Salmonid alphavirus non-structural protein 2 is a key protein that activates the NF-κB signaling pathway to mediate inflammatory responses.

Salmonid alphavirus (SAV) infection of Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) causes pancreas disease (PD) with typical inflammatory responses, such as necrosis of the exocrine pancreas, cardiomyopathy and skeletal myopathy. However, the pathogenic mechanism underlying SAV infection is still unclear. Inflammation may cause damage to the body, but it is a defense response against infection by pathogenic microorganisms, of which nuclear factor-kappa B (NF-κB) is the main regulator. This study revealed that SAV can activate NF-κB, of which the viral nonstructural protein Nsp2 is the major activating protein. SAV activates the NF-κB signaling pathway by simultaneously up-regulating TLR3, 7, 8 and then the expression of the signaling molecule myeloid differentiation factor 88 (Myd88) and tumor necrosis factor receptor-associated factor 6 (TRAF6). We found that Nsp2 can induce IκB degradation and p65 phosphorylation and transnucleation, and activate NF-κB downstream inflammatory cytokines. Nsp2 may simultaneously activate NF-κB through TLR3,7,8-dependent signaling pathways. Overexpression of Nsp2 can up-regulate mitochondrial antiviral signaling protein (MAVS) and then promote the expression of IFNa1 and antiviral protein Mx, which inhibits viral replication. This study shows that Nsp2 acts as a key activator protein for the NF-κB signaling pathway, which induces inflammation post-SAV infection. This study systematically analyzes the molecular mechanism of SAV activation of the NF-κB signaling pathway, and provides a theoretical basis for revealing the mechanism of innate immune response and inflammatory injury caused by SAV.

Structural Insights into Alphavirus Assembly Revealed by the Cryo-EM Structure of Getah Virus.

Getah virus (GETV) is a member of the alphavirus genus, and it infects a variety of animal species, including horses, pigs, cattle, and foxes. Human infection with this virus has also been reported. The structure of GETV has not yet been determined. In this study, we report the cryo-EM structure of GETV at a resolution of 3.5 Å. This structure reveals conformational polymorphism of the envelope glycoproteins E1 and E2 at icosahedral 3-fold and quasi-3-fold axes, which is believed to be a necessary organization in forming a curvature surface of virions. In our density map, three extra densities are identified, one of which is believed a "pocket factor"; the other two are located by domain D of E2, and they may maintain the stability of E1/E2 heterodimers. We also identify three N-glycosylations at E1 N141, E2 N200, and E2 N262, which might be associated with receptor binding and membrane fusion. The resolving of the structure of GETV provides new insights into the structure and assembly of alphaviruses and lays a basis for studying the differences of biology and pathogenicity between arthritogenic and encephalitic alphaviruses.

Establishment of an enzyme-linked immunosorbent assay for Getah virus infection in horses using a 20-mer synthetic peptide for the E2 glycoprotein as an antigen.

An enzyme-linked immunosorbent assay (ELISA) using a synthetic peptide for the E2 glycoprotein was developed for the serodiagnosis of Getah virus infection in horses. To identify an immunogenic epitope, a series of 20-mer peptides (n = 22) for the E2 protein was screened with pooled sera from horses infected with Getah virus. Peptide P11 (PTEEEIDMHTPPDIPDITLL) showed the strongest reaction. ELISA using P11 (E2-P11-ELISA) detected increased antibody levels in all seven experimentally infected horses and in five out of nine vaccinated horses. Out of 28 naturally infected horses, 25 were seronegative in their acute sera but turned seropositive in their convalescent sera. For the remaining three horses whose acute sera were seropositive, an endpoint method with serial dilutions detected a ≥ 4-fold increase in titer between paired sera. The concordance between E2-P11-ELISA and a virus-neutralization test in terms of seropositivity was assessed using a series of 220 horse sera, resulting in almost perfect agreement, with a kappa coefficient value of 0.865. E2-P11-ELISA had a sensitivity of 93.3% (95% CI 86.6-97.1%) and a specificity of 95.0% (95% CI 92.5-96.4%). This highly sensitive and specific E2-P11-ELISA should be useful for serodiagnosis of Getah virus infection in horses.

Development of an enzyme-linked immunosorbent assay for Getah virus infection in horses using recombinant E2 protein as an antigen.

