Co-Circulation and Persistence of Genetically Distinct Saffold Viruses, Denmark

To the Editor: Cardioviruses are positive-sense, single-stranded RNA viruses of the family Picornaviridae, genus Cardiovirus. Until recently, cardioviruses were primarily known for their ability to infect rodents. In 2007, findings of a retrospective study of undiagnosed enteric illnesses in the United States were published, including results from analysis of a fecal sample from an infant girl whose symptoms were diagnosed as fever of unknown origin in 1981. The novel human cardiovirus that was isolated was designated Saffold virus (SAFV) (1). Eight genotypes of SAFV have been described (1–4), and a ninth was recently isolated in Nigeria (O. Blinkova, unpub. data). Serologic studies indicate that infection with SAFV genotypes 2 and 3 generally occurs in early life (5), although the clinical significance of these findings remains unclear. 
 
The first SAFV infection in Denmark was recorded in 2009 (6). To elucidate the molecular epidemiology of SAFV, we performed a 3-year surveillance study of SAFV in Denmark. During 2009–2011, we tested 1,393 fecal samples from 454 children. Surveillance included collection of fecal samples from children at 6, 10, and 15 months of age; additional fecal samples were collected when the children had gastroenteritis. Most of the SAFV-positive samples reported in this study were obtained from a randomized trial in the pediatric department of University Hospital of Holbaek (Holbaek) on the effect of probiotic therapy on the incidence of infection during early childhood (M. Gyhrs, unpub. data). The study was approved by the local ethics committee; Den Regionale Videnskabsetiske Komite for Region Sjaelland, Denmark. 
 
Nucleic acids were extracted from 200-µL fecal suspension (10% in phosphate-buffered saline) by using the Cobas AmpliPrep Total Nucleic Acid Isolation Kit (Roche Diagnostics, Ltd., Mannheim, Germany) on the MagnaPure LC instrument (Roche Diagnostics). We used 5 µL of extracted nucleic acid for reverse transcription PCR (RT-PCR) (total volume 25 µL) using the OneStep RT-PCR Kit (QIAGEN, Hilden, Germany). The samples were tested for SAFV by using real-time RT-PCR primer/probe, and all positive samples were genotyped by partial sequencing of the viral protein (VP) 1 gene (6). Overall, 38 (2.8%) of the clinical samples were positive for SAFV (Technical Appendix), all of which fell into genotype 2 (SAFV-2), which is most prevalent in Western nations. Of these samples, 31 had sequence information of sufficient length for additional analyses. All SAFV-2 sequences were submitted to GenBank (accession nos. {"type":"entrez-nucleotide-range","attrs":{"text":"JX048000-JX048030","start_term":"JX048000","end_term":"JX048030","start_term_id":"397787700","end_term_id":"397787760"}}JX048000-JX048030). 
 
To determine the evolutionary history of strains of SAFV identified in persons in Denmark, we combined the VP1 sequences collected here with all others available on GenBank. We aligned sequences as described using MUSCLE software (7), then checked the alignments using manual calculations. We performed phylogenetic analysis using the maximum likelihood method as described in PhyML 3.0 (8), on the basis of the best-fit GTR+Γ nucleotide model as determined by jModelTest (9). Phylogenetic robustness was determined by using 1,000 bootstrap replicates. 
 
Our phylogenetic analysis places the strains isolated in Denmark within the SAFV-2 group (Technical Appendix). These SAFV-2 strains were further subdivided into 2 strongly supported clusters: DK-A, which comprised viruses isolated during 2009–2011, demonstrating probable persistence in Denmark during this period; and DK-B, a smaller group that included viruses from United States, Germany, and the Netherlands, indicating widespread viral gene flow (Figure). The DK-B strains were identified in samples collected during 2009–2010, supporting probable sustained viral persistence within Denmark. Within DK-B, strain 115883 is phylogenetically distinct from the other DK-B viruses. 
 
 
 
Figure 
 
Phylogenetic analyses of Saffold viruses (SAFVs). Phylogenetic analysis of SAFV-2. Strains from Denmark are named by using the isolation numbers assigned for the study, then the country of origin and year of sampling in parentheses. The 2 subgroups (DK-A ... 
 
