Specific nucleotides at the 3′-terminal promoter of viral hemorrhagic septicemia virus are important for virulence in vitro and in vivo

AbstractViral hemorrhagic septicemia virus (VHSV), a member of the Novirhabdovirus genus, contains an 11-nucleotide conserved sequence at the terminal 3′- and 5′-untranslated regions (UTRs) that are complementary. To study the importance of nucleotides in the 3′-UTR of VHSV for replication of novirhabdoviruses, we performed site-directed mutagenesis of selected residues at the 3′-terminus and generated mutant viruses using a reverse genetics approach. Assessment of growth kinetics and in vitro real-time cytopathogenicity studies showed that the order of two nucleotides (A4G5) of the 3′-terminus of VHSV directly affects growth kinetics in vitro. The mutant A4G-G5A virus has reduced total positive-strand RNA synthesis efficiency (51% of wild-type) at 48h post-transfection and 70h delay in causing complete cytopathic effect in susceptible fish cells, as compared to the WT-VHSV. Furthermore, when the A4G-G5A virus was used to challenge zebrafish, it exhibited reduced pathogenicity (54% lower end-point mortality) compared to the WT-VHSV. From these studies, we infer that specific residues in the 3′-UTR of VHSV have a promoter function and are essential to modulate the virulence in cells and pathogenicity in fish

Salmonid alphavirus infection in Atlantic salmon : viral properties and host responses to infection

Pancreas disease (PD) is a contagious viral disease in salmonid aquaculture in Europe and North America. PD is caused by salmon pancreas disease virus also referred to as salmonid alphavirus (SAV), which belongs to the genus alphavirus within the family Togaviridae. Up to now, at least six subtypes of SAV have been reported. In Norway SAV3 was the only subtype detected in PD diseased fish, but from 2010, a marine variant of subtype 2 has been on an increase, particularly in the Møre and Romsdal and Sør-Trøndelag counties. A better understanding of host-pathogen interactions will provide the basis for improved disease control and vaccine development. Currently, there is one commercial vaccine licensed in Norway and UK/Ireland against PD, based on inactivated whole virus. The efficacy of the vaccine under field conditions has been questioned. In this thesis, the focus was first on the elicited immune response induced by SAV3 infection both in vivo and in vitro. Fish challenged in lab-experiments with SAV3 developed classical, pathological changes as seen under a natural infection. The sequence of pathological changes in the primary target organs, pancreas and subsequently heart, coincides with virus replication levels. Despite a strong innate immune response was detected in the affected organs, the virus infection progressed. The same phenomenon was observed when SAV3 was inoculated onto susceptible cell lines. However, when cells were pre-treated with recombinant IFN-α from 24 to 4 hours prior to infection, viral replication was halted, suggesting the timing of initiation of IFN system is imperative for the antiviral activity. The work was followed by the development of an infectious SAV3 cDNA clone by use of reverse genetics. The constructed cDNA clone containing the full-length genome can be manipulated by deletion, insertion or substitution via genetic engineering. This constitutes a powerful tool for studying viral pathogenesis and developing nucleic acid based vaccines. The recovery of recombinant SAV3 was successful in three different cell lines (CHH-1, CHSE- 214 and BF-2), though the recovery was dependent on the IFN response in the transfected cells. The 6K-deleted cDNA clone failed to generate infectious virus despite production of viral proteins was detected in the cytoplasm. RNA recombination was observed when the 6Kdeleted cDNA was co-transfected into cells together with a helper cDNA encoding all structural genes, resulting in rescued, full-length viral RNA. A SAV3 replicon based vector vaccine was developed and modified by inserting a hammerhead (HH) ribozyme sequence upstream the 