Getah virus causes fever, skin eruptions, and limb edema in horses. For a high-throughput and time-saving method for serodiagnosis, we explored immunogenic antigens of Getah virus, and established an enzyme-linked immunosorbent assay (ELISA) using a recombinant protein. Western blot analysis using sera from infected horses showed strong reaction with viral antigens around 46 kDa corresponding to E1 or E2 glycoproteins. Recombinant E2 (rE2) protein reacted more strongly with infected horse sera than did rE1 protein in both Western blotting and ELISA. In ELISA using rE2 protein (rE2-ELISA), for all horses experimentally infected with Getah virus (n = 7), optical density (OD) exceeded the cutoff value at 14 days post-infection. ODs in five of nine vaccinated horses also slightly exceeded the cutoff value after vaccination. Among naturally infected horses (n = 28), 24 were seronegative in the acute sera, which turned seropositive in the convalescent sera. For the four horses seropositive in the acute sera, an endpoint method with serial dilutions of paired sera detected a ≥4-fold increase in titer. In conclusion, we established rE2-ELISA that could detect horse antibodies against Getah virus after experimental and natural infections; this should be useful in the diagnosis and surveillance of Getah virus infection.

Transcriptome Analysis Shows That IFN-I Treatment and Concurrent SAV3 Infection Enriches MHC-I Antigen Processing and Presentation Pathways in Atlantic Salmon-Derived Macrophage/Dendritic Cells.

Type I interferons (IFNs) have been shown to play an important role in shaping adaptive immune responses in addition to their antiviral properties in immune cells. To gain insight into the impact of IFN-I-induced pathways involved in early adaptive immune responses, i.e., antigen-presenting pathways, in an Atlantic salmon-derived (Salmo salar L.) macrophage cell line (TO-cells), we used a comparative de novo transcriptome analysis where cells were treated with IFN-I or kept untreated and concurrently infected with salmonid alphavirus subtype 3 (SAV3). We found that concurrent treatment of TO-cells with IFN-I and SAV3 infection (SAV3/IFN+) significantly enriched the major histocompatibility complex class I (MHC-I) pathway unlike the non-IFN-I treated TO-cells (SAV3/IFN-) that had lower expression levels of MHC-I pathway-related genes. Genes such as the proteasomal activator (PA28) and ß-2 microglobulin (ß2M) were only differentially expressed in the SAV3/IFN+ cells and not in the SAV3/IFN- cells. MHC-I pathway genes like heat shock protein 90 (Hsp90), transporter of antigen associated proteins (TAPs) and tapasin had higher expression levels in the SAV3/IFN+ cells than in the SAV3/IFN- cells. There were no MHC-II pathway-related genes upregulated in SAV3/IFN+-treated cells, and cathepsin S linked to the degradation of endosomal antigens in the MHC-II pathway was downregulated in the SAV3/IFN- cells. Overall, our findings show that concurrent IFN-I treatment of TO-cells and SAV3 infection enriched gene expression linked to the MHC-I antigen presentation pathway. Data presented indicate a role of type I IFNs in strengthening antigen processing and presentation that may facilitate activation particularly of CD8+ T-cell responses following SAV3 infection, while SAV3 infection alone downplayed MHC-II pathways.

Protective effect and antibody response of DNA vaccine against salmonid alphavirus 3 (SAV3) in Atlantic salmon.

This work reports the effect of two DNA vaccines against salmonid alphavirus 3 (SAV3) in Atlantic salmon. Presmolts were vaccinated by intramuscular injection of plasmids encoding the SAV3 structural polyprotein C-E3-E2-6K-E2 (pCSP), E2 only (pE2), or plasmid without insert (pcDNA3.3). E2 is expressed at the surface of cells transfected with pCSP and internally in cells transfected with pE2. A commercial vaccine based on inactivated SAV (NCPD) was used for comparison. At 10 weeks post-vaccination, only fish vaccinated with pCSP showed antibody against E2 and virus-neutralizing activity. Vaccinated fish were infected with SAV3 to determine protection by virus quantitation in serum after 7 days and scoring of pathological changes after 21 days. Fish vaccinated with both pCSP and NCPD vaccines showed significant virus reduction in serum, while fish vaccinated with pE2 did not. All fish vaccinated with pcDNA3.3 and pE2 showed pathological changes in organs typical of PD, 60% of fish vaccinated with NCPD showed PD pathology, while fish vaccinated with pCSP did not show PD pathology. Taken together, DNA vaccination with pCSP provided strong protection for salmon against SAV3 infection, which in part may be due to production of virus-neutralizing antibodies.

