Intra-Host Diversity of SARS-Cov-2 Should Not Be Neglected: Case of the State of Victoria, Australia

Since the identification of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as the etiological agent of the current COVID-19 pandemic, a rapid and massive effort has been made to obtain the genomic sequences of this virus to monitor (in near real time) the phylodynamic and diversity of this new pathogen. However, less attention has been given to the assessment of intra-host diversity. RNA viruses such as SARS-CoV-2 inhabit the host as a population of variants called quasispecies. We studied the quasispecies diversity in four of the main SARS-CoV-2 genes (ORF1a, ORF1b, S and N genes), using a dataset consisting of 210 next-generation sequencing (NGS) samples collected between January and early April of 2020 in the State of Victoria, Australia. We found evidence of quasispecies diversity in 68% of the samples, 76% of which was nonsynonymous variants with a higher density in the spike (S) glycoprotein and ORF1a genes. About one-third of the nonsynonymous intra-host variants were shared among the samples, suggesting host-to-host transmission. Quasispecies diversity changed over time. Phylogenetic analysis showed that some of the intra-host single-nucleotide variants (iSNVs) were restricted to specific lineages, highlighting their potential importance in the epidemiology of this virus. A greater effort must be made to determine the magnitude of the genetic bottleneck during transmission and the epidemiological and/or evolutionary factors that may play a role in the changes in the diversity of quasispecies over time.

Bovine viral diarrhoea virus loses quasispecies diversity rapidly in culture

Bovine viral diarrhoea (BVD) is an important disease of cattle with significant impacts on animal health and welfare. The wide host range of the causative pestiviruses may lead to formation of virus reservoirs in other ruminant or wildlife species, presenting a concern for the long-term success of BVD eradication campaigns. It is likely that the quasispecies nature of these RNA viruses contributes to their interspecies transmission by providing genetic plasticity. Understanding the spectrum of sequence variants present in BVD persistently infected (PI) animals is therefore essential for studies of virus transmission. To analyse quasispecies diversity without amplification bias, we extracted viral RNA from serum of a PI cow, and from cell culture fluid after three passages of the same virus in culture, to produce cDNA without amplification. Sequencing of this material using Illumina 250bp paired-read technology produced full-length virus consensus sequences from both sources and demonstrated the quasispecies diversity of this Pestivirus A type 1a field strain within serum and after culture. We report the distribution and diversity of over 800 single nucleotide polymorphisms and provide evidence for a loss of diversity after only three passages in cell culture, implying that cultured viruses cannot be used to understand quasispecies diversity and may not provide reliable molecular markers for source tracing or transmission studies.Additionally, both serum and cultured viruses could be sequenced as a set of 25 overlapping PCR amplicons that demonstrated the same consensus sequences and the presence of many of the same quasispecies variants. The observation that aspects of the quasispecies structure revealed by massively parallel sequencing are also detected after PCR and Sanger sequencing suggests that this approach may be useful for small or difficult to analyse samples.Impact statementBovine viral diarrhoea viruses are globally important cattle pathogens, which impact performance due to acute infection and BVD-induced immunosuppression. Eradication of BVD in cattle is widely pursued but is hampered by the production of persistently infected (PI) calves – the offspring of cows infected in early pregnancy – which shed virus constantly and drive BVD spread. Genetic variation in BVD viruses is an important feature of their biology, allowing them to adapt to changing conditions and to infect different hosts. Inaccurate virus replication produces a population of viruses with slightly different sequences, a quasispecies, some of which may grow better in other hosts or in culture. Analysing virus sequence variation may help us understand how the virus evolves within and between its hosts. In this paper we show that a BVD virus strain loses quasispecies diversity quickly when cultured and that these changes can be detected even in small diagnostic samples, implying that cultured viruses do not perfectly represent the field strains they were isolated from and therefore may not provide reliable molecular markers for source tracing or transmission studies.Data SummaryPestivirus A genome sequences used in this article are as follows:Sequence data associated with this manuscript has been submitted to the European Nucleotide Archive (www.ebi.ac.uk/ena/) with accession numbers as follows:Consensus genome sequences:MRI103 serum NGS: LR699799MRI103 culture NGS: LR699800MRI103 serum Sanger: LR699801MRI103 culture P3 Sanger: LR699802MRI103 culture P5 Sanger: LR699803NGS raw dataSerum dataset: ERR3624580Culture dataset: ERR3624581