Analysis of Virus Population Profiles within Pigs Infected with Virulent Classical Swine Fever Viruses: Evidence for Bottlenecks in Transmission but Absence of Tissue-Specific Virus Variants

Abstract

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.

A Deep Sequencing Strategy for Investigation of Virus Variants within African Swine Fever Virus-Infected Pigs

“…Hence, the properties of the minority variant virus can become the properties of the circulating virus, and the nature of the disease will reflect the properties of the new variant. Transmission of the virus from one host animal to another will often involve some sort of bottleneck event, and a minority variant can then become a major component within some of the newly infected animals even without a major selection advantage (as observed here and previously with classical swine fever virus (CSFV) [ 44 ]).…”

“…MiSeq reads were mapped to the ASFV/POL/2015/Podlaskie reference sequence using BWA-MEM [ 40 ], and aligned reads were filtered for mapping quality (mapq) 60; secondary and supplementary reads were removed using Samtools [ 41 ]. Variant calling and annotation was performed using a combination of Lo-Freq [ 42 ] and SnpEFF [ 43 ] as described previously [ 44 ]. Briefly, quality scores were recalibrated by Lo-Freq, and variants were filtered for a minimum coverage of 100, frequency above 2%, and strand-bias Phred Score below 12.…”

A Deep Sequencing Strategy for Investigation of Virus Variants Within African Swine Fever Virus-infected Pigs

“…MiSeq reads were mapped to the ASFV/POL/2015/Podlaskie reference sequence using BWA-MEM [40], and aligned reads were filtered for mapping quality (mapq) 60; secondary and supplementary reads were removed using Samtools [41]. Variant calling and annotation was performed using a combination of Lo-Freq [42] and SnpEFF [43] as described previously [44]. Briefly, quality scores were recalibrated by Lo-Freq, and variants were filtered for a minimum coverage of 100, frequency above 2%, and strand-bias Phred Score below 12.…”

Pestiviruses infection: Interferon-virus mutual regulation

“…The genome of Pestivirus viruses is approximately 12.3 kb, which can encode at least 11 viral proteins (Figure 1) (Neill et al, 2019;Johnston et al, 2020;Walz et al, 2020). N pro is a nonstructural protein unique to pestivirus with autoprotease activity and is often used for classifying pestiviruses.…”