Molecular Evolution and Phylogeography of Co-circulating IHNV and VHSV in Italy

Abstract

Infectious haematopoietic necrosis virus (IHNV) and viral haemorrhagic septicaemia virus (VHSV) are the most important viral pathogens impacting rainbow trout farming. These viruses are persistent in Italy, where they are responsible for severe disease outbreaks (epizootics) that affect the profitability of the trout industry. Despite the importance of IHNV and VHSV, little is known about their evolution at a local scale, although this is likely to be important for virus eradication and control. To address this issue we performed a detailed molecular evolutionary and epidemiological analysis of IHNV and VHSV in trout farms from northern Italy. Full-length glycoprotein gene sequences of a selection of VHSV (n = 108) and IHNV (n = 89) strains were obtained. This revealed that Italian VHSV strains belong to sublineages Ia1 and Ia2 of genotype Ia and are distributed into 7 genetic clusters. In contrast, all Italian IHNV isolates fell within genogroup E, for which only a single genetic cluster was identified. More striking was that IHNV has evolved more rapidly than VHSV (mean rates of 11 and 7.3 × 10(-4) nucleotide substitutions per site, per year, respectively), indicating that these viruses exhibit fundamentally different evolutionary dynamics. The time to the most recent common ancestor of both IHNV and VHSV was consistent with the first reports of these pathogens in Italy. By combining sequence data with epidemiological information it was possible to identify different patterns of virus spread among trout farms, in which adjacent facilities can be infected by either genetically similar or different viruses, and farms located in different water catchments can be infected by identical strains. Overall, these findings highlight the importance of combining molecular and epidemiological information to identify the determinants of IHN and VHS spread, and to provide data that is central to future surveillance strategies and possibly control.

Keywords: IHNV; VHSV; evolution; molecular epidemiology; phylogeny.

Figure 1

Sampling area. The infected farms studied here are located in the northeastern part of Italy, in Trentino Alto Adige, Veneto, and Friuli Venezia Giulia regions. Farms are represented by dots colored according to the disease etiology (blue: IHN; green: VHS; red: IHN + VHS).

Figure 2

ML phylogenetic tree of IHNV based on the complete G gene. The Italian strains are highlighted in yellow and labeled according to their water basin of origin as indicated in the “Water catchments” column. IHNV genotype subdivision is designated by vertical bars. The colored box represents the identified genetic group (A) within genogroup E. For the Italian strains under investigation, the water basin of origin has been indicated. The numbers at nodes represent bootstrap values (only values >70% are reported), while branch lengths are scaled according to the number of nucleotide substitutions per site. The scale bar is reported. The numbers within the red boxes represent the amino acid substitutions occurring along the branches. The tree is mid-point rooted for clarity only.

Figure 3

ML phylogenetic tree of VHSV based on the complete G gene. The Italian strains are highlighted in yellow and labeled according to their water basin of origin as indicated in the “Water catchments” column. VHSV genotype subdivision is designated by vertical bars. The colored boxes represent the 7 different genetic clusters (A,B,C,D,E,F,F1) identified within genotype Ia, sublineages Ia1 and Ia2. For the Italian strains under investigation, the water basin of origin has been indicated. The numbers at nodes represent bootstrap values (only values >70% are reported), while branch lengths are scaled according to the number of nucleotide substitutions per site. The scale bar is reported. The numbers within the red boxes represent the amino acid substitutions occurring along the branches. The tree is mid-point rooted for clarity only.

Figure 4

Examples of the proposed patterns of IHNV and VHSV spread in Italy. (i) neighboring farms located in the same water system experiencing disease outbreaks caused by genetically related strains (A,B); (ii) genetically diverse viruses in neighboring farms (C,D); and (iii) infection by identical viruses in farms with no connection via water (E). The time period considered is reported for each map. Infected trout farms are represented by dots colored according to the assignment of the IHNV and VHSV strains to the genetic groups identified in the phylogenetic analysis. Non-infected trout farms are designated in black. The number associated to each infected farm corresponds to the isolates reported in Table 2.