Calicivirus from Novel Recovirus Genogroup in Human Diarrhea, Bangladesh

To identify unknown human viruses in the enteric tract, we examined 105 stool specimens from patients with diarrhea in Bangladesh. A novel calicivirus was identified in a sample from 1 patient and subsequently found in samples from 5 other patients. Phylogenetic analyses classified this virus within the proposed genus Recovirus.

D iarrhea, characterized by frequent liquid or loose stools, commonly results from gastroenteritis caused by infection with bacteria, parasites, or viruses. Patients with mild diarrhea do not require medical attention; the illness is typically self-limited, and disease symptoms usually resolve quickly. However, diarrheal diseases can result in severe illness and death worldwide and are the second leading cause of death around the world in children <5 years of age, particularly in low-and middle-income countries (1). For many cases of diarrhea in humans, no causative agent is identifi ed.
In recent years, many novel viruses have been identifi ed in human and animal blood, respiratory secretions, and fecal material through viral metagenomic studies consisting of random amplifi cation in combination with next-generation sequencing methods (2)(3)(4)(5). To identify unknown human viruses in the enteric tracts of persons with diarrhea, we performed sequence-independent amplifi cation on purifi ed viral nucleic acid from fecal samples obtained from patients with diarrhea in Bangladesh (6,7). We identifi ed a novel calicivirus and classifi ed it in the proposed genus Recovirus. Caliciviruses, which are nonenveloped, positive-stranded RNA viruses with a polyadenylated genome οf ≈6. 4-8.4 kb, cause illness in animals and humans (8,9), including gastroenteritis in humans. The family Caliciviridae consists of 5 genera, Norovirus, Sapovirus, Lagovirus, Vesivirus, and Nebovirus, and 3 proposed genera, Recovirus, Valovirus, and chicken calicivirus (8)(9)(10).

The Study
Each year, >100,000 diarrhea patients are admitted to the Dhaka hospital of the International Centre for Diarrheal Disease Research, Bangladesh (ICDDR,B). Fecal samples from 2% of these patients are collected and examined as part of systematic routine surveillance system for the presence of enteric pathogens (11). All procedures were performed in compliance with relevant laws and institutional guidelines and in accordance with the Declaration of Helsinki. Stool specimens from a subset of patients from routine surveillance during 2007-2009 (1,614 samples total) were available for further studies. These specimens were prescreened for adenovirus and rotavirus A by using Taq-Man EZ RT-PCR Core Reagents (Applied Biosystems, Foster City, CA, USA), rotavirus primers RVNSP3R and RVNSP3F and probe 5′-FAM-AGTTAAAAGCTAA-CACTGTCAAA-TAMRA-3′ (12), and TaqMan Universal Mastermix (Applied Biosystems) (13). Sequence-independent nucleic acid amplifi cation and next-generation sequencing were performed on 105 stool specimens from diarrhea patients enrolled during 2007 by using a 454 GS Junior Instrument (Roche, Indianapolis, IN, USA) as described (6,7). More than 725,000 trimmed reads were assembled by using de novo assembly and analyzed according to BLAST searches (online Technical Appendix Figure 1 and Table 1, wwwnc.cdc.gov/EID/pdfs/12-0344-Techapp.pdf) (6,7). Sequences were classifi ed on the basis of the taxonomic origin of the best-hit sequence (6,7). An E (expect) value of 0.001 was used as cutoff value of signifi cant virus hits. The largest proportion of virus-related sequences in human diarrhea samples from Bangladesh in 2007 was related to known bacteriophages and mammalian viruses (online Technical Appendix Figure 1).
One sample, no. 289, yielded a novel mammalian virus from the family Caliciviridae that we further characterized by near full-length genome sequencing using random amplifi cation with next-generation sequencing, specifi c reverse transcription PCRs, and 3′ rapid amplifi cation of cDNA ends PCR (Figure 1, panel A) as described (6,7). We named the virus isolate calicivirus Bangladesh/289/2007 (GenBank accession no. JQ745645).
We performed a diagnostic real-time recovirus PCR targeting the RdRp of all 1,614 available samples from patients with diarrhea (11). Reverse transcription PCR-grade viral nucleic acid was extracted by using the MagNA Pure LC Total Nucleic Acid Isolation Kit (Roche) and amplifi ed by using reverse transcription PCR with primers VS665 and VS666 and probe VS664 (online Technical Appendix Table 2) and TaqMan EZ RT-PCR Core Reagents (Applied Biosystems). The cycling program consisted of 50°C for 2 min, 60°C for 30 min, 95°C for 5 min, and 50 cycles of 95°C for 20 s and 59°C for 1 min, resulting in a 164-bp amplicon.
In addition to sample 289, 5 other human diarrhea samples ( Table 2) were sequence-confi rmed to be positive for a recovirus, with high homology (>98%) to calicivirus Bangladesh/289/2007; this fi nding indicates that recovirus Bangladesh infects humans. Clinical data indicate that all patients with a recovirus present in their feces had 6 to >21 watery stools in the fi rst 24 hours after illness onset ( Table 2); 4 patients experienced vomiting and 2 patients had fever. Patient ages ranged from 3 months to 50 years. Three recovirus-positive patients showed evidence of coinfection with other pathogens that are known to cause di-arrhea in humans, such as rotavirus A, adenovirus, Vibrio cholerae, or Salmonella spp.; the other 3 patients did not. Although viruses such as norovirus, sapovirus, and astrovirus were not detected in the recovirus-positive samples by sequence-independent amplifi cation assays, all samples were not analyzed for all known enteric pathogens. Of 514 diarrhea samples gathered by the Diagnostic Unit, Department of Virology, Erasmus Medical Center, Rotterdam, the Netherlands, for diagnosis of gastrointestinal infections during 2007 and 2009, none was positive for Recovirus Bangladesh (data not shown).

Conclusions
For a large proportion of human diarrhea cases, no etiologic agent can be identifi ed, despite multiple metagenomic studies of viruses in human stool aimed at identifying new etiologic agents (3)(4)(5). In addition, it cannot be known when and where emerging and reemerging viruses will appear in the human population. To identify potential etiologic agents of diarrhea in humans, we performed a metagenomic viral inventory in diarrhea samples from Bangladesh, which led to the identifi cation of a novel calicivirus.
Although no species demarcation criteria have been defi ned for the family Caliciviridae by the International Committee on the Taxonomy (15). Larger epidemiologic studies using genetic and serologic screening will be necessary to provide more insight into the distribution and pathogenic potential of recoviruses in humans. Dr Smits works at the Virology Department, Erasmus Medical Center, and Viroclinics Biosciences BV, Rotterdam, the Netherlands. Her research interests are hepatitis C virus and severe acute respiratory syndrome coronavirus.