Molecular cloning of MDA5, phylogenetic analysis of RIG-I-like receptors (RLRs) and differential gene expression of RLRs, interferons and proinflammatory cytokines after in vitro challenge with IPNV, ISAV and SAV in the salmonid cell line TO.

The RIG-I receptors RIG-I, MDA5 and LGP2 are involved in viral recognition, and they have different ligand specificity and recognize different viruses. Activation of RIG-I-like receptors (RLRs) leads to production of cytokines essential for antiviral immunity. In fish, most research has focused on interferons, and less is known about the production of proinflammatory cytokines during viral infections. In this study, we have cloned the full-length MDA5 sequence in Atlantic salmon, and compared it with RIG-I and LGP2. Further, the salmonid cell line TO was infected with three fish pathogenic viruses, infectious pancreatic necrosis virus (IPNV), infectious salmon anaemia virus (ISAV) and salmonid alphavirus (SAV), and differential gene expression (DEG) analyses of RLRs, interferons (IFNa-d) and proinflammatory cytokines (TNF-α1, TNF-α2, IL-1ß, IL-6, IL-12 p40s) were performed. The DEG analyses showed that the responses of proinflammatory cytokines in TO cells infected with IPNV and ISAV were profoundly different from SAV-infected cells. In the two aforementioned, TNF-α1 and TNF-α2 were highly upregulated, while in SAV-infected cells these cytokines were downregulated. Knowledge of virus recognition by the host and the immune responses during infection may help elucidate why and how some viruses can escape the immune system. Such knowledge is useful for the development of immune prophylactic measures.

De novo assembly and transcriptome analysis of Atlantic salmon macrophage/dendritic-like TO cells following type I IFN treatment and Salmonid alphavirus subtype-3 infection.

BACKGROUND: Interferons (IFN) are cytokines secreted by vertebrate cells involved in activation of signaling pathways that direct the synthesis of antiviral genes. To gain a global understanding of antiviral genes induced by type I IFNs in salmonids, we used RNA-seq to characterize the transcriptomic changes induced by type I IFN treatment and salmon alphavirus subtype 3 (SAV-3) infection in TO-cells, a macrophage/dendritic like cell-line derived from Atlantic salmon (Salmo salar L) head kidney leukocytes. RESULTS: More than 23 million reads generated by RNA-seq were de novo assembled into 58098 unigenes used to generate a total of 3149 and 23289 differentially expressed genes (DEGs) from TO-cells exposed to type I IFN treatment and SAV-3 infection, respectively. Although the DEGs were classified into genes associated with biological processes, cellular components and molecular function based on gene ontology classification, transcriptomic changes reported here show upregulation of genes belonging to the canonical type I IFN signaling pathways together with a broad spectrum of antiviral genes that block virus replication in host cells. In addition, the transcriptome shows a profile of genes associated with apoptosis as well as genes that activate adaptive immunity. Further, our findings show that the profile of genes expressed by TO-cells is comparable to orthologous genes expressed by mammalian macrophages and dendritic cells in response to type I IFNs. Twenty DEGs randomly selected for qRT-PCR confirmed the validity of the transcriptomic changes detected by RNA-seq by showing that the genes upregulated by RNA-seq were also upregulated by qRT-PCR and that genes downregulated by RNA-seq were also downregulated by qRT-PCR. CONCLUSIONS: The de novo assembled transcriptome presented here provides a global description of genes induced by type I IFNs in TO-cells that could serve as a repository for future studies in fish cells. Transcriptome analysis shows that a large proportion of IFN genes expressed in this study are comparable to IFNs genes expressed in mammalia. In addition, the study shows that SAV-3 is a potent inducer of type I IFNs and that the responses it induces in TO-cells could serve as a model for studying IFN responses in salmonids.

Enveloped virus-like particles as vaccines against pathogenic arboviruses.

