the picornavirus 5 untranslated region ( 5 utr ) has proven the most suitable genomic target for molecular detection ( 8,9 ) . at the time of this study ,
only 5 aiv complete genomes were available in genbank , complicating selection of reliable oligonucleotides for universal detection .
a nested rt - pcr encompassing the aiv 5 utr was developed ( table 1 ) and used for screening of stool samples collected in northern germany from outpatients with gastroenteritis .
the first subcohort of this collection consisted of 499 patients with gastroenteritis ; samples were collected evenly from january through december 2004 in a prospective study on acute community - acquired diarrhea by 47 general practitioners in bremen , northern germany ( 10 ) .
the second subcohort consisted of 39 control patients without symptoms of gastroenteritis seen by the same physicians .
the third subcohort consisted of 118 patients with diarrhea linked to outbreak scenarios involving canteen food ( n = 36 ) , kindergartens ( n = 54 ) , or retirement homes ( n = 28 ) .
* aiv , aichi virus ; i d , identification ; rt - pcr , reverse transcription pcr ; utr , untranslated region . 
25-l qiagen onestep rt - pcr reactions as described by the manufacturer ( qiagen , hilden , germany ) used 400 nmol / l each of 1st - round primers , 1 g bovine serum albumin , and 5 l rna extract .
amplification involved 30 min at 50c ; 15 min at 95c ; 10 cycles of 20 s at 94c , 30 s starting at 60c with a decrease of 1c per cycle , and 50 s at 72c ; and 40 cycles of 20 s at 95c , 30 s at 54c , and 50 s at 72c ; and a final elongation step of 5 min at 72c .  
50-l platinum taq reactions as described by the manufacturer ( invitrogen , karlsruhe , germany ) used 1 l of 1st - round pcr product , 2.5 mmol / l mgcl2 , and 400 nmol / l each of 2nd - round primers . amplification involved 3 min at 94c and 45 cycles of 20 s at 94c , 30 s at 60c , and 40 s at 72c . 
25-l qiagen onestep rt - pcr reactions used 3 l of rna extract , 600 nmol / l of each primer , and 320 nmol / l of the probe .
( darmstadt , german ) 7700 sds instrument involved the following steps : 55c for 15 min , 95c for 15 min , and 45 cycles of 95c for 15 s and 58c for 30 s ( fluorescence measured ) .
we purified viral rna from stool samples by using the qiagen viral rna and dna stool mini kits ( qiagen , hilden , germany ) as described ( 8) ; 9 samples were positive for aiv by nested rt - pcr .
the 5 utr sequences of these viruses were determined and deposited in genbank ( accession nos .
gq927704gq927712 ) . with additionally available sequence data , real - time rt - pcr targeting conserved regions of the viral 5 utr was developed ( table 1 ) .
assay sensitivity was determined to be 1.5 copies per reaction by using photometrically quantified in vitro crna transcripts , as described ( 9 ) .
retesting of all samples with this highly sensitive assay increased the aiv detection rate , yielding 10 positive samples .
all case - patients were part of the subcohort of symptomatic outpatients seen by general practitioners ( figure 1 ) . in this cohort ,
no patients from the control group ( n = 39 ) or from foodborne outbreaks ( n = 118 ) had positive test results for aiv . however , this difference was not statistically significant for any group comparison ( fisher exact test , 2-tailed p>0.05 for all ) . as shown in table 2 ,
no other virus commonly associated with diarrhea was detected in any patient , including norovirus , rotavirus , adenovirus , astrovirus , parechovirus , or enterovirus .
a bacterial cause of disease was ruled out by using standard culture methods ( 10 ) .
no clear association with a foodborne etiology , sociologic risk factor ( including travel history ) , or contact with animals was observed .
as shown in table 2 , high rna copy numbers of up to 1.32  10 per gram of stool ( mean 1.32  10 , median = 1.82  10 ) were found in patients with positive test results .
age distribution of cohorts tested in study of aichi virus in patients with acute diarrhea , germany .
a ) patients from food - associated diarrhea outbreaks ( kindergartens , canteens , or retirement homes ) ; b ) outpatients seen for gastroenteritis by general practitioners ; c ) nongastroenteritis control patients for the outpatient study cohort .
