Vaccines against classical swine fever have proven very effective in protecting pigs from this deadly disease. However, little is known about how vaccination impacts the selective pressures acting on the classical swine fever virus (CSFV). Here we use high-throughput sequencing of viral genomes to investigate evolutionary changes in virus populations following the challenge of naïve and vaccinated pigs with the highly virulent CSFV strain “Koslov”. The challenge inoculum contained an ensemble of closely related viral sequences, with three major haplotypes being present, termed A, B, and C. After the challenge, the viral haplotype A was preferentially located within the tonsils of naïve animals but was highly prevalent in the sera of all vaccinated animals. We find that the viral population structure in naïve pigs after infection is very similar to that in the original inoculum. In contrast, the viral population in vaccinated pigs, which only underwent transient low-level viremia, displayed several distinct changes including the emergence of 16 unique non-synonymous single nucleotide polymorphisms (SNPs) that were not detectable in the challenge inoculum. Further analysis showed a significant loss of heterogeneity and an increasing positive selection acting on the virus populations in the vaccinated pigs. We conclude that vaccination imposes a strong selective pressure on viruses that subsequently replicate within the vaccinated animal.

Members of the ZFY and ZNF6 gene families have been cloned from species representing different taxa and different modes of sex determination. Comparisons of these genes show the ZFY-like and ZNF6 sequences to be strongly conserved across marsupials, birds, and lepidosaurians. Sequence analyzed by neighbor-joining indicated that both gene families are monophyletic with a high bootstrap value. Pairing of sequences from males and females of nonmammalian species showed there to be no significant difference between male and female sequences from a single species, consistent with autosomal locations. The molecular distances between murine Zfy-1, Zfy-2, and other ZFY-like sequences suggested that Zfy genes have undergone a period of rapid evolutionary change not seen in human ZFY.

Many reptiles do not have heteromorphic sex chromosomes and for these species sex is determined during embryogenesis by the temperature of egg incubation rather than at conception. The phenomenon of temperature-dependent sex determination (TSD) was discovered almost thirty years ago, but few advances have been made towards the elucidation of its mechanism. In the past few years substantial progress has been made in the understanding of the molecular basis of XY chromosomal (genetic) sex determination (GSD) through the discovery of SRY. It is now possible to start comparing TSD with GSD. TSD is found in some evolutionarily ancient vertebrates and has been postulated to be the ancestral process from which GSD has evolved. If this is true then the two mechanisms may share a common molecular basis. This paper details the current knowledge of TSD, our progress on the investigation of the involvement of SRY-type proteins, and finally presents some of the problems that need to be resolved to gain an understanding of the molecular basis of TSD.

Classical swine fever virus (CSFV) contains a specific motif within the E2 glycoprotein that varies between strains of different virulence. In the highly virulent CSFV Koslov, this motif comprises residues S763/L764 in the polyprotein. However, L763/P764 represent the predominant alleles in published CSFV genomes. In this study, changes were introduced into the CSFV Koslov (here called vKos_SL), to generate modified CSFVs with substitutions at residues 763/764 (vKos_LL, vKos_SP and vKos_LP). The properties of these mutant viruses, in comparison to vKos_SL, were determined in pigs. Each of the viruses was virulent and induced typical clinical signs of CSF but the vKos_LP produced them significantly earlier. Full-length CSFV cDNA amplicons (12.3kb) derived from sera of infected pigs were deep sequenced and cloned to reveal the individual haplotypes that contributed to the single nucleotide polymorphism (SNP) profiles observed in the virus population. The SNP profiles for vKos_SL and vKos_LL displayed low-level heterogeneity across the entire genome, whereas vKos_SP and vKos_LP displayed limited diversity with a few high frequency SNPs. This indicated that vKos_SL and vKos_LL exhibited a higher level of fitness in the host and more stability at the consensus level, whereas several consensus changes were observed in the vKos_SP and vKos_LP sequences, pointing to adaptation. For each virus, only a subset of the variants present within the virus inoculums were maintained in the infected pigs. No clear tissue dependent quasispecies differentiation occurred within inoculated pigs, however, clear evidence for transmission bottle-necks to contact animals was observed, with subsequent loss of sequence diversity. IMPORTANCE The surface exposed E2 protein of classical swine fever virus is required for its interaction with host cells. A short motif within this protein varies between strains of different virulence. The importance of two particular amino acid residues in determining the properties of a highly virulent strain of the virus has been analyzed. Each of the different viruses tested proved highly virulent but one of them produced earlier, but not more severe, disease. By analyzing the virus genomes present within infected pigs, it was found that the viruses which replicated within inoculated animals were only a subset of those within the virus inoculum. Furthermore, following contact transmission, it was shown that a very restricted set of viruses had transferred between animals. There were no significant differences in the virus populations present in various tissues of the infected animals. These results indicate mechanisms of virus population change during transmission between animals.

BackgroundDirect molecular cloning of full-length cDNAs derived from viral RNA is an approach to identify the individual viral genomes within a virus population. This enables characterization of distinct viral haplotypes present during infection.ResultsIn this study, we recover individual genomes of classical swine fever virus (CSFV), present in a pig infected with vKos that was rescued from a cDNA clone corresponding to the highly virulent CSFV Koslov strain. Full-length cDNA amplicons (ca. 12.3 kb) were made by long RT-PCR, using RNA extracted from serum, and inserted directly into a cloning vector prior to detailed characterization of the individual viral genome sequences. The amplicons used for cloning were deep sequenced, which revealed low level sequence variation (< 5%) scattered across the genome consistent with the clone-derived origin of vKos. Numerous full-length cDNA clones were generated using these amplicons and full-genome sequencing of individual cDNA clones revealed insights into the virus diversity and the haplotypes present during infection. Most cDNA clones were unique, containing several single-nucleotide polymorphisms, and phylogenetic reconstruction revealed a low degree of order.ConclusionsThis optimized methodology enables highly efficient construction of full-length cDNA clones corresponding to individual viral genomes present within RNA virus populations.Electronic supplementary materialThe online version of this article (10.1186/s12864-018-4971-8) contains supplementary material, which is available to authorized users.