5’UTR sequence and incorporating N-terminal 102 nt of capsid gene downstream the internal UTR and upstream the foreign gene. This construct confers a significant increase of the foreign antigen expression. In conclusion, we have established an in vivo infection model of SAV3 and documented the antiviral role of IFN-α against SAV3 replication. We have also produced SAV3 recombinant virus using reverse genetics and a replicon construct for expression of heterologous proteins in fish cells. These tools can be used in future studies of viral pathogenesis and development of future virus vaccine.Pancreas disease (PD) er en smittsom virussykdom hos laksefisk i akvakultur i Europa og Nord-Amerika. PD er forårsaket av salmon pancreas disease virus (SPDV) også betegnet salmonid alfavirus (SAV), tilhørerende slekten alfavirus i familien Togaviridae. Hittil har minst seks subtyper av SAV (SAV1-6) blitt rapportert, og innenfor disse er SAV3 funnet utelukkende i Norge. Bedre forståelse av vert-agens interaksjoner vil gi grunnlag for bedre sykdomskontroll og danne grunnlaget for vaksineutvikling. For tiden er det en kommersielt tilgjengelig vaksine mot PD, basert på inaktivert hel-virus, men den gir ikke tilfredsstillende beskyttelse i felt. I denne avhandlingen fokuserte vi først på immunresponsen ved SAV3 infeksjon både in vivo og in vitro. Fisk eksperimentelt smittet med SAV3 utviklet klassiske patologiske forandringer lik de vi finner ved naturlige infeksjoner. Rekkefølgen av patologiske forandringer i primære målorganer, bukspyttkjertel og senere hjerte, sammenfalt med nivå av virusreplikasjon. Til tross for at infeksjonen induserte sterke medfødte, immunresponser i affiserte organer, begrenset ikke dette infeksjonen. Det samme så vi når SAV3 ble inokulert på mottakelige cellelinjer. Imidlertid, når cellene ble forbehandlet med rekombinant IFN-α (fra 24 til 4 timer før infeksjon), hindret det replikasjonen av virus. Dette tyder på at tidspunkt for aktivering av IFN-systemet er avgjørende for antiviral aktivitet. Vi etablerte deretter et revers genetikk system for SAV3 slik at vi kunne produsere rekombinant virus fra en cDNA klon som inneholdt (kodet for) hele SAV3 genomet. Dette muliggjør at man senere kan modifisere sekvensen ved å endre, slette eller legge til elemenenter i virusgenomet, for slik å lage virusmutanter. Dette utgjør et svært viktig verktøy når en skal studere egenskaper hos virus, virus patogenese og fremtidig utvikling av DNA baserte vaksiner. Produksjon av rekombinant SAV3 var vellykket i tre forskjellige cellelinjer (CHH-1, CHSE-214 og BF-2 ), selv om effektiviteten varierte avhengig av i hvor stor grad de ulike cellelinjene produserte IFN. Vi forsøkte deretter å lage en attenuert SAV3 stamme uten genet som koder for 6K proteinet. Til tross for at virusets proteiner ble uttrykt i cellenes cytoplasma, klarte vi ikke å produsere rekombinant SAV3 uten 6K. Vi oppdaget derimot at SAV3 viste stor evne til å RNA rekombinasjon. Rekombinasjon ble observert når cDNA kodende for SAV3 uten 6K ble ko-transfektert inn i celler sammen med et hjelpe-cDNA som inneholder kun strukturelle gener (full-lengde). Dette resulterte i hyppig reversjon til fullengde virus. Med utgangspunkt i den infeksiøse cDNA klonene laget vi et SAV3 replicon for uttrykk av heterologe proteiner i cellekultur, og deretter ble effekt av ulike modifiseringer testet. Blant annet undersøkte vi effekten av å sette inn en «hammer-head» (HH) ribozyme sekvens oppstrøms for replikonet. Videre hvordan innlemming av N – terminale deler (102 nt) av kapsid genet nedstrøms for intern UTR og oppstrøms for innsatt fremmed gen gjorde at man fikk et signifikant høyere uttrykk av fremmed protein. Som konklusjonen har vi etablert en in vivo infeksjon modell for SAV3 og dokumentert IFN-α sin viktige antivirale effekt mot SAV3. Vi har videre produsert rekombinant SAV3 virus ved hjelp av revers genetikk samt et SAV replikon konstrukt for uttrykk av heterologt protein i fiskeceller. Disse verktøyene kan brukes i videre studier av virusets patogenese og utvikling av fremtidige virusvaksiner

