Virulence of a chimeric recombinant infectious haematopoietic necrosis virus expressing the spring viraemia of carp virus glycoprotein in salmonid and cyprinid fish.

Infectious haematopoietic necrosis virus (IHNV) and spring viraemia of carp virus (SVCV) are both rhabdoviruses of fish, listed as notifiable disease agents by the World Organization for Animal Health. Recombinant rhabdoviruses with heterologous gene substitutions have been engineered to study genetic determinants and assess the potential of these recombinant viruses for vaccine development. A recombinant IHNV (rIHNV), containing the full-length genome of a European IHNV strain, was modified by deleting the glycoprotein (G) gene and replacing it with a European SVCV G-gene to make the rIHNV-Gsvcv. The chimeric rIHNV-Gsvcv level of virulence in rainbow trout, common carp and koi was assessed, and its ability to induce a protective immune response in surviving koi against wild-type SVCV infection was tested. The rIHNV-Gsvcv infection of trout led to high mortality, ranging from 78% to 92.5%, after immersion. In contrast, no deaths occurred in juvenile common carp after infection with rIHNV-Gsvcv by either immersion or intraperitoneal (IP) injection. Similarly, koi infected with rIHNV-Gsvcv via IP injection had little to no mortality (≤9%). Koi that survived initial infection with a high dose of recombinant virus rIHNV-Gsvcv were protected against a virulent SVCV challenge resulting in a high relative per cent survival of 82.5%.

Comparative susceptibility among three stocks of yellow perch, Perca flavescens (Mitchill), to viral haemorrhagic septicaemia virus strain IVb from the Great Lakes.

The Great Lakes strain of viral haemorrhagic septicaemia virus IVb (VHSV-IVb) is capable of infecting a wide number of naive species and has been associated with large fish kills in the Midwestern United States since its discovery in 2005. The yellow perch, Perca flavescens (Mitchill), a freshwater species commonly found throughout inland waters of the United States and prized for its high value in sport and commercial fisheries, is a species documented in several fish kills affiliated with VHS. In the present study, differences in survival after infection with VHSV IVb were observed among juvenile fish from three yellow perch broodstocks that were originally derived from distinct wild populations, suggesting innate differences in susceptibility due to genetic variance. While all three stocks were susceptible upon waterborne exposure to VHS virus infection, fish derived from the Midwest (Lake Winnebago, WI) showed significantly lower cumulative % survival compared with two perch stocks derived from the East Coast (Perquimans River, NC and Choptank River, MD) of the United States. However, despite differences in apparent susceptibility, clinical signs did not vary between stocks and included moderate-to-severe haemorrhages at the pelvic and pectoral fin bases and exophthalmia. After the 28-day challenge was complete, VHS virus was analysed in subsets of whole fish that had either survived or succumbed to the infection using both plaque assay and quantitative PCR methodologies. A direct correlation was identified between the two methods, suggesting the potential for both methods to be used to detect virus in a research setting.

Development of an aquatic pathogen database (AquaPathogen X) and its utilization in tracking emerging fish virus pathogens in North America.

The AquaPathogen X database is a template for recording information on individual isolates of aquatic pathogens and is freely available for download (http://wfrc.usgs.gov). This database can accommodate the nucleotide sequence data generated in molecular epidemiological studies along with the myriad of abiotic and biotic traits associated with isolates of various pathogens (e.g. viruses, parasites and bacteria) from multiple aquatic animal host species (e.g. fish, shellfish and shrimp). The cataloguing of isolates from different aquatic pathogens simultaneously is a unique feature to the AquaPathogen X database, which can be used in surveillance of emerging aquatic animal diseases and elucidation of key risk factors associated with pathogen incursions into new water systems. An application of the template database that stores the epidemiological profiles of fish virus isolates, called Fish ViroTrak, was also developed. Exported records for two aquatic rhabdovirus species emerging in North America were used in the implementation of two separate web-accessible databases: the Molecular Epidemiology of Aquatic Pathogens infectious haematopoietic necrosis virus (MEAP-IHNV) database (http://gis.nacse.org/ihnv/) released in 2006 and the MEAP- viral haemorrhagic septicaemia virus (http://gis.nacse.org/vhsv/) database released in 2010.

DNA vaccine protects ornamental koi (Cyprinus carpio koi) against North American spring viremia of carp virus.

The emergence of spring viremia of carp virus (SVCV) in the United States constitutes a potentially serious alien pathogen threat to susceptible fish stocks in North America. A DNA vaccine with an SVCV glycoprotein (G) gene from a North American isolate was constructed. In order to test the vaccine a challenge model utilizing a specific pathogen-free domestic koi stock and a cold water stress treatment was also developed. We have conducted four trial studies demonstrating that the pSGnc DNA vaccine provided protection in vaccinated fish against challenge at low, moderate, and high virus doses of the homologous virus. The protection was significant (p < 0.05) as compared to fish receiving a mock vaccine construct containing a luciferase reporter gene and to non-vaccinated controls in fish ranging in age from 3 to 14 months. In all trials, the SVCV-G DNA immunized fish were challenged 28-days post-vaccination (546 degree-days) and experienced low mortalities varying from 10 to 50% with relative percent survivals ranging from 50 to 88%. The non-vaccinated controls and mock construct vaccinated fish encountered high cumulative percent mortalities ranging from 70 to 100%. This is the first report of a SVCV DNA vaccine being tested successfully in koi. These experiments prove that the SVCV DNA (pSGnc) vaccine can elicit specific reproducible protection and validates its potential use as a prophylactic vaccine in koi and other vulnerable North American fish stocks.

