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The genetic distance between the two viruses was confirmed by phylogenetic analysis based on complete genome sequences (Figure 1).

These analyses indicate that Cy CV-VN and Cy CV-VS5700009 represent two distinct cycloviral species..

However, it cannot be ruled out that related cycloviruses (including Cy CV-VS5700009) may have gone undetected since our PCR method was designed for specific detection of Cy CV-VN.

As part of standard operating procedures applied in our laboratory, CSF samples were spiked with an optimal amount of phocid herpesvirus and equine arteritis virus as (reverse transcription) PCR internal controls, and were then subjected to an automatic extraction procedure with use of the Viral NA Small Volume Kit (Roche applied sciences, UK) and a Mag NA Pure 96 system (Roche applied sciences). Sequence comparison and phylogenetic analysis between Cy CV-VN and another novel cyclovirus recently identified in CSF from Malawian patients indicated that these represent distinct cycloviral species, albeit phylogenetically closely related. The data suggest that Cy CV-VN has a limited geographic distribution within southern and central Vietnam. We previously reported a prevalence of 4% (28/644) among children and adults with confirmed or suspected CNS infections in overlapping time periods with the current study, hence failure to detect Cy CV-VN in this study is unlikely due to sample size restrictions or temporal bias. This is supported by observed statistically significant differences in prevalence (Table 1). Pairwise comparisons showed that the degree of sequence similarity between Cy CV-VN and Cy CV-VS5700009 was 60% at the nucleotide level of the complete genome sequence and 36% and 76% at the amino acid level of the capsid protein and replication association proteins, respectively.

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