using metagenomic deep sequencing , we analyzed fecal samples from 180 infants and children ages 7 days96 months ( mean 18.7 months ) in tunisia who had unexplained diarrhea that tested negative for rotavirus , norovirus , astrovirus , sapovirus , adenovirus types 40 and 41 , and aichi virus by reverse transcription pcr ( 7 ) .
the fecal supernatants were filtered through a 0.45-m filter ( millipore , darmstadt , germany ) to remove bacterium - sized particles , and the filtrates were digested with a mixture of dnases ( turbo dnase from ambion , carlsbad , ca , usa ; baseline - zero from epicenter , madison , wi , usa ; and benzonase from novagen , san diego , ca , usa ) and rnase ( fermentas , pittsburgh , pa , usa ) to digest unprotected nucleic acids . enriched viral nucleic acids ( rna and dna ) were then extracted and amplified by using scriptseq v2 rna - seq library preparation kit ( epicenter ) and analyzed in pools of 10 specimens in 2 illumina miseq run of 250-bp end reads , yielding 20,693,619 unique sequences .
we compared the illumina sequences with the genbank nonredundant protein databases using blastx ( http://blast.ncbi.nlm.nih.gov/blast.cgi ) . using a blastx e - score cutoff of 10
, we identified , in decreasing frequency , sequences related to the mammalian viruses : sapovirus ( 120,177 reads ) , anelloviridae ( 14,841 reads ) , parechovirus ( 10,557 reads ) , norovirus ( 4,551 reads ) , enterovirus ( 3,857 reads ) , circoviridae ( 2,127 reads ) , group a rotavirus ( 839 reads ) , adeno - associated virus ( 812 reads ) , picobirnavirus ( 274 reads ) , bufavirus ( 168 reads ) , wu polyomavirus ( 136 reads ) , bocavirus ( 62 reads ) , adenovirus ( 58 reads ) , papillomavirus ( 22 reads ) , cosavirus ( 20 reads ) , group c rotavirus ( 17 reads ) , human astrovirus 1 ( 14 reads ) , salivirus ( 4 reads ) , and aichi virus ( 2 reads ) .
one pool showed a single read encoding a parvovirus - like protein segment with high levels of genetic similarity ( blastx e - score of 5  10 ) to the nonstructural protein ( ns ) 1 of rat parvovirus ( genbank accession no .
was then identified by using pcr and underwent further deep sequencing as above , generating 11 more parovirus sequences .
no other eukaryotic viral sequences were identified from 260,000 unique sequence reads from this patient .
the near complete parvovirus genome was then acquired by filling genome gaps by pcr and amplifying 5 and 3 extremities using race ( rapid amplification of cdna ends , life technologies ) .
we named this virus tusavirus 1 for tunisian stool - associated parvovirus . 
  a nearly complete 4,424-bp genome ( tusavirus 1 , genbank accession no .
kj495710 ) was successfully acquired with partial 5 untranslated region ( 243 bp ) , complete ns1 open reading frame ( 625 aa ) , complete viral protein ( vp ) 1 open reading frame ( 715 aa ) , and a partial 3 untranslated region ( 68 bp ) .
tusavirus has a potential upstream start codon mss in a weaker kozak consensus sequence than maq ( figure , panel a ) , which we selected as the start codon . the walker loop gpattgks [ gxxxxgk(t / s ) ] , which is an atp- or gtp - binding motif , was found in the ns1 .
potential splicing signals to express vp1 were identified on the basis of alignments to other protoparvoviruses and classic rna splicing motifs ( figure , panel a ) .
the phospholipase a2 ( pla2 ) motif was identified in vp1 n - termini with expected calcium - binding site and catalytic residues .
the methionine codon of vp2 was located upstream of glycine - rich sequence ( gggaraggvg ) .
an unusual serine - rich sequence ( sssdsgpsss ) was also seen near vp1 n - termini .
