp (Cyprinus carpio) and other cyprinid fish species. The aim of the present study was to develop an in vivo therapeutic measure against SVCV using artificial microRNA (AmiRNA) targeting the SVCV P gene transcript. Three candidates of AmiRNAs (AmiR-P1, -P2, and -P3) were selected, and their ability to downregulate SVCV P gene transcript was analyzed by both synthesized AmiRNA mimics and AmiRNA-expressing vector system, in which AmiR-P3 showed the strongest inhibitory activity among the three candidates. To overcome in vivo limitation of miRNA mimics or plasmid-based miRNA expression systems, we rescued recombinant snakehead rhabdoviruses (SHRVs) expressing SVCV P gene-targeting AmiRNA (rSHRV-AmiR-P3) or control AmiRNA (rSHRV-AmiR-C) using reverse genetic technology. The successful expression of AmiR-P3 and AmiR-C in cells infected with the rescued viruses was verified by quantitative PCR. To evaluate the availability of rSHRV-AmiR-P3 for in vivo control of SVCV, zebrafish (Danio rerio) were (i) infected with either rSHRV-AmiR-C or rSHRV-AmiR-P3 followed by SVCV infection or (ii) infected with SVCV followed by either rSHRV-AmiR-C or rSHRV-AmiR-P3 infection. Fish infected with rSHRVs before and after SVCV infection showed significantly higher survival rates than fish infected with SVCV alone. There was no significant difference in survival rates between groups of fish infected with rSHRV-AmiR-C and rSHRV-AmiR-P3 before SVCV infection; however, fish infected with SVCV followed by infection with rSHRV-AmiR-P3 showed significantly higher survival rates than fish infected with rSHRV-AmiR-C. These results suggest that rSHRV-AmiR-P3 has therapeutic potential against SVCV in fish when administered after SVCV infection, and rSHRVs expressing artificial microRNAs targeting SVCV transcripts could be used as a tool to control SVCV infection in fish for a therapeutic purpose.

Communicating the risks of handling bats: analysing approaches used by Australian stakeholders in the context of Australian bat lyssavirus.

Australian bat lyssavirus (ABLV) is a member of the Lyssavirus genus of the Rhabdoviridae family and is found in Australian bat species. It is of public health concern because of the rabies-like syndrome it causes in humans, resulting in government health and wildlife agencies using varied communication approaches to inform targeted audiences about zoonotic risks associated with handling bats. Despite these warnings, the number of reports of human-bat interactions remains high. This paper details a survey conducted to analyse the approaches utilised by a range of stakeholders to educate and communicate warnings to their target audiences. The survey focused on identifying the target audiences, communication methods used, along with the message frequency, content, and perceived effectiveness. Analysis of the top three messages delivered by stakeholders revealed that over half were information-focused messages and over a third, instruction-focused. Stakeholders identified the need to balance messaging about bat handling risks with information regarding the vulnerable status of bats and their environmental significance. Whilst the most common and (perceived) effective method of communication was one-on-one discussions, it was also identified to be ineffective for targeting mass audiences leading stakeholders to recognise the need to adapt to more efficient means of communication. The outcomes of this study may be useful to improve risk communication strategies regarding ABLV in Australia.

Untargeted LC-MS metabolomics reveals the metabolic responses in olive flounder subjected to hirame rhabdovirus infection.