Detection of spring viraemia of carp virus (SVCV) by loop-mediated isothermal amplification (LAMP) in koi carp, Cyprinus carpio L.

Spring viraemia of carp virus (SVCV) is a rhabdovirus associated with systemic illness and mortality in cyprinids. Several diagnostic tests are available for detection of SVCV. However, most of these tests are time consuming and are not well adapted for field-based diagnostics. In this study, a diagnostic tool for SVCV detection based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) has been developed. Based on the nucleotide sequence of the glycoprotein (G) gene of SVCV North Carolina (NC) isolate, four sets (each set containing two outer and two inner) of primers were designed. Temperature and time conditions were optimized to 65 degrees C and 60 min, respectively, for LAMP and RT-LAMP using one primer set. In vitro specificity was evaluated using four different strains of fish rhabdoviruses and RT-LAMP was found to be specific to SVCV. Serial dilutions of SVCV NC isolate was used to evaluate the in vitro sensitivity of RT-LAMP. Sensitivity of the assays was similar to RT-PCR and detected SVCV even at the lowest dilution of 10(1) TCID50 mL(-1). The ability of RT-LAMP to detect SVCV from infected carp was also tested and the assay detected SVCV from all infected fish. The isothermal temperature requirements, high specificity and sensitivity, and short incubation time of the RT-LAMP assay make it an excellent choice as a field diagnostic test for SVCV.

Genetic diversity and epidemiology of infectious hematopoietic necrosis virus in Alaska.

Forty-two infectious hematopoietic necrosis virus (IHNV) isolates from Alaska were analyzed using the ribonuclease protection assay (RPA) and nucleotide sequencing. RPA analyses, utilizing 4 probes, N5, N3 (N gene), GF (G gene), and NV (NV gene), determined that the haplotypes of all 3 genes demonstrated a consistent spatial pattern. Virus isolates belonging to the most common haplotype groups were distributed throughout Alaska, whereas isolates in small haplotype groups were obtained from only 1 site (hatchery, lake, etc.). The temporal pattern of the GF haplotypes suggested a 'genetic acclimation' of the G gene, possibly due to positive selection on the glycoprotein. A pairwise comparison of the sequence data determined that the maximum nucleotide diversity of the isolates was 2.75% (10 mismatches) for the NV gene, and 1.99% (6 mismatches) for a 301 base pair region of the G gene, indicating that the genetic diversity of IHNV within Alaska is notably lower than in the more southern portions of the IHNV North American range. Phylogenetic analysis of representative Alaskan sequences and sequences of 12 previously characterized IHNV strains from Washington, Oregon, Idaho, California (USA) and British Columbia (Canada) distinguished the isolates into clusters that correlated with geographic origin and indicated that the Alaskan and British Columbia isolates may have a common viral ancestral lineage. Comparisons of multiple isolates from the same site provided epidemiological insights into viral transmission patterns and indicated that viral evolution, viral introduction, and genetic stasis were the mechanisms involved with IHN virus population dynamics in Alaska. The examples of genetic stasis and the overall low sequence heterogeneity of the Alaskan isolates suggested that they are evolutionarily constrained. This study establishes a baseline of genetic fingerprint patterns and sequence groups representing the genetic diversity of Alaskan IHNV isolates. This information could be used to determine the source of an IHN outbreak and to facilitate decisions in fisheries management of Alaskan salmonid stocks.

Immune response to synthetic peptides representing antigenic sites on the glycoprotein of infectious hematopoietic necrosis virus.

Monoclonal antibodies against infectious hematopoietic necrosis virus have been used to react with recombinant expression products in immunoblots and to select neutralization-resistant mutants for sequence analysis. These strategies identified neutralizing and non-neutralizing antigenic sites on the viral glycoprotein. Synthetic peptides based upon the amino acid sequences of these antigenic sites were synthesized and were injected together with an adjuvant into rainbow trout. The constructs generally failed to stimulate neutralizing antibodies in the fish. These results indicate that we need to understand more about the ability of peptide antigens to stimulate fish immune systems.

Comparison of the polymerases (L genes) of spring viremia of carp virus and infectious hematopoietic necrosis virus.

We have determined the partial nucleotide sequences of the polymerase genes of the fish rhabdoviruses, spring viremia of carp virus (SVCV) and infectious hematopoietic necrosis virus (IHNV). At this point we have deduced the amino acid sequences and analysed the first 1,400 amino acids comprising two thirds of the polymerase genes of SVCV and IHNV. We have compared sequence similarities of SVCV and IHNV polymerases with other rhabdovirus and paramyxovirus polymerases. The SVCV polymerase showed the closest relationship with the vesicular stomatitis virus polymerases and also shared significant sequence identity with the polymerase of rabies virus. Other rhabdovirus and paramyxovirus polymerases showed lower sequences identities with the SVCV polymerase. The IHNV polymerase shared a relatively low amino acid sequence identity with the rabies virus polymerase, and similar low identities with other rhabdovirus and paramyxovirus polymerases. Several domains of various lengths were conserved in the virus polymerases included in this study. These domains were less conserved in the IHNV polymerase than in the SVCV polymerase, and some of the domains present in the other polymerases were not identified in the IHNV. These preliminary results indicate that SVCV is closely related to mammalian vesiculoviruses and that IHNV may be only distantly related to mammalian lyssa and vesiculotype rhabdoviruses.