Introduction
Cyprinid herpesvirus 3 (CyHV-3) is the archetype of fish alloherpesviruses. It causes severe economic loss within the carp culture industry worldwide. Genomic and biological comparisons of CyHV-3 strains have revealed a negative correlation among strains, suggesting the existence of genovariants conferring advantages in vitro but reduced fitness in vivo, and vice versa. We identified such genovariants of the CyHV-3 ORF131 gene. This gene is essential for viral growth in cell culture and encodes a 429 amino acid type 1 membrane protein. We demonstrated by mutagenesis that the genetic determinant of the phenotypic trait related to ORF131 depends on a single nucleotide polymorphism (SNP) (C225791T mutation) that results in codon 183 encoding either an alanine (183A) or a threonine (183T) residue. Understanding the key factors that determine how purifying (negative) selection operates on herpesvirus genomes would provide useful insights into the evolution of these viruses. In the present study, pairs of viruses differing only by the C225791T SNP were generated and compared for fitness in vitro and in vivo by infection with single viruses or co-infection with both viruses. This study illustrates how the host_virus interactions and the fundamental biological properties of some viruses and their hosts may have a profound impact on the degree of diversity that arises within viral populations.
Materials and methods
Common carp brain (CCB) cells were cultured. A total of seven CyHV-3 strains from various geographic origins were used and indirect immunofluorescence staining was done. Multiple DNA sequence alignments were made using MAFFT online version 7 and then processed using MEGA X software. CyHV-3 strain FL was isolated in Belgium from a fish that died from CyHV-3 infection and used to produce the FL BAC plasmid. FL EGFP rec ORF131-A (or) -T and FL mCherry rec ORF131-A (or) -T were produced by transfecting the FL BAC plasmid into CCB cells. The recombinant strain was cloned by three successive steps of plaque picking. All recombinant strains were confirmed by monitoring SacI restriction fragment length polymorphism (RFLP) and full-length genome sequencing. Growth curves, plaque size assay and syncytial plaque assay were investigated. Live cell images were collected using Incucyte to ensure that cells detected as double positive represented cells co-infected by EGFP and mCherry recombinants. In vivo infection was carried out either by immersion of uninfected fish in water containing virus or by cohabitation of uninfected fish with infected fish. The experiments, maintenance and care of fish complied with the guidelines of the European Convention CETS 123. Viral genome copies were quantified by real-time TaqMan qPCR. Fish were analyzed using an IVIS Spectrum in vivo imaging system.
Results
The results demonstrate that among the strains studied, FL, Cavoy and T strains were the most fit in cell culture but the least virulent in vivo. The opposite was the case for the M3, I, E and GZ11-SC strains. The strains with the 183A genovariant formed syncytia, whereas the strains with the 183T genovariant did not. This suggests that the ORF131 183A genovariant is responsible for syncytial plaque formation. The experiment involving simultaneous or delayed infection (one genovariant, then infection with another after) of monolayer revealed that the primary infection of monolayer reduces its ability to be superinfected by a second virus and that this phenomenon increases with the length of delay between the first and second infection. The ORF131 183T genovariant confers higher fitness in vivo than the 183A genovariant but in the case of in vitro it is vice-versa.
Conclusion
The present study indicates that CyHV-3 may have an intrinsic ability to actively contribute to the purifying selection of less fit variants by stimulation of superinfection inhibition at both the cellular and the host levels. However, more widely, our observations demonstrate how the fundamental biology of some (perhaps many) viruses and their hosts may have a profound impact on the degree of diversity that arises within viral populations.