Arthropod-borne arboviruses form a continuous threat to human and animal health, but few arboviral vaccines are currently available. Advances in expression technology for complex, enveloped virus-like particles (eVLPs) create new opportunities to develop potent vaccines against pathogenic arboviruses. In this short review, I highlight the successes and challenges in eVLP production for members of the three major arbovirus families: Flaviviridae (e.g., dengue, West Nile, Japanese encephalitis); Bunyaviridae (e.g., Rift Valley fever); and Togaviridae (e.g., chikungunya). The results from pre-clinical testing will be discussed as well as specific constraints to the large-scale manufacture and purification of eVLPs, which are complex assemblies of membranes and viral glycoproteins. Insect cells emerge as ideal substrates for correct arboviral glycoprotein folding and posttranslational modification to yield high quality eVLPs. Furthermore, baculovirus expression in insect cell culture is scalable and has a proven safety record in industrial human and veterinary vaccine manufacturing. In conclusion, eVLPs produced in insect cells using modern biotechnology have a realistic potential to be used in novel vaccines against arboviral diseases.

An experimental means of transmitting pancreas disease in Atlantic salmon Salmo salar L. fry in freshwater.

A challenge model for pancreas disease in Atlantic salmon, Salmo salar L. fry, was developed comparing two salmonid alphavirus (SAV) subtypes: SAV1 and SAV5. Viral doses of 3 × 10(5) TCID50  mL(-1) for SAV1 and 3 × 10(4) for SAV5 were tested in triplicate tanks, each containing 450 salmon fry. Cumulative mortalities of 1.2% were recorded. Titres of virus recovered from the mortalities ranged from 10(2) to 10(7) TCID50  mL(-1) . Fry were sampled at 3, 5 and 7.5 weeks post-challenge. Sampling after 3 weeks revealed a high prevalence of infection in the absence of clinical signs, and infectious virus was recovered from 80% and 43% of sampled fry infected with SAV1 and SAV5, respectively. After 5 weeks pancreas, heart and red skeletal muscle lesions were generally observed, whilst degeneration in white skeletal muscle was observed only in fish infected with SAV1. In situ hybridisation confirmed the presence of viral genome in infected pancreas, heart and muscle. After 7.5 weeks, infectious virus (both isolates) was recovered from 13.3% of the fish sampled, with a viral titre of 10(2) TCID50  mL(-1) . Clearly, salmon fry are susceptible to SAV infection and pancreas disease.

Salmonid alphavirus glycoprotein E2 requires low temperature and E1 for virion formation and induction of protective immunity.

Salmonid alphavirus (SAV; also known as Salmon pancreas disease virus; family Togaviridae) causes pancreas disease and sleeping disease in Atlantic salmon and rainbow trout, respectively, and poses a major burden to the aquaculture industry. SAV infection in vivo is temperature-restricted and progeny virus is only produced at low temperatures (10-15 °C). Using engineered SAV replicons we show that viral RNA replication is not temperature-restricted suggesting that the viral structural proteins determine low-temperature dependency. The processing/trafficking of SAV glycoproteins E1 and E2 as a function of temperature was investigated via baculovirus vectors in Sf9 insect cells and by transfection of CHSE-214 fish cells with DNA constructs expressing E1 and E2. We identified SAV E2 as the temperature determinant by demonstrating that membrane trafficking and surface expression of E2 occurs only at low temperature and only in the presence of E1. Finally, a vaccination-challenge model in Atlantic salmon demonstrates the biological significance of our findings and shows that SAV replicon DNA vaccines encoding E2 elicit protective immunity only when E1 is co-expressed. This is the first study that identifies E2 as the critical determinant of SAV low-temperature dependent virion formation and defines the prerequisites for induction of a potent immune response in Atlantic salmon by DNA vaccination.

Novel vaccination approaches against equine alphavirus encephalitides.