Experimental piscine alphavirus RNA recombination in vivo yields both viable virus and defective viral RNA

RNA recombination in non-segmented RNA viruses is important for viral evolution and documented for several virus species through in vitro studies. Here we confirm viral RNA recombination in vivo using an alphavirus, the SAV3 subtype of Salmon pancreas disease virus. The virus causes pancreas disease in Atlantic salmon and heavy losses in European salmonid aquaculture. Atlantic salmon were injected with a SAV3 6K-gene deleted cDNA plasmid, encoding a non-viable variant of SAV3, together with a helper cDNA plasmid encoding structural proteins and 6K only. Later, SAV3-specific RNA was detected and recombination of viral RNA was confirmed. Virus was grown from plasmid-injected fish and shown to infect and cause pathology in salmon. Subsequent cloning of PCR products confirming recombination, documented imprecise homologous recombination creating RNA deletion variants in fish injected with cDNA plasmid, corresponding with deletion variants previously found in SAV3 from the field. This is the first experimental documentation of alphavirus RNA recombination in an animal model and provides new insight into the production of defective virus RNA

Gene expression studies of host response to Salmonid alphavirus subtype 3 experimental infections in Atlantic salmon

Abstract Salmonid alphavirus subtype-3 (SAV-3) infection in Atlantic salmon is exclusively found in Norway. The salmonid alphaviruses have been well characterized at the genome level but there is limited information about the host-pathogen interaction phenomena. This study was undertaken to characterize the replication and spread of SAV-3 in internal organs of experimentally infected Atlantic salmon and the subsequent innate and adaptive immune responses. In addition, suitability of a cohabitation challenge model for this virus was also examined. Groups of fish were infected by intramuscular injection (IM), cohabited (CO) or kept uninfected in a separate tank. Samples of pancreas, kidney, spleen, heart and skeletal muscles were collected at 2, 4 and 8 weeks post infection (wpi). Pathological changes were assessed by histology concurrently with viral loads and mRNA expression of immune genes by real time RT-PCR. Pathological changes were only observed in the pancreas and heart (target organs) of both IM and CO groups, with changes appearing first in the pancreas (2 wpi) in the former. Lesions with increasing severity over time coincided with high viral loads despite significant induction of IFN-α, Mx and ISG15. IFN-γ and MHC-I were expressed in all tissues examined and their induction appeared in parallel with that of IL-10. Inflammatory genes TNF-α, IL-12 and IL-8 were only induced in the heart during pathology while T cell-related genes CD3ε, CD4, CD8, TCR-α and MHC-II were expressed in target organs at 8 wpi. These findings suggest that the onset of innate responses came too late to limit virus replication. Furthermore, SAV-3 infections in Atlantic salmon induce Th1/cytotoxic responses in common with other alphaviruses infecting higher vertebrates. Our findings demonstrate that SAV-3 can be transmitted via the water making it suitable for a cohabitation challenge model.</p

A 6K-deletion variant of salmonid alphavirus is non-viable but can be rescued through RNA recombination.

Pancreas disease (PD) of Atlantic salmon is an emerging disease caused by Salmonid alphavirus (SAV) which mainly affects salmonid aquaculture in Western Europe. Although genome structure of SAV has been characterized and each individual viral protein has been identified, the role of 6K protein in viral replication and infectivity remains undefined. The 6K protein of alphaviruses is a small and hydrophobic protein which is involved in membrane permeabilization, protein processing and virus budding. Because these common features are shared across many viral species, they have been named viroporins. In the present study, we applied reverse genetics to generate SAV3 6K-deleted (Δ6K) variant and investigate the role of 6K protein. Our findings show that the 6K-deletion variant of salmonid alphavirus is non-viable. Despite viral proteins of Δ6K variant are detected in the cytoplasm by immunostaining, they are not found on the cell surface. Further, analysis of viral proteins produced in Δ6K cDNA clone transfected cells using radioimmunoprecipitation (RIPA) and western blot showed a protein band of larger size than E2 of wild-type SAV3. When Δ6K cDNA was co-transfected with SAV3 helper cDNA encoding the whole structural genes including 6K, the infectivity was rescued. The development of CPE after co-transfection and resolved genome sequence of rescued virus confirmed full-length viral genome being generated through RNA recombination. The discovery of the important role of the 6K protein in virus production provides a new possibility for the development of antiviral intervention which is highly needed to control SAV infection in salmonids

Alpha Interferon and Not Gamma Interferon Inhibits Salmonid Alphavirus Subtype 3 Replication In Vitro▿

Salmonid alphavirus (SAV) is an emerging virus in salmonid aquaculture, with SAV-3 being the only subtype found in Norway. Until now, there has been little focus on the alpha interferon (IFN-α)-induced antiviral responses during virus infection in vivo or in vitro in fish. The possible involvement of IFN-γ in the response to SAV-3 is also not known. In this study, the two IFNs were cloned and expressed as recombinant proteins (recombinant IFN-α [rIFN-α] and rIFN-γ) and used for in vitro studies. SAV-3 infection in a permissive salmon cell line (TO cells) results in IFN-α and IFN-stimulated gene (ISG) mRNA upregulation. Preinfection treatment (4 to 24 h prior to infection) with salmon rIFN-α induces an antiviral state that inhibits the replication of SAV-3 and protects the cells against virus-induced cytopathic effects (CPE). The antiviral state coincides with a strong expression of Mx and ISG15 mRNA and Mx protein expression. When rIFN-α is administered at the time of infection and up to 24 h postinfection, virus replication is not inhibited, and cells are not protected against virus-induced CPE. By 40 h postinfection, the alpha subunit of eukaryotic initiation factor 2 (eIF2α) is phosphorylated concomitant with the expression of the E2 protein as assessed by Western blotting. Postinfection treatment with rIFN-α results in a moderate reduction in E2 expression levels in accordance with a moderate downregulation of cellular protein synthesis, an approximately 65% reduction by 60 h postinfection. rIFN-γ has only a minor inhibitory effect on SAV-3 replication in vitro. SAV-3 is sensitive to the preinfection antiviral state induced by rIFN-α, while postinfection antiviral responses or postinfection treatment with rIFN-α is not able to limit viral replication

cAMP induction post PGE2 treatment.

<p>Three cell lines, CHSE-214, RTG-2 and TO were used in this experiment. Cells were either treated with PGE2 (P) or left without treatment (C).</p

cAMP induction post forskolin treatment.

<p>Three cell lines, CHSE-214, RTG-2 and TO were used in this experiment. Cells were either treated with forskolin or left untreated (controls). * = p<0.01 between control and the two treatments. Average ± SEM is shown (n = 3).</p

List of primers used for PCR and cloning.

<p>List of primers used for PCR and cloning.</p

Nucleotide sequences of two EP4 subtypes.

<p>Comparison of nucleotide sequence of two subtypes of EP4 gene identified in Atlantic salmon.</p