Molecular Epidemiology of Novirhabdoviruses Emerging in Iranian Trout Farms

Abstract

Novirhabdoviruses cause large epizootics and economic losses of farmed trout. In this study, we surveyed Viral hemorrhagic septicemia virus and Infectious hematopoietic and necrosis virus (VHSV and IHNV) through both monitoring and investigation of clinical outbreaks reported by farmers in the regions with major rainbow trout production in Iran from 2015 to 2019. RT-PCR assays of the kidney samples and cell culture (EPC/FHM cells) samples confirmed the presence of the viruses, with 9 VHSV and 4 IHNV isolates, in both endemic and new areas of Iran. Sequence analysis of the G gene revealed that VHSV isolates belonged to genogroup Ia, and IHNV isolates were clustered into genogroup E, both typical for isolates from European countries. A haplotype analysis based on non-homologous amino acids of the G gene supports the emergence of two lineages of IHNV from clade 1 (E-1), as well as VHSV clade 2 (Ia-2) of the European genogroups, confirming that VHSV and IHNV isolates in Iran, have originated from Europe possibly via imported eggs.

Keywords: IHN; VHS; glycoprotein (G) gene; haplotype analysis; phylogeny; trout.

Figure 1

Cytopathic effect (CPE) caused by Viral hemorrhagic septicemia virus and Infectious hematopoietic and necrosis virus (VHSV and IHNV) in EPC and FHM cell lines at different days post-inoculations (dpi). The upper row shows control uninfected EPC cells (40×), IHNV, and VHSV infected EPC cells showing CPE at 5 dpi (100×). The bottom row shows FHM control cells (40×) and infected FHM cells with VHSV at 5 dpi (40× and 100×, respectively).

Figure 2

Phylogenetic analysis of Iranian isolate of VHSV based on the partial nucleotide sequences of G gene (513 bp). The phylogenetic trees were constructed using the Geneious Prime (Neighbor-joining with the Hasegawa-Kishino-Yano (HKY) model and 1000 bootstrap replicates). The Iranian isolates of VHSV (S.AV-IR-VHSV to S.AV-IR-VHSV8) were classified as an Ia genogroup (European origin). GenBank accession numbers are listed in Table 1.

Figure 3

Neighbor network of non-homologous amino acids of VHSV glycoprotein fragment (513 bp). Left panel genotype Ia sequences only. Right panel all genotype sequences as indicated in Figure 2. Green circles indicate Iranian sequences.

Figure 4

Phylogenetic analysis of Iranian isolate of IHNV based on the partial nucleotide sequences of G gene (693 bp). The phylogenetic trees were constructed using the Geneious Prime (Neighbor-joining with the HKY model and bootstrap replicates of 1000). The Iranian isolate of IHNV (S.AV-IR-IHNV1- S.AV-IR-IHNV4) was classified as a European (E) genogroup (GenBank accession numbers are listed in Table 1).

Figure 5

Neighbor network of non-homologous amino acids of IHNV glycoprotein fragment (303 bp mid G gene). IHNV sequences as indicated in Figure 4. Green circles indicate Iranian sequences.