Cosavirus is a proposed new genus in the family Picornaviridae
) originally identified in 2008 in the feces of South Asian children with non-polio acute flaccid paralysis (AFP) 
. HCoSV were found at high prevalence in feces of both healthy (44%) and paralyzed (49%) children from Pakistan 
. Cosavirus was also detected in an Australian child with acute diarrhea 
and in Chinese children with (3.2%) and without (1.6%) diarrhea 
. Analysis of untreated sewage water also showed cosaviruses to be present in the United States 
. Low viral loads of cosavirus were also recently reported in the feces of healthy Brazilian children from a community child-care center (49% in 2008 and 6.5% in 2011) and with gastroenteritis in a pediatrics department (3.6%) 
. A single case of cosavirus infection was also reported in an adult with diarrhea in Thailand 
Cosaviruses, whose closest picornavirus relatives are cardioviruses and senecavirus have been tentatively classified into four distinct species labeled HCoSV-A to -D 
. A fifth species (HCoSV-E) has also been reported in Australia 
The wide genetic diversity of this recently characterized viral genus complicates disease association studies since different viral species and serotypes may be expected (as is the case for other picornaviruses such as the extensively studied enteroviruses) 
to have very different clinical impact on infected subjects. Here we extend our knowledge of the genetic diversity of human cosaviruses causing a highly common enteric infection of children in developing countries 
Biological samples and methods
Stool samples used for cosavirus sequencing were collected by the WHO Collaborating Centers for Poliovirus and Enterovirus Surveillance in Pakistan (n
2), Nigeria (n
26), Tunisia (n
5) and from the Armed Forces Research Institute of Medical Sciences, Enteric disease Department in Bangkok for Nepalese stool samples (n
10). Feces samples from WHO collaborating group were from children under 15 years of age with acute flaccid paralysis or from healthy contacts of such patients. Nepalese samples were from adults with unexplained gastroenteritis and healthy adults.
cDNA were generated with Superscript III (Invitrogen) and random primers. RT-PCR reactions were performed with LATaq
(Takara) to bridge pairs of separated cosavirus sequences previously derived by viral metagenomics 
. The 5′ and 3′ RACE was used to extend sequences towards the viral genome extremities. PCR products were directly sequenced by primer walking.
Initial cosavirus detection was done using 5′UTR RT-nested PCR on nucleic acids extracted from feces as previously described 
. The capsid sequences were generated by RT nested PCR using primers over the most conserved nucleotide regions. Upstream primers were positioned over VP3 and downstream primers were positioned over the 3′ end of VP1 as the immediate downstream region was too highly variable for consensus primer design. Primers for the first PCR round were VP1-INO-F1 (GAICARGCIATGATGGGIAC) and VP1-INO-R2 (GCIGGICCIGGRTTKGWYTC) in standard PCR conditions with first 15 cycles annealing at 60°C to 45°C (1°C decrease every cycle) followed by 25 cycles annealing at 54°C. Second PCR round used PCR primers VP1-INO-F1-2 (GCCATGATGGGIACITWYDCIATITGGGA) and VP1-INO-R3 (TARTCIGGRTAICCRTCRAA) in standard PCR conditions with first 10 cycles annealing at 60°C to 50°C (1°C decrease every cycle) followed by 30 cycles annealing at 50°C. I is for inosine, mixtures of bases are from the IUB nucleotide codes. A 904 bases amplicon was generated (from nucleotide position 2628 to 3531 on cosavirus reference genome HCoSV-A1, accession number NC_012800.1) and directly sequenced. Protein sequences aligned for phylogenetic analysis ranged in size from 272 to 281 amino acids spanning a portion of VP3 (VP3 amino acid 167 to 233 and VP1 (VP1 amino acid 1 to 206) in reference genome HCoSV-A1. The VP1 segment is 87 amino acids short of its carboxyl termini. We refer to these sequences as VP1*. Amplification products were extracted by QIAquick Gel Extraction kit (QIAGEN) and were directly sequenced by primer walking. Confirmation of the recombination site in NG385 was performed by direct RT-PCR and re-sequencing over the recombination point.
Sequence analysis and recombination detection
DNA fragments were assembled into genome using Sequencher 5.0 program (Genecodes Corp.). Sequence alignments were constructed by MUSCLE 
with a maximum of 64 iterations. Phylogenetic trees was generated using Maximum likelihood analysis, WAG substitution model and Gamma distribution estimated in Mega 5.0 
. Identity plot analyses were performed in BioEdit.
Similarity plots among the aligned nucleotide sequences were generated using SimPlot, version 3.5.1. 
. The level of similarity in window of 200 bp was calculated by the Kimura two-parameter method. To detect potential recombination aligned sequences were subsequently analyzed by using the Bootscanning method; the neighbor-joining algorithm was run with 100 pseudoreplicates implemented in SimPlot.
These studies were approved by the UCSF Committee on Human Research. Informed consent was not required as samples were pre-existing and anonymized.