This study presents the discovery and characterization of a novel picornavirus, human klassevirus 1. Klassevirus has a typical picornavirus organization with a ~700–800 bp 5' UTR, long open reading frame and, ~100 bp 3' UTR. The phylogenetic relationship of the new genus to other picornaviruses by amino amino acid sequence is shown in Figure . Given that the klassevirus genome possesses <40% amino acid identity in the P1 and P2 regions and <50% amino acid in the P3 region to the nearest picornavirus, this strain qualifies for designation as a new picornavirus genus, as per ICTV standards [12
Phylogenetic tree of klassevirus genome versus strains of other picornavirus genomes from genera based on coding region amino acid identity using clustalw.
Similar to cosavirus, this virus was identified through deep sequencing of stool, a strategy to identify novel viruses that are too divergent to be identified by other methods. Without filtering or selecting for viral particles, we were able to obtain sequence for 75% of the klassevirus genome based on TBLASTX against Aichi virus. Aligning all the pyrosequencing reads to the complete recovered genome of klassevirus indicated that 95% of the viral genome could be identified from the deep sequencing run (Figure ). This indicates that deep sequencing is a feasible strategy for rapidly identifying entire genomes of novel viruses.
Unlike previously identified kobuviruses, the first 140 nt of human klassevirus 1 is highly divergent. Published studies of Aichi virus suggests the first three stem-loop structures are required for positive and negative strand replication as well as encapsidation [7
]. The three known kobuviruses share a very high degree of homology in the first 50 bp and all have the three stem-loop structures with pseudoknot originally described in Aichi virus [4
]. Multiple attempts were made using 5' RACE to detect the conserved elements at the 5' end of known kobuvirus genomes and all failed. Similar sequence was recovered from both cases of human klassevirus 1 infection and RNAse protection demonstrated that the divergent 5' UTR sequence was part of the klassevirus genome and not an artifact of PCR amplification. We cannot rule out the existence of further 5' nucleotides beyond our current 5' end.
Despite the sequence divergence at the 5' end of its genome compared to known kobuviruses, human klassevirus 1 contains two stem-loops and a pseudoknot structure within the first 140 bp of its genome. Human klassevirus 1 also shares a high degree of sequence identity with Aichi virus throughout the remainder of the 5' UTR, indicating that IRES structure and function is likely preserved between the two viruses. This is especially interesting when compared to porcine kobuvirus which shares the conserved first 50 bp to the kobuvirus 5' UTR but has a hepacivirus/pestivirus-like type IV IRES [4
]. Though the exact secondary structure of the Aichi virus and bovine kobuvirus IRES are not known, it has been suggested that they contain type II IRES based on the position of the initial start codon of the polyprotein relative to the upstream polypyrimidine tract [2
]. The sequence of human klassevirus 1 3' UTR demonstrated almost no homology to other kobuvirus 3' UTR sequences or any other sequence in GenBank.
Although it remains to be determined whether human klassevirus 1 causes bona fide human infection, the data are suggestive. Screening using a newly developed PCR primer pair designed to amplify any klassevirus or kobuvirus found klassevirus only in two young children from the same family. The virus was present in relatively high copy number in both samples, suggesting that replication occurs in the gut and that human klassevirus 1 is not merely a passenger virus. However, both infants were asymptomatic at the time virus was present in their stool. The low prevalence rate is akin to that of Aichi virus, which is a rare known cause human gastroenteritis. Bovine and porcine kobuvirus, on the other hand, have both been found in healthy stool and bovine kobuvirus has been found in the serum of infected cattle [13
]. It remains to be determined whether klasseviruses are present in other human tissues or animal hosts.
Future studies to determine a possible link to disease in humans and any unique characteristics of the viral life cycle will be required. Viral culture on human cell lines, especially those from the gastrointestinal tract, could be suggestive that the virus is competent to replicate in human cells and that humans could be a bona fide host of klassevirus. Culture would also help elucidate the importance of different secondary structures in the divergent 5' UTR as well as determine cleavage sites of the polyprotein. Further epidemiological screening and serological assays will be necessary to understand the diversity within this possible genus, the prevalence of klassevirus, and the average age of those infected. Notably, both of the cases in this study were 11 months old, which is approximately the age at which maternal antibodies decline.