The current production of inactivated vaccines for the prevention of equine alphavirus encephalitides caused by Eastern, Western and Venezuelan Equine Encephalitis viruses (EEEV, WEEV, VEEV) involves the manipulation of large quantities of infectious viral particles under biosafety level 3 containment laboratories with the potential risk of transmission to the operators. Moreover, these vaccines are not capable of inducing a long-lasting immunity. Modified live vaccines, which were also attempted, maintain residual virulence and neurotropism, causing disease in both horses and humans. Therefore, the production of an efficacious second generation vaccine which could be used in the prevention of alphavirus infection without the need to manipulate infectious viral particles under high biocontainment conditions could be of great benefit for the worldwide horse industry. Furthermore, equine alphaviruses are considered as biological threat agents. Subunit, chimeric, gene-deleted live mutants, DNA and adenovirus-vectored alphavirus vaccines have been evaluated; such approaches are reviewed in this work. Climate changes, together with modifications in bird and vector ecology, are leading to the arise of emerging pathogens in new geographical locations, and these zoonotic New World arboviruses are gaining concern. Novel vaccine development does show a promising future for prevention of these infections in both horses and humans.

Geographical distribution of salmonid alphavirus subtypes in marine farmed Atlantic salmon, Salmo salar L., in Scotland and Ireland.

Sequence data from salmonid alphavirus (SAV) strains obtained from farmed marine Atlantic salmon, Salmo salar L. , over a 20-year period between 1991 and 2011 was reviewed to examine the geographical distribution of the genetically defined SAV subtypes in twelve regions across Ireland and Scotland. Of 160 different Atlantic salmon SAV strains examined, 62 belonged to subtype 1, 28 to subtype 2, 34 to subtype 4, 35 to subtype 5 and 1 to subtype 6. SAV subtypes 1, 4 and 6 were found in Ireland, while subtypes 1, 2, 4 and 5 were found in Scotland. In the majority of regions, there was a clear clustering of subtypes, with SAV subtype 1 being the dominant subtype in Ireland overall, as well as in Argyll and Bute in Scotland. SAV subtype 2 predominated in the Shetland and Orkney Islands. The emergence in Atlantic salmon of subtype 2 strains typically associated with sleeping disease in rainbow trout in Argyll and Bute, strongly suggesting transmission of infection between these species, was noted for the first time. SAV subtype 4 was the most common subtype found in the southern Western Isles, while SAV subtype 5 predominated in the northern Western Isles and north-west mainland Scotland. No single strain was dominant on sites in the western Highlands, with a number of sites in this region in particular having more than one subtype detected in different submissions. The significance of these results in relation to aspects of the epidemiology of infection, including transmission, biosecurity and wildlife reservoirs are discussed and knowledge gaps identified.

Immunoprotective activity of a Salmonid Alphavirus Vaccine: comparison of the immune responses induced by inactivated whole virus antigen formulations based on CpG class B oligonucleotides and poly I:C alone or combined with an oil adjuvant.

CpG oligonucleotides and polyinosinic:polycytidylic acid (poly I:C) are toll-like receptor (TLR) agonists that mimic the immunostimulatory properties of bacterial DNA and double-stranded viral RNA respectively, and which have exhibited potential to serve as vaccine adjuvants in previous experiments. Here, a combination of CpGs and poly I:C together with water- or oil-formulated Salmonid Alphavirus (SAV) antigen preparations has been used for a vaccine in Atlantic salmon and tested for protection in SAV challenge trial. The results demonstrate that vaccination with a high dose of the SAV antigen induced protection against challenge with SAV which correlated with production of neutralizing antibodies (NAbs). As the high antigen dose alone induced full protection, no beneficial effect from the addition of CpG and poly I:C could be observed. Nevertheless, these TLR ligands significantly enhanced the levels of NAbs in serum of vaccinated fish. Interestingly, gene expression analysis demonstrated that while addition of oil suppressed the CpG/poly I:C-induced expression of IFN-γ, the upregulation of IFNa1 was substantially enhanced. A low dose of the SAV antigen combined with oil did not induce any detectable levels of NAbs either with or without TLR ligands present, however the addition of CpG and poly I:C to the low SAV antigen dose formulation significantly enhanced the protection against SAV suggesting that CpG/poly I:C may have enhanced a cytotoxic response - a process which is dependent on the up-regulation of type I IFN. These results highlight the immunostimulatory properties of the tested TLR ligands and will serve as a ground for further, more detailed studies aimed to investigate their capacity to serve as adjuvants in vaccine formulations for Atlantic salmon.

Superior protection conferred by inactivated whole virus vaccine over subunit and DNA vaccines against salmonid alphavirus infection in Atlantic salmon (Salmo salar L.).