 
 
We next measured the selection pressures acting on these lineages through the mean number of nonsynonymous (dN) to synonymous (dS) nucleotide substitutions per site using the single-likelihood ancestor counting, fixed effects likelihood, and random effects likelihood methods available in the Datamonkey HyPhy package as described (10). The DK-A and DK-B groups differed significantly in selection pressure: DK-A, dN/dS ratio = 0.195 (95% CI 0.105–0.328); and DK-B, dN/dS ratio = 0.033 (95% CI 0.015–0.062), which indicates stronger purifying selection on the DK-B group. Ancestral state reconstruction, performed by using Datamonkey (10), revealed that the ancestors of DK-A and DK-B differ only at aa 135 in VP1: Val in DK-A and Ala in DK-B. Notably, aa 135 was positively selected in DK-A (random effects likelihood: dN/dS = 3.53, Bayes factor = 50: fixed effects likelihood: dN/dS >>1; cutoff p = 0.1), with more tentative evidence for adaptation at aa 135 in DK-B: dN/dS ratio >>1 by using fixed effects likelihood (p = 0.2). The functions of aa 135 in VP1, and what it means for the fitness of SAFV, merit further consideration. 
 
We conclude that SAFV-2 has been introduced into Denmark in 3 groups: DK-A, viral strain 115883 and strains of DK-B reported in Denmark; all have recently co-circulated in this country. We have demonstrated the entry and persistence of 3 phylogenetically distinct lineages of SAFV-2 in Denmark. That SAFV-2 can persist between years suggests that it might be common, yet underreported, in Denmark, which provides the opportunity for spread to additional localities. Increased awareness of improved laboratory protocols for SAFV detection is needed among clinicians in Denmark and neighboring countries. 
 
Technical Appendix: 
Epidemiologic and sequence information of Saffold virus isolated from specimens from children, Denmark, 2009–2011, and phylogenetic analyses of Saffold viruses. 
 
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Co-Circulation and
Persistence of Genetically Distinct Saffold Viruses, Denmark To the Editor: Cardioviruses are positive-sense, single-stranded RNA viruses of the family Picornaviridae, genus Cardiovirus. Until recently, cardioviruses were primarily known for their ability to infect rodents. In 2007, fi ndings of a retrospective study of undiagnosed enteric illnesses in the United States were published, including results from analysis of a fecal sample from an infant girl whose symptoms were diagnosed as fever of unknown origin in 1981. The novel human cardiovirus that was isolated was designated Saffold virus (SAFV) (1). Eight genotypes of SAFV have been described (1)(2)(3)(4), and a ninth was recently isolated in Nigeria (O. Blinkova, unpub. data Ltd., Mannheim, Germany) on the MagnaPure LC instrument (Roche Diagnostics). We used 5 μL of extracted nucleic acid for reverse transcription PCR (RT-PCR) (total volume 25 μL) using the OneStep RT-PCR Kit (QIAGEN, Hilden, Germany). The samples were tested for SAFV by using realtime RT-PCR primer/probe, and all positive samples were genotyped by partial sequencing of the viral protein (VP) 1 gene (6). Overall, 38 (2.8%) of the clinical samples were positive for SAFV (online Technical Appendix, wwwnc.cdc.gov/EID/ pdfs/12-0793-Techapp.pdf), all of which fell into genotype 2 (SAFV-2), which is most prevalent in Western nations. Of these samples, 31 had sequence information of suffi cient length for additional analyses. All SAFV-2 sequences were submitted to GenBank (accession nos. JX048000-JX048030).
To determine the evolutionary history of strains of SAFV identifi ed in persons in Denmark, we combined the VP1 sequences collected here with all others available on GenBank. We aligned sequences as described using MUSCLE software (7), then checked the alignments using manual calculations. We performed phylogenetic analysis using the LETTERS maximum likelihood method as described in PhyML 3.0 (8), on the basis of the best-fi t GTR+Γ nucleotide model as determined by jModelTest (9). Phylogenetic robustness was determined by using 1,000 bootstrap replicates.
Our phylogenetic analysis places the strains isolated in Denmark within the SAFV-2 group (online Technical Appendix). These SAFV-2 strains were further subdivided into 2 strongly supported clusters: DK-A, which comprised viruses isolated during 2009-2011, demonstrating probable persistence in Denmark during this period; and DK-B, a smaller group that included viruses from United States, Germany, and the Netherlands, indicating widespread viral gene fl ow (Figure). The DK-B strains were identifi ed in samples collected during 2009-2010, supporting probable sustained viral persistence within Denmark. Within DK-B, strain 115883 is phylogenetically distinct from the other DK-B viruses.
We next measured the selection pressures acting on these lineages through the mean number of nonsynonymous (d N ) to synonymous (d S ) nucleotide substitutions per site using the single-likelihood ancestor counting, fi xed effects likelihood, and random effects likelihood methods available in the Datamonkey HyPhy package as described (10) We conclude that SAFV-2 has been introduced into Denmark in 3 groups: DK-A, viral strain 115883 and strains of DK-B reported in Denmark; all have recently co-circulated in this country. We have demonstrated the entry and persistence of 3 phylogenetically distinct lineages of SAFV-2 in Denmark. That SAFV-2 can persist between years suggests that it might be common, yet underreported, in Denmark, which provides the opportunity for spread to additional localities. Increased awareness of improved laboratory protocols for SAFV detection is needed among clinicians in Denmark and neighboring countries.