arrows indicate patients who had positive test results for aichi virus by real - time reverse transcription pcr . * no patients had fever ( > 38.5c ) or co - infections .
all samples were tested for norovirus , rotavirus , adenovirus , astrovirus , parechovirus , enterovirus , and common bacterial pathogens .
although samples had been collected throughout 2004 , all aiv - positive cases occurred during 8 weeks from october to december and originated from a geographically restricted area within the city of bremen . to verify if this temporal and geographic accumulation of cases represented a point - source outbreak , we amplified and sequenced the entire viral protein ( vp ) 1 gene , which is commonly used for picornavirus typing , from 9/10 samples ( genbank accession nos .
failure of vp1 amplification in sample d / vi2591 was probably caused by low virus concentration .
as shown in figure 2 , a total of 8 samples formed a distinct phylogenetic cluster within aiv genotype b. the first 3 strains , sampled from october 19 through november 10 ( table 2 ) , were almost identical in vp1 , with only 1 strain ( d / vi2244 ) diverging by 2 synonymous substitutions .
all other samples ( november 16december 7 ) showed a vp1 nucleotide diversity of up to 0.8% ( 27/864 nt ) and an amino acid diversity of up to 1.4% ( 14/288 residues ) in comparison to the 3 initially sampled specimens and to each other .
strain d / vi2287 , sampled november 8 , belonged to aiv genotype a , with nucleotide and amino acid differences of up to 13.2% and 5.4% from the genotype b strains .
neighbor - joining phylogeny of aichi virus ( aiv ) viral protein 1 gene of strains from study of aiv in patients with acute diarrhea ( boldface ) , germany , compared with strains from genbank .
the tree was generated by using mega4 ( www.megasoftware.net ) using the maximum - composite likelihood nucleotide substitution model and complete deletion option .
porcine kobuvirus was used as an outgroup ( branch truncated as indicated by slashed lines ) .
for many of the recently described picornaviruses , human pathogenicity is still under study . with the advent of metagenomics
although it is generally difficult to generate sufficiently large and appropriately sampled control groups in studies on respiratory and enteric diseases , quantitative data on virus shedding and proof of monocausality can contribute to confirm the link of novel viruses to human disease .
for aiv , the high concentrations found in several samples in this study provide support for viral replication in humans . however , virus shedding appeared unrelated to the severity of symptoms , and clinical presentations were generally mild .
contrary to the findings of previous studies from france and tunisia ( 5,6 ) , no food association could be observed in this study . in agreement with some , but not all , published reports ( 5,11 ) , no case of aiv - associated gastroenteritis from this study had apparent co - infections , further supporting aiv pathogenicity in humans
. the overall 2.0% detection rate of aiv in stool samples from outpatients with gastroenteritis is compatible with detection rates in recent studies from several european and asian countries ( 5,12 ) .
this was in sharp contrast to parechoviruses and cardioviruses detected predominantly in patients < 6 years of age in the same study cohorts ( 8,9 ) and indicated different modes of transmission and maintenance of these genetically related picornaviruses at the population level . similarly , the lower aiv infection rate described here was consistent with the 51.0% seroprevalence rate described in german infants < 2 years of age ( 13 ) compared with > 75.0% described for cardioviruses and parechoviruses ( 14,15 ) .
the geographic and temporal accumulation of cases , together with the observed sequence variation , supports locally and temporally restricted circulation of aiv with human - to - human transmission , rather than a point - source epidemic pattern as observed in foodborne infections .
oral human - to - human transmission would be facilitated by the high fecal virus concentrations in some patients .
our data indicate that aiv can be considered an authentic human pathogen that can be transmitted from human to human .
 we assessed aichi virus shedding in patients with gastroenteritis and negative test results for other viral and bacterial infections . 
 high concentrations of up to 1.32  1012 rna copies / g stool were found in 10 ( 2.0% ) of 499 outpatients sampled in northern germany , 2004 . 
 these data substantiate aichi virus pathogenicity in humans . 
The Study
Conclusions