the alignment of the pla2 regions of representatives of 5 protoparvovirus species show the calcium - binding region and catalytic residues in tusavirus .
pairwise sliding window of percentage nucleotide similarity of tusavirus aligns with the genetically closest kilham rat parvovirus .
b ) phylogenetic trees generated with nonstructural protein ( ns ) 1 and vp1 of tusavirus and of the 5 international committee on taxonomy of viruses  designated species in the protoparvovirus genus .
fpv , feline parvovirus ; mev , mink enteritis virus ; cpv , canine parvovirus ; rapv , raccoon parvovirus ; rpv1 , rat parvovirus 1 ; mvmp , minute virus of mice , prototype ; hapv , hamster parvovirus ; ppv - kr , porcine parvovirus kresse ; simian bupv , simian bufavirus ; bupv1 , bufavirus 1 ; bupv2 , bufavirus 2 ; amdv , aleutian mink disease virus ; gfav , gray fox amdovirus ; b19v - lali , human parvovirus b19-lali .
bootstrap values ( based on 100 replicates ) for each node are given if > 70% .
protein sequence alignments were made by using clustalx version 2.0.3 ( http://www.clustal.org ) with the default settings ; a phylogenetic tree with 100 bootstrap resamples of the alignment datasets was generated by using the neighbor - joining method based on the jones - taylor - thornton matrix - based model in mega5 ( http://www.megasoftware.net ) .
phylogenetic analysis showed that tusavirus 1 was distinct from known members of the protoparvovirus genus ( figure , panel b ) .
vp1 and vp2 shared identities of 39% and 37% , respectively , to those of kilham rat parvovirus .
according to the international committee on taxonomy of viruses , the members of the same parvovirus genus should share > 30% and members of the same species > 85% aa identity in ns1 .
the protoparvovirus genus currently comprises species infecting carnivores , rodents , pigs , and humans ( 1 ) .
tusavirus 1 is proposed as prototype for primate protoparvovirus 2 species that would join bufaviruses as human viruses in this genus .
we used a nested pcr targeting ns1 to determine the prevalence of this virus in the 180 tunisia diarrhea samples .
primers tusa - f1 ( 5-gaagaagctggaaactgtggtca-3 ) and tusa - r1 ( 5-ctcgtctttctcccaggcatct-3 ) were used for the first round of pcr , and primers tusa - f2 ( 5-attgctccaacaccagtcatca-3 ) and tusa - r2 ( 5-tctggtctggtccaatcttcttc-3 ) for the second round of pcr .
the pcr conditions were : 95c for 5 min , 35 cycles 95c for 30 s , 52c or 51c ( for the first or second round , respectively ) for 30 s , and 72c for 1 min , a final extension at 72c for 10 min .
no samples except the one initially detected by deep sequencing were pcr positive , yielding a low prevalence of 0.56% ( 1/180 ) in this tunisian population .
we detected fecal shedding of a previously uncharacterized parvovirus in a child with unexplained diarrhea .
the 18-month - old girl showed twice daily liquid and greenish feces over 3 days but no fever ( 37.4c ) , vomiting , or dehydration . to identify other viral infections in this patient ,
no other mammalian virus was detected , suggesting a possible role for tusavirus 1 in this patient s gastrointestinal illness , although the lack of testing for pathogenic bacteria and parasites does not enable us to exclude these alternative explanations .
control pcr studies of unexplained diarrhea and serologic tests , are needed to define the prevalence and disease association of this new parvovirus species in different age groups and populations .
 a divergent parvovirus genome was the only eukaryotic viral sequence detected in feces of a tunisian child with unexplained diarrhea . 
 tusavirus 1 shared 44% and 39% identity with the nonstructural protein 1 and viral protein 1 , respectively , of the closest genome , kilham rat parvovirus , indicating presence of a new human viral species in the protoparvovirus genus . 
The Study
Conclusions