Saffold Cardioviruses of 3 Lineages in Children with Respiratory Tract Infections, Beijing, China

To clarify the potential for respiratory transmission of Saffold cardiovirus (SAFV) and characterize the pathogen, we analyzed respiratory specimens from 1,558 pediatric patients in Beijing. We detected SAFV in 7 (0.5%) patients and identified lineages 1–3. However, because 3 patients had co-infections, we could not definitively say SAFV caused disease.

To clarify the potential for respiratory transmission of Saffold cardiovirus (SAFV) and characterize the pathogen, we analyzed respiratory specimens from 1,558 pediatric patients in Beijing. We detected SAFV in 7 (0.5%) patients and identifi ed lineages 1-3. However, because 3 patients had co-infections, we could not defi nitively say SAFV caused disease.
Because SAFVs are mainly detected in fecal samples, virus transmission is thought to occur by the fecal-oral route (1,(3)(4)(5)(6)(7). However, 2 research groups also found SAFV-2 lineage in respiratory secretions (2,4). Thus, we investigated whether the respiratory tract route could be an additional transmission route and whether SAFV lineages other than SAFV-2 may also infect the respiratory tract. We identifi ed and characterized 7 SAFV strains, which belonged to 3 distinct lineages, from respiratory samples of children with lower and upper respiratory tract infections (LRTIs and URTIs, respectively).

The Study
We assessed 2 cohorts. Cohort 1 comprised 1,032 children (617 boys and 415 girls) with acute LRTIs, hospitalized in Beijing Children's Hospital (BCH), from whom nasopharyngeal aspirates were collected from May 2007 through March 2009. The patients ranged in age from 2 weeks to 16 years (mean age 31.3 months, median 9 months). Cohort 2 comprised 506 BCH outpatient children (277 boys and 229 girls) with acute URTIs, from whom throat swabs were collected from May through August 2009. These patients ranged in age from 4 months to 16 years (mean age 46.7 months, median 37 months).
Virus nucleic acids in clinical samples were extracted by using the NucliSens easyMAG system (bioMérieux, Marcy-l'Etoile, France) according to the manufacturer's instructions. SAFV RNA was detected by nested reverse transcription-PCR (RT-PCR) by using primers selective for the 5′ untranslated region (UTR) (3). The viral protein (VP) 1 gene was amplifi ed by using 3 pairs of primers as previously described (3,5,6). The full genomic sequences were obtained by a genome walking method (7). The 5′ and 3′ UTR sequences were determined by using the RACE System (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. After being cloned into the pGEM-T Easy vector (Promega, Madison, WI, USA), all PCR products were verifi ed by sequencing. In addition, all screened specimens were tested for known respiratory viruses as previously described (8,9). Mycoplasma pneumoniae was detected by using the gelatin particle agglutination test kit (SERODIA-MYCO II, Fujirebio, Japan).
For phylogenetic analysis, we constructed neighborjoining trees based on the distances of SAFV nucleotide or amino acid sequences by using MEGA 4.0 (10). We used SimPlot (version 3.5.1) to analyze possible recombination between viral genome sequences (11).
LLC-MK2 cells were used to isolate SAFV as previously described (12). Cells were collected either when cytopathic effects were observed or after 12 days postinoculation, and then they were tested for SAFV by RT-PCR.
We detected SAFV RNA in 4 (0.4%) of the 1,032 nasopharyngeal aspirates from patients with LRTIs and in 3 (0.6%) of the 506 throat swab specimens from outpatients with URTIs. The SAFV-positive patients (4 girls and 3 boys) were 5 months to 9 years of age (Table). SAFV infection did not appear to have a predominant time for occurrence: cases were detected in a range of months for the periods covered (   SAFV-positive patients with LRTIs, 2 had underlying illnesses, i.e., tuberculosis, hepatic dysfunction, or respiratory failure (Table). All SAFV-positive patients recovered within 7-11 days. No major differences were found in disease duration between patients who had underlying diseases and those who did not. Co-infections with additional respiratory pathogens were detected for 3 of 7 SAFV-positive patients, all in the fi rst cohort. These pathogens were respiratory syncytial virus (1 patient), enterovirus (1 patient), and M. pneumoniae (1 patient) (Table). A SAFV-3 strain was isolated from sample BCH1031. Starting at 5 days postinoculation, cytopathic effects were observed. Virus was not isolated from other SAFV-positive samples (data not shown).
We identifi ed 3 genetic lineages, SAFV-1, -2 and -3, on the basis of phylogenetic analysis of VP1, the most diverse protein of picornaviruses (6) (Figure 1). Multiplealignment analysis, based on reference sequences of each lineage available in GenBank, showed that the identity of the VP1 amino acid sequences among the strains in the same lineage was 92.6%-100% and among strains belonging to different lineages, 63.3%-100% (online Appendix  Table, www.cdc.gov/EID/content/16/7/1158-appT.htm). We found no obvious differences in amino acid sequences, nor any new motifs in these sequences, between strains detected in respiratory samples and those in fecal samples within the same lineage.
To further characterize the variation of SAFVs, we amplifi ed the P1 region sequences of 6 strains identifi ed in this study. The identity of all available nucleotide sequences of the P1 region among SAFVs was 68.5%-97.2%, whereas that of amino acid sequences was 74.9%-99.4%. The VP1 CD and VP2 EF loop structures, which display the greatest amino acid divergence among different SAFVs and are associated with tropism and virulence (6,7), were analyzed by amino acid alignment. Similar to previously reported fi ndings (6,7), we found that the sequences in both loops among SAFVs were highly diverse among SAFVs (Figure 2). The amino acid identities among all known SAFVs were 53.1%-100% in CD loops and 40.3%-100% in EF loops; amino acid identities of SAFVs versus animal cardioviruses were 29.1%-41.1% in CD loops and 19.2%-32.6% in EF loops. We did not fi nd any major differences among the available amino acid sequences of CD loops (91.1%-100.0%) or EF loops (97.9%-100.0%) of SAFVs within the same lineage for samples collected from either the gastrointestinal or respiratory tracts.

Conclusions
Although 8 lineages of SAFV have been detected in fecal samples worldwide, only SAFV-2 has been detected in respiratory samples (2,4). In addition, only SAFV-1 had been reported in China (5,13). In this study, we found that SAFV lineages 1, 2, and 3 co-circulated in patients with respiratory infections in Beijing.
Although SAFV is known to be transmitted by the fecal-oral route (1,(3)(4)(5)(6)(7), as are other picornaviruses (14), our detection of SAFV in respiratory samples suggests that various SAFV lineages may also be transmitted through the respiratory tract and may be associated with disease. However, other respiratory viruses and M. pneumoniae were codetected in 3 of the 7 SAFV-positive patients. Given that we did not conduct assays for common respiratory bacteria (and the number of SAFV-positive cases was limited), whether SAFV actually caused the observed symptoms in the patients cannot be defi nitively determined.
The genetic diversity of SAFVs in respiratory samples can complicate the relationship between SAFVs and disease because different genotypes of the same picornavirus species may cause different clinical signs and symptoms (6,14). Further investigations are needed to clarify any possible link between the pathogenicity and genetic diversity of SAFV.