during an epidemiologic survey conducted from october 1 , 2008 , through september 30 , 2009 , of viral infections of the respiratory tract , in which we collected respiratory samples from all patients admitted to our university hospital , fondazione istituto di ricovero e cura a carattere scientifico ( irccs ) policlinico san matteo , we detected 5 strains of the new ev-104 genotype . the 5 pavia ( pav ) strains were neither temporally nor epidemiologically related to each other and were recovered from 2 immunocompetent patients ( pav-2 and pav-4 ) and 3 immunocompromised patients ( pav-1 , pav-3 , and pav-5 ) ( table ) . * hev , human enterovirus ; ct , cycle threshold ; hrv , human rhinovirus ; npa , nasopharyngeal aspirate ; hsct , hematopoietic stem cell transplant ; aml , acute myelocytic leukemia ; rsv , respiratory syncytial virus . all respiratory secretion samples ( 1,500 ) were routinely tested for respiratory viruses ( 10 ) . pcr ( rt - pcr ) for amplification and quantification of both hevs ( 11 ) and hrvs ( 12 ) . primers and probes used for hrv and hev detection in this study were the following : hrv - forward 5-cpxgcczgcgtggc-3 , hrv - reverse 5-gaaacacggacacccaaagta-3 , hrv - probe 5-tcctccggcccctgaatgyggc-3 , hev - forward 5-cctccggcccctga-3 , hev - reverse 5-gattgtcaccataagcagcc-3 , and hev - probe 5-cggaaccgactactttgggt-3. however , the hev assay ( 11 ) does exhibit several mismatches with ev-104 target . using these 2 assays , we detected 5 strains from 5 patients ; these strains were amplified to a comparable degree . when these 5 strains were tentatively amplified in the viral protein ( vp ) 1 region ( 13 ) , no amplicon was obtained , as reported by others ( 8) . amplification products were obtained when typing was attempted by amplifying the vp4/vp2 region ( 14 ) , which yielded a sequence resembling hev - c genotypes but did not match any of the sequences published in genbank . upon request , the new ev-104 strain ( 9 ) sequence was received from the researchers in switzerland ( genbank accession no . comparison of the sequences from the strains from switzerland and the pav strains showed that the 5 new pav strains belonged to genotype ev-104 within the hev - c species ( genbank accession nos . pav-5 , gu722097 ; pav-4 , gu722098 ; pav-3 , gu722099 ; pav-2 , gu722100 ; pav-1 , gu722101 ) . from the 5 new pav strains , 181 nt were within the 5 noncoding region , 207 in vp4 , and 249 in vp2 . the nucleotide identity within the 5 pav strains was in the range of 97.9%99.3% , and the identity with the reference strain from switzerland was 95.4%97.0% . amino acid identity among the pav strains was 100% for 4 strains ; pav-5 strain showed a 2-aa difference , and the amino acid identity for all pav strains with the strain found in switzerland was 98.1%99.4% . within the hev - c species , the closest genotypes are cv - a19 , cv - a1 , and cv - a22 with a nucleotide identity of 63% , 59% , and 62% and an amino acid identity of 81% , 80% , and 81% with the pav strains , respectively ( figure 1 ) . phylogenetic analysis of the viral protein ( vp ) 4/vp2 region of the 5 enterovirus 104 ( ev-104 ) strains belonging to the human enterovirus c ( hev - c ) species ( delimited by circular dotted line ) , along with the reference strain from switzerland ( genbank accession no . prototype strains are also reported for the different hev and human rhinovirus ( hrv ) species . ev-104 was associated with episodes of chronic rhinopharyngitis , whereas 3 immunocompromised patients exhibited symptoms of acute respiratory tract infection ( table ) . however , in the patient infected with pav-5 , a hematopoietic stem cell transplant recipient , at the beginning of a 4-month follow - up after the transplant , respiratory syncytial virus was detected in association with ev-104 and was likely responsible for the acute respiratory symptoms ( figure 2 ) in the immediate posttransplant period . subsequently , respiratory syncytial virus disappeared , while ev-104 remained at the same stable level ( cycle threshold 2429 ) during the entire follow - up period . in the last month of the follow - up period , ev-104 was not associated with any clinical or respiratory symptoms . because we did not grow the virus in cell cultures , we can not exclude that our real - time rt - pcr assay may have detected only rna left over from the ev-104 infection . even in the patient infected with pav-5 , we could not detect any mutation in several samples examined during the 4 months of follow - up . virologic and clinical follow - up of immunocompromised patient infected with pavia strain ( pav-5 ) showing the kinetics of enterovirus 104 ( ev-104 ) and respiratory syncytial virus ( rsv ) viral loads , along with respiratory symptoms . starting on day 90 after transplantation , the patient s clinical symptoms began to disappear in the presence of a substantially unchanged ev-104 viral load in respiratory secretions . the 5 ev-104 strains from italy , along with the reference strain from switzerland , form a separate clade within the hev - c species , which includes 3 polioviruses , several cv - a genotypes , ev-99 , and ev-102 ( figure 1 ) . the new hev genotype confirms the close relationship between hrvs and hevs , and these findings suggest that both species of viruses should be sought in respiratory syndromes ( 8) . although the ev-104 strains detected in switzerland were in children from different regions and covered a period of 4 years ( 8) , our 5 strains were detected during a short period of the same year and in the same geographic area ; the patients , however , had presumably not been in contact with one another . however , population - based studies are needed to infer the actual prevalence of ev-104 infections . in the patients reported here , only the upper respiratory tract was involved in the ev-104 infection , whereas in the study in switzerland , otitis media and pneumonia were also reported . obviously , the range of ev-104 pathogenicity will have to be defined in an extended clinical and epidemiologic survey . however , virus detection in patient pav-5 in the absence of clinical symptoms indicates a potential nonpathogenic role for this virus , as is already known for hevs , hrvs , and other respiratory viruses . in contrast , the sustained persistence of ev-104 in the respiratory tract of patient pav-5 indicates that , at least in immunocompromised patients , virus can be shed for a long period , as has been shown for other respiratory viruses in this patient population ( 15 ) . we found also that the 2 immunocompetent persons ( pav-2 and pav-4 ) had a chronic respiratory infection ; however , because of limited sampling , the persistence of ev-104 was not shown . from a methodologic standpoint , we believe it is reasonable to conclude that , although only vp1 has been fully validated in multiple laboratories as the optimal typing region , other genome regions that code for capsid proteins may be amplified to enable typing of known or even unknown hev genotypes .
in an epidemiologic investigation of respiratory infections in italy , october 2008september 2009 , we tested samples from patients for respiratory viruses . 
 human enterovirus genotype ev-104 ( identified in switzerland ) was found in 3 immunocompromised and 2 immunocompetent patients . 
 ev-104 is closely related to human rhinoviruses ; thus , both types of viruses should be sought in respiratory syndromes .