Salmonid alphavirus 3 (SAV-3) is an emerging pathogen in Norwegian salmon farming and causes severe annual losses. We studied the immunogenicity and protective ability of subunit and DNA vaccines based on E1 and E2 spike proteins of salmonid alphavirus subtype 3 (SAV-3), and compared these to an experimental inactivated, whole virus (IWV) vaccine in Atlantic salmon. The antigens were delivered as water-in-oil emulsions for the subunit and inactivated vaccines and non-formulated for the DNA vaccines. The IWV and the E2 subunit prime-boost groups had circulating neutralizing antibodies at challenge, correlating with high protection against lethal challenge and 3-log(10) reduction of virus titer in heart for the IWV group. Prime-boost with E1 subunit vaccine also conferred significant protection against mortality, but did not correlate with neutralizing antibody levels. Protection against pathology in internal organs was only seen for the IWV group. Prime-boost with E1 and E2 DNA vaccines showed marginal protection in terms of reduction of viral replication in target organs and protection against mortality was not different from controls. The IWV group showed significant upregulation of IFNγ and IL2 mRNA expression at 4 weeks post challenge possibly indicating that other mechanisms in addition to antibody responses play a role in mediating protection against infection. This is the first report comparing the immunogenicity and protection against mortality for IWV vaccines and spike protein subunit and DNA vaccines against salmonid alphavirus infection in Atlantic salmon. The IWV vaccine has superior immunogenicity over sub-unit and DNA vaccines.

Formulations combining CpG containing oliogonucleotides and poly I:C enhance the magnitude of immune responses and protection against pancreas disease in Atlantic salmon.

Both CpG oligodeoxynucleotides and double-stranded RNA (poly I:C) have documented effects as treatments against several viral diseases in fish. However, as stand-alone treatments their effects have been modest. We have tested here whether CpG and poly I:C, alone or in combination induce protection against Salmonid Alphavirus (SAV), the causative agent of pancreas disease in Atlantic salmon. Our results revealed a significant reduction of viraemia 2 weeks after ip injection of the combined treatment and 1 week after challenge with SAV subtype 3, followed by reduced SAV induced heart pathology 3 weeks later. The SAV titers in blood samples from the combination group were lower as compared to single treatments with either CpG or poly I:C. Surprisingly, reduced SAV levels could also be found in fish as long as 7 weeks after receiving the combination treatment. The expression of IFNγ and to a lesser extent IFNa and Mx was up-regulated in head kidney and spleen 5 days after the fish had been treated with CpG and poly I:C. Furthermore, the complement factor C4 was depleted in serum 8 weeks post treatment, suggesting complement activation leading to C4 consumption. We hypothesize that the CpG/poly I:C-induced protection against SAV3 is mediated by mechanisms involving type I and type II IFN induced antiviral activity and complement mediated protective responses.

Development and evaluation of a one-step SYBR-Green I-based real-time RT-PCR assay for the detection and quantification of Chikungunya virus in human, monkey and mosquito samples.

This paper reports the development of a one-step SYBR-Green I-based realtime RT-PCR assay for the detection and quantification of Chikungunya virus (CHIKV) in human, monkey and mosquito samples by targeting the E1 structural gene. A preliminary evaluation of this assay has been successfully completed using 71 samples, consisting of a panel of negative control sera, sera from healthy individuals, sera from patients with acute disease from which CHIKV had been isolated, as well as monkey sera and adult mosquito samples obtained during the chikungunya fever outbreak in Malaysia in 2008. The assay was found to be 100-fold more sensitive than the conventional RT-PCR with a detection limit of 4.12x10(0) RNA copies/µl. The specificity of the assay was tested against other related viruses such as Dengue (serotypes 1-4), Japanese encephalitis, Herpes Simplex, Parainfluenza, Sindbis, Ross River, Yellow fever and West Nile viruses. The sensitivity, specificity and efficiency of this assay were 100%, 100% and 96.8% respectively. This study on early diagnostics is of importance to all endemic countries, especially Malaysia, which has been facing increasingly frequent and bigger outbreaks due to this virus since 1999.

Complete nucleotide sequence of Middelburg virus, isolated from the spleen of a horse with severe clinical disease in Zimbabwe.

