Uncovering the first occurrence of Tilapia parvovirus in Thailand in tilapia during co-infection with Tilapia tilapinevirus.

The recently discovered Tilapia parvovirus (TiPV) was the first Parvovirus confirmed to infect fish, causing mortality outbreaks in farmed adult Nile tilapia in China. Severe mortality outbreaks caused by Tilapia tilapinevirus (TiLV) to farmed tilapia in Thailand revealed the concomitant occurrence of TiPV. Out of ten fish farms screened, TiPV was detected in one site rearing juvenile red hybrid tilapia. Clinical signs included abnormal swimming, scale protrusion, skin and muscle haemorrhaging, exophthalmia and generalized anaemia. Histological findings showed extensive infiltration of lymphocytes, with increased melanomacrophage centres in the anterior kidney and spleen, erythrocyte depletion in the spleen and hepatic syncytial cells. Both TiLV and TiPV were systemically distributed in the body of moribund fish. The analysis of the near-complete TiPV genome isolated from Thailand revealed 98.74% sequence identity to the formerly isolated from China, together with a highly conserved and comparable genomic organization and with a 3 nucleotides deletion in the 5-UTR. The viral genome structure was highly conserved for each of its components, with nucleotide and amino acid identity ranging from 100% for ORF1 to 97% for ORF2, and with conserved HuH and Walker loop motifs within NS1. Taken together, our results document the first detection of TiPV outside China, thus for the first time in Thailand. Moreover, TiPV was detected for the first time during a natural occurrence in farmed red hybrid tilapia and involved in co-infection pattern with TiLV. Diagnostic investigations during tilapia disease outbreaks should include the screening for TiPV. Further studies are needed to elucidate TiPV genomic variance, pathobiology, including focussing on the outcomes of TiLV-TiPV co-infection patterns, necessary to enable risk assessment for the worldwide spreading of TiPV and to design adequate control measures against these emerging viruses in tilapia.

Comparative effects of Novirhabdovirus genes on modulating constitutive transcription and innate antiviral responses, in different teleost host cell types.

BACKGROUND: Infectious hematopoietic necrosis virus (IHNV) and viral hemorrhagic septicemia virus (VHSV) are highly contagious, pathogenic Novirhabdoviruses affecting fish and are thusly notifiable diseases with the World Organization for Animal Health. This study assessed the relative capacities of IHNV and VHSV genes to modulate host general transcription and explores the abilities of specific IHNV genes to interfere with the interferon pathway in heterogenous teleost cell-lines. METHODS: Optimized protocols allowed for efficient transient transfections in EPC, BF-2, RTG-2 and RTgill-W1 cell lines of plasmids encoding IHNV (M genogroup) and VHSV (-IVb genotype) genes, including N, P, M, G and NV. Their impact on general cellular transcription was measured 48 hours post transfection (hpt) with luciferase constructs driven by a modified ß-Actin promoter (pCAG). Their modulation of the innate antiviral immune response was characterized 72 hpt, using luciferase constructs measuring rainbow trout Type I IFN or MX-1 promoter augmentation, upon MAVS co-transfection. RESULTS: M was generally confirmed as the strongest constitutive transcriptional suppressor while IHNV P, but not VHSV P, augmented constitutive transcription in fibroblastic cell types. Cell-specific effects were observed for viral G gene, with VHSV G exhibiting suppression of basal transcription in EPC and BF-2 but not in trout cells; while IHNV G was stimulatory in RTG-2, but inhibitory in RTgill-W1. NV consistently stimulated constitutive transcription, with higher augmentation patterns seen in fibroblastic compared to epithelial cells, and for IHNV NV compared to VHSV NV. The innate antiviral immune response, focusing on the IFN pathway, was silenced by IHNV M in all cell lines tested. IHNV N showed a dose-dependent suppression of type I IFN, but with minor effects on MX-1. IHNV P and G played minor IFN-inhibitory roles, consistent and dose-dependent only for G in rainbow trout cells. IHNV NV mediated a consistent stimulatory effect on either Type I IFN or MX-1, but much less pronounced in RTgill-W1. CONCLUSIONS: This study extends our understanding of Novirhabdoviruses-host interaction, showing differential innate immune responses in heterogenous cell types. Viral regulators of innate immune signaling are identified, either as dose-dependent suppressors (such as M and N) or stimulators (mainly NV), indicating novel targets for the design of more efficient vaccination strategies.

Role of Viral Hemorrhagic Septicemia Virus Matrix (M) Protein in Suppressing Host Transcription.

