Molecular characterization of the virulent infectious hematopoietic necrosis virus (IHNV) strain 220-90

Abstract

Background: Infectious hematopoietic necrosis virus (IHNV) is the type species of the genus Novirhabdovirus, within the family Rhabdoviridae, infecting several species of wild and hatchery reared salmonids. Similar to other rhabdoviruses, IHNV has a linear single-stranded, negative-sense RNA genome of approximately 11,000 nucleotides. The IHNV genome encodes six genes; the nucleocapsid, phosphoprotein, matrix protein, glycoprotein, non-virion protein and polymerase protein genes, respectively. This study describes molecular characterization of the virulent IHNV strain 220-90, belonging to the M genogroup, and its phylogenetic relationships with available sequences of IHNV isolates worldwide.

Results: The complete genomic sequence of IHNV strain 220-90 was determined from the DNA of six overlapping clones obtained by RT-PCR amplification of genomic RNA. The complete genome sequence of 220-90 comprises 11,133 nucleotides (GenBank GQ413939) with the gene order of 3'-N-P-M-G-NV-L-5'. These genes are separated by conserved gene junctions, with di-nucleotide gene spacers. An additional uracil nucleotide was found at the end of the 5'-trailer region, which was not reported before in other IHNV strains. The first 15 of the 16 nucleotides at the 3'- and 5'-termini of the genome are complementary, and the first 4 nucleotides at 3'-ends of the IHNV are identical to other novirhadoviruses. Sequence homology and phylogenetic analysis of the glycoprotein genes show that 220-90 strain is 97% identical to most of the IHNV strains. Comparison of the virulent 220-90 genomic sequences with less virulent WRAC isolate shows more than 300 nucleotides changes in the genome, which doesn't allow one to speculate putative residues involved in the virulence of IHNV.

Conclusion: We have molecularly characterized one of the well studied IHNV isolates, 220-90 of genogroup M, which is virulent for rainbow trout, and compared phylogenetic relationship with North American and other strains. Determination of the complete nucleotide sequence is essential for future studies on pathogenesis of IHNV using a reverse genetics approach and developing efficient control strategies.

Figure 1

Genetic map of the IHNV genome and cDNA clones used for sequence analysis. The location and relative size of the IHNV ORFs are shown; the numbers indicate the starts and ends of the respective ORFs. Six cDNA fragments (F1 to F6) were synthesized from the genomic RNA by RT-PCR. The primers used for RT-PCR fragments are shown at the end of each fragment. The RNA genome is 11,133 nucleotides long and contains a leader (L) and trailer (T) sequences at its 3'-end and 5'-end, respectively. The coding regions of N, P, M, G, NV and L genes are separated by intergenic sequences, which have gene-start and gene-end signals.

Figure 2

Analysis of the gene junctions and complementarities in the IHNV genome. A) Seven identified gene junctions of IHNV in the negative-sense of the genomic RNA are shown. 3'/N, junction of 3'-leader and nucleocapsid gene; N/P, junction of nucleocapsid and phosphoprotein gene; P/M, junction of phosphoprotein and matrix gene; M/G, junction of matrix and glycoprotein gene; G/NV, junction of glycoprotein and non-virion gene; NV/L, junction of non-virion and polymerase gene; L/5'-, junction of polymerase gene and 5' trailer. GE = Gene end; IG = Intergenic di-nucleotide; GS = Gene start. The stop codon of NV ORF is merged with gene end sequence and is shown in red box. B) Complementarities of the 3'- and 5'-ends of the IHNV genome. The first 15 of the 16 nucleotides at the 3'-end are complementary to the 5'-end nucleotides of genomic RNA.

Figure 3

Phylogenetic tree analysis of sequences of nucleocapsid (N), matrix (M), phosphoprotein (P), and non-virion protein (NV) of various IHNV strains. Information about the IHNV strains used in this analysis is described in additional file 1. IHNV 220-90 strain is marked with blue diamond. Phylogenetic tree analysis was conducted by neighbor-joining method using 1000 bootstrap replications. The scale at the bottom indicates the number of substitution events and bootstrap confidence values are shown at branch nodes.

Figure 4

Phylogenetic relationship of the full-length glycoprotein (G) sequences of 28 IHNV strains with IHNV 220-90. Genogroups are depicted by vertical lines, as described by [10]. Brackets indicate the three major genogroups, U, M and L. IHNV 220-90 (blue diamond) is grouped under M genogroup. Data of virus isolates used here are available in additional file 1. Phylogenetic tree analysis was conducted by neighbor-joining method using 1000 bootstrap replications. The scale at the bottom indicates the number of substitution events and bootstrap confidence values are shown at branch nodes.

Figure 5

Kozak sequence context of each gene of IHNV 220-90. Sequences shown here are positive-sense anti-genome. * Conserved adenosine (A) at position -3. ** Start codon (ATG)