Hirame novirhabdovirus (HIRRV), which mainly infects the olive flounder (Paralichthys olivaceus), is considered to be one of the most serious viral pathogens threatening the global fish culture industry. However, little is known about the mechanism of host-pathogen interactions at the metabolomic level. In this study, in order to explore the metabolic response of olive flounder to HIRRV infection, liquid chromatography mass spectrometry (LC-MS) was used to detect the changes of endogenous compounds of the olive flounder after HIRRV infection. A total of 954 unique masses were obtained, including 495 metabolites and 459 lipids. Among them, 7 and 173 qualified differential metabolites were identified at 2 days and 7 days post-infection, respectively. Distinct metabolic profiles were observed along with viral infection. At the early stage of infection, only a few metabolites were perturbed. Among them, the level of inosine and carnosine were increased and the potential antiviral ability of these two metabolites was further confirmed by exogenous addition experiment. At the late stage of HIRRV infection, the metabolic profiles changed remarkably. The changes in amino acids and nucleotides especially the 7-methylguanine also accelerated the amplification of viral particles. And the down-regulation of glutathione (GSH) implied an elevated level of ROS (reactive oxygen species) that attenuated the immune system of flounders. HIRRV also induced the accumulation of purine and reduction of pyrimidine, and elevated LPC and LPE levels. The unbalanced purine/pyrimidine and altered lipid profile may be beneficial for the replication and infection of HIRRV at the late stage of infection. These findings provide new insights into the pathogenic mechanism of HIRRV infection in olive flounder.

Phylogenetic analysis of spring viraemia of carp virus isolated in Serbia.

Spring viraemia of carp (SVC) is an infectious disease responsible for severe economic losses for various cyprinid species, particularly common carp (Cyprinus carpio carpio). The causative agent is the Rhabdovirus carpio or SVC virus (SVCV), a member of the Sprivivirus genus, within the Rhabdoviridae family. Phylogenetically, SVCV is divided into four genogroups (SVCV a, SVCV b, SVCV c and SVCV d), which have a reasonable correlation with the geographical distribution of the virus. In the late twentieth century, the disease was widespread in Serbian aquaculture and caused massive deaths in common carp. This study aimed to molecularly characterize the circulating SVCV isolates in Serbia over a 17-year period. The genetic relationships between 21 SVCV isolates from common carp and rainbow trout in Serbia between 1992 and 2009 were determined based on the partial nucleotide sequence of the glycoprotein gene (G gene). The phylogenetic analysis showed that the dominant SVCV isolates in Serbia belong to the SVCV d genogroup, with only one isolate belonging to genogroup SVCV b. The SVCV strains circulating in Serbia exhibited high homogeneity, as several isolates shared 100% similarity within these genogroups. Most Serbian isolates belonged to SVCV d1 and d2 subgroups, with one isolate notably different and included in a new subgroup SVCV d5. Understanding the SVCV genetic variants circulating in Serbia would be helpful in future epizootic investigations.

Differences between DNA vaccine and single-cycle viral vaccine in the ability of cross-protection against viral hemorrhagic septicemia virus (VHSV) and infectious hematopoietic necrosis virus (IHNV).

Vaccination procedures can be stressful for fish and can bring severe side effects. Therefore, vaccines that can minimize the number of administrations and maximize cross-protection against multiple serotypes, genotypes, or even different species would be highly advantageous. In the present study, we investigated the cross-protective ability of two types of vaccines - viral hemorrhagic septicemia virus (VHSV) G protein-expressing DNA vaccine and G gene-deleted single-cycle VHSV genotype IVa (rVHSV-ΔG) vaccine - against both VHSV genotype Ia and infectious hematopoietic necrosis virus (IHNV) in rainbow trout (Oncorhynchus mykiss). The results showed that rainbow trout immunized with VHSV genotype Ia G gene- or IVa G gene-expressing DNA vaccine were significantly protected against VHSV genotype Ia, but were not protected against IHNV. In contrast to the DNA vaccine, the single-cycle VHSV IVa vaccine induced significant protection against not only VHSV Ia but also IHNV. Considering no significant increase in ELISA titer and serum neutralization activity against IHNV in fish immunized with single-cycle VHSV IVa, the protection might be independent of humoral adaptive immunity. The scarcity of cytotoxic T cell epitopes between VHSV and IHNV suggested that the possibility of involvement of cytotoxic T cell-mediated cellular adaptive immunity would be low. The role of trained immunity (innate immune memory) in cross-protection should be further investigated.