Viral hemorrhagic septicemia virus (VHSV) is a pathogenic fish rhabdovirus found in discrete locales throughout the Northern Hemisphere. VHSV infection of fish cells leads to upregulation of the host's virus detection response, but the virus quickly suppresses interferon (IFN) production and antiviral gene expression. By systematically screening each of the six VHSV structural and nonstructural genes, we identified matrix protein (M) as the virus' most potent antihost protein. Only M of VHSV genotype IV sublineage b (VHSV-IVb) suppressed mitochondrial antiviral signaling protein (MAVS) and type I IFN-induced gene expression in a dose-dependent manner. M also suppressed the constitutively active simian virus 40 (SV40) promoter and globally decreased cellular RNA levels. Chromatin immunoprecipitation (ChIP) studies illustrated that M inhibited RNA polymerase II (RNAP II) recruitment to gene promoters and decreased RNAP II C-terminal domain (CTD) Ser2 phosphorylation during VHSV infection. However, transcription directed by RNAP I to III was suppressed by M. To identify regions of functional importance, M proteins from a variety of VHSV strains were tested in cell-based transcriptional inhibition assays. M of a particular VHSV-Ia strain, F1, was significantly less potent than IVb M at inhibiting SV40/luciferase (Luc) expression yet differed by just 4 amino acids. Mutation of D62 to alanine alone, or in combination with an E181-to-alanine mutation (D62A E181A), dramatically reduced the ability of IVb M to suppress host transcription. Introducing either M D62A or D62A E181A mutations into VHSV-IVb via reverse genetics resulted in viruses that replicated efficiently but exhibited less cytotoxicity and reduced antitranscriptional activities, implicating M as a primary regulator of cytopathicity and host transcriptional suppression.IMPORTANCE Viruses must suppress host antiviral responses to replicate and spread between hosts. In these studies, we identified the matrix protein of the deadly fish novirhabdovirus VHSV as a critical mediator of host suppression during infection. Our studies indicated that M alone could block cellular gene expression at very low expression levels. We identified several subtle mutations in M that were less potent at suppressing host transcription. When these mutations were engineered back into recombinant viruses, the resulting viruses replicated well but elicited less toxicity in infected cells and activated host innate immune responses more robustly. These data demonstrated that VHSV M plays an important role in mediating both virus-induced cell toxicity and viral replication. Our data suggest that its roles in these two processes can be separated to design effective attenuated viruses for vaccine candidates.

Viral and bacterial septicaemic infections modulate the expression of PACAP splicing variants and VIP/PACAP receptors in brown trout immune organs.

Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) and PACAP-Related Peptide (PRP) are structurally similar peptides encoded in the same transcripts. Their transcription has been detected not only in the brain but also in a wide range of peripheral tissues, even including organs of the immune system. PACAP exerts pleiotropic activities through G-protein coupled membrane receptors: the PACAP-specific PAC-1 and the VPAC-1 and VPAC-2 receptors that exhibit similar affinities for the Vasoactive Intestinal Peptide (VIP) and PACAP. Recent findings added PACAP and its receptors to the growing list of mediators that allow cross-talk between the nervous, endocrine and immune systems in fish. In this study the expression of genes encoding for PACAP and PRP, as well as VIP/PACAP receptors was studied in laboratory-reared brown trout (Salmo trutta) after septicaemic infections. Respectively Viral Haemorrhagic Septicaemia Virus (VHSV-Ia) or the Gram-negative bacterium Yersinia ruckeri (ser. O1 - biot. 2) were used in infection challenges. Kidney and spleen, the teleost main lymphopoietic organs, were sampled during the first two weeks post-infection. RT-qPCR analysis assessed specific pathogens burden and gene expression levels. PACAP and PRP transcription in each organ was positively correlated to the respective pathogen burden, assessed targeting the VHSV-glycoprotein or Y. ruckeri 16S rRNA. Results showed as the transcription of PACAP splicing variants and VIP/PACAP receptors is modulated in these organs during an acute viral and bacterial septicaemic infections in brown trout. These gene expression results provide clues as to how the PACAP system is modulated in fish, confirming an involvement during active immune responses elicited by both viral and bacterial aetiological agents. However, further experimental evidence is still required to fully elucidate and characterize the role of PACAP and PRP for an efficient immune response against pathogens.

Salmonids have an extraordinary complex type I IFN system: characterization of the IFN locus in rainbow trout oncorhynchus mykiss reveals two novel IFN subgroups.

Fish type I IFNs are classified into two groups with two (group I) or four (group II) cysteines in the mature peptide and can be further divided into four subgroups, termed IFN-a, -b, -c, and -d. Salmonids possess all four subgroups, whereas other teleost species have one or more but not all groups. In this study, we have discovered two further subgroups (IFN-e and -f) in rainbow trout Oncorhynchus mykiss and analyzed the expression of all six subgroups in rainbow trout and brown trout Salmo trutta. In rainbow trout RTG-2 and RTS-11 cells, polyinosinic-polycytidylic acid stimulation resulted in early activation of IFN-d, whereas the IFN-e subgroup containing the highest number of members showed weak induction. In contrast with the cell lines, remarkable induction of IFN-a, -b, and -c was detected in primary head kidney leukocytes after polyinosinic-polycytidylic acid treatment, whereas a moderate increase of IFNs was observed after stimulation with resiquimod. Infection of brown trout with hemorrhagic septicemia virus resulted in early induction of IFN-d, -e, and -f and a marked increase of IFN-b and IFN-c expression in kidney and spleen. IFN transcripts were found to be strongly correlated with the viral burden and with marker genes of the IFN antiviral cascade. The results demonstrate that the IFN system of salmonids is far more complex than previously realized, and in-depth research is required to fully understand its regulation and function.