Pathogenic Leptospira spp. in Bats, Madagascar and Union of the Comoros
To the Editor: Leptospirosis is a zoonosis of global distribution; incidence rates are particularly high in tropical areas (1). Leptospirosis is a major public health problem on islands in the southwestern Indian Ocean, particularly La Réunion, Mayotte, and the Seychelles (where incidence rates are among the highest in the world) (1). In contrast, no human case has been reported on the nearby islands of Madagascar and Union of the Comoros. However, the recent demonstration of pathogenic Leptospira spp. in small mammals introduced to Madagascar suggests possible transmission from free-living animals to humans (2).
In addition to the fact that incidence rates vary among humans, clinical bacterial isolates from different islands belong to different serogroups and serovars and show diverse molecular features (3,4). This diversity might be correlated with that of the reservoir hosts; the islands in the southwestern Indian Ocean are a hot spot of biodiversity with extraordinary levels of vertebrate endemism. Most studies investigating wild-animal reservoirs of Leptospira spp. on the islands in the southwestern Indian Ocean have focused on small mammals that had been introduced to the islands (2,5), although bats infected with pathogenic Leptospira spp. have been identifi ed in other regions (6). Whether bats are a reservoir of Leptospira spp. on these islands remains unknown. Therefore, we looked for this bacterium in bats from Madagascar and Union of the Comoros and characterized associated genetic diversity.
As part of an ongoing program aimed at identifying viral and Total nucleic acids were extracted from a pool of kidney, spleen, and lung tissue by using the Biorobot EZ1 and EZI Virus Mini Kit version 2.0 (QIAGEN, Les Ulis, France). Reverse transcription was then performed with GoScript reverse transcriptase (Promega, Charbonnières-les-Bains, France) to obtain cDNA. We screened pathogenic Leptospira spp. with a probe-specifi c real-time PCR (7). The 25 positive samples were subsequently subjected to a PCR procedure that amplifi ed fragments from 682 to 1,293 bp of the 16S rRNA gene (depending on the amplifi cation success) by using published primers (8)(9)(10). Resulting PCR products from 7 samples were sequenced and compared with available sequences in GenBank by using phylogenetic construction with PhyML 3.0.
Of the 12 bat species tested, 11 were positive for Leptospira spp. (the only H. anchietae bat tested was negative). Among 52 bats from Madagascar, 18 (34.6%) were infected; detection rates were often high, e.g., 8 (80%) of 10 T. menamena bats. In contrast, among 77 bats from Union of the Comoros, only 9 (11.7%) were infected. Leptospira spp. seem to be ubiquitous in the study areas; infected bats were found at 7 of 8