The complete nucleotide sequence of Middelburg virus (MIDV) was determined for strain MIDV-857 from Zimbabwe. The isolation of this virus in 1993 from a horse that died showing severe clinical signs represents the first indication that MIDV can cause severe disease in equids. Full-length cDNA copies of the viral genome were successfully synthesized by an innovative RT-PCR amplification approach using an 'anchor primer' combined with the SMART methodology described previously for the synthesis of full-length cDNA copies from genome segments of dsRNA viruses. The MIDV-857 genome is 11,674 nt, excluding the 5'-terminal cap structure and poly(A) tail (which varies in length from approximately 180 to approximately 220 residues). The organization of the genome is like that of other alphaviruses, including a read-through stop codon between the nsP3 and nsP4 genes. However, phylogenetic analyses of the structural protein amino acid sequences suggested that the MIDV E1 gene was generated by recombination with a Semliki Forest virus-like virus. This hypothesis was supported by bootscanning analysis using a recombination-detection program. The 3' untranslated region of MIDV-857 also contains a 112 nt duplication. This study reports the first full-length sequence of MIDV, which was obtained from a single RT-PCR product.

Phylogenetic analysis of Buggy Creek virus: evidence for multiple clades in the Western Great Plains, United States of America.

We present the first detailed phylogenetic analysis of Buggy Creek virus (BCRV), a poorly known alphavirus with transmission cycles involving a cimicid swallow bug (Oeciacus vicarius) vector and cliff swallows (Petrochelidon pyrrhonota) and house sparrows (Passer domesticus) as the principal avian hosts. Nucleotide sequences of a 2,075-bp viral envelope glycoprotein-coding region, covering the entire PE2 gene, were determined for 33 BCRV isolates taken from swallow bugs at cliff swallow colonies in Nebraska and Colorado in the summer of 2001 and were compared with the corresponding region of BCRV isolates collected from Oklahoma in the 1980s. We also analyzed isolates of the closely related Fort Morgan virus (FMV) collected from Colorado in the 1970s. Phylogenetic analysis indicated that BCRV falls into the western equine encephalomyelitis complex of alphaviruses, in agreement with antigenic results and a previous alphavirus phylogeny based on the E1 coding region. We found four distinct BCRV/FMV clades, one each unique to Nebraska, Colorado, and Oklahoma and one containing isolates from both Nebraska and Colorado. BCRV isolates within the two clades from Nebraska showed 5.7 to 6.2% nucleotide divergence and 0.7 to 1.9% amino acid divergence, and within these clades, we found multiple subclades. Nebraska subclades tended to be confined to one or a few cliff swallow colonies that were close to each other in space, although in some cases, near-identical isolates were detected at sites up to 123 km apart. Viral gene flow occurs when cliff swallows move (bugs) between colony sites, and the genetic structure of BCRV may reflect the limited dispersal abilities of its insect vector.

Nucleotide sequence variation in salmonid alphaviruses from outbreaks of salmon pancreas disease and sleeping disease.

We compared 18 salmonid alphaviruses (SAV) including the reference F93-125 salmon pancreas disease virus (SPDV) and S49p sleeping disease virus (SDV) isolates by nucleotide sequence analyses of regions within the E1, nsP4 and nsP3 genes, and found these to comprise 3 distinct groups, which we have designated Subtypes 1, 2 and 3: Subtype 1, which comprised SAVs with sequences closely similar to the reference SPDV isolate, included SAVs from pancreas disease (PD) outbreaks in farmed salmon in Ireland and Scotland over a 10 yr period; viruses from recent outbreaks of sleeping disease (SD) in freshwater-reared trout farmed in England, Scotland and France were closely similar to and were grouped with the reference SDV isolate in Subtype 2; 3 viruses isolated from PD-affected salmon in Norway were genetically different from viruses belonging to Subtypes 1 and 2 and have been assigned to Subtype 3; 1 virus isolated from PD-affected salmon in the Western Isles, Scotland, in 2003 showed consistent nucleotide sequence differences from SAV Subtypes 1, 2 and 3, but was more closely related to the Subtype 1 SAVs. The occurrence of the different subtype SAVs appeared to have a geographical basis, which may prove useful in future molecular epidemiology studies of SAV-induced disease outbreaks.