Detection and Characterization of Human Caliciviruses Associated with Sporadic Acute Diarrhea in Adults in Djibouti (Horn of Africa)

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1 Am. J. Trop. Med. Hyg., 78(3), 2008, pp Copyright 2008 by The American Society of Tropical Medicine and Hygiene Detection and Characterization of Human Caliciviruses Associated with Sporadic Acute Diarrhea in Adults in Djibouti (Horn of Africa) Jérôme Maslin,* Elisabeth Nicand, Katia Ambert-Balay, Christine Fouet, Jérôme Kaplon, Rachel Haus, Pierre Pothier, and Evelyne Kohli Laboratoire de Biologie Clinique, Groupement Médico Chirurgical Bouffard, République de Djibouti; Laboratoire de Biologie Clinique, Hôpital d Instruction des Armées du Val de Grâce, Paris, France; Laboratoire de Virologie, Reference Laboratory for Enteric Viruses, Hopital du Bocage, Dijon, France; Département Epidémiologie et Santé Publique Nord, Ecole d Application du Service de Santé des Armées, St Mandé, France Abstract. Recent advances in molecular diagnostics have allowed us to recognize Human caliciviruses (HuCVs) as important agents of acute diarrhea in industrialized countries. Their prevalence and genetic diversity in developing countries remains unknown. We report on the characterization of HuCVs among adults presenting acute diarrheas in Djibouti; 108 stool samples collected were screened by EIA, RTPCR, or cell cultures for the group A Rotaviruses, Adenoviruses, Astroviruses, and HuCVs, which were further characterized by genotyping. Among stool samples screened for HuCVs, 25.3% were positive. The other enteric viruses were less prevalent. The 11 HuCV strains sequenced revealed a large diversity (3 sapoviruses and 8 noroviruses). GII strains noroviruses were predominant, five were newly described genotypes, and two were recombinant with a pol gene related to GGIIb strains with the particularity to associate a unique pol gene to different capsid genes. These results could help to the knowledge of HuCV infections in Tropical Africa. INTRODUCTION Acute infectious diarrhea represents a public health concern throughout the world. In industrialized countries, the role and the diversity of the 4 main viruses causing gastroenteritis, group A rotaviruses (RV), human caliciviruses (HuCVs), astroviruses (AsV), and enteric adenoviruses (AdV) have now been well documented. HuCVs have notably been identified as the second leading cause of acute gastroenteritis in young children 1 and as a major cause of nonbacterial gastroenteritis in sporadic and outbreak cases in all age groups. 2 4 HuCVs are a very diverse group of singlestranded RNA viruses of the Caliciviridae family. They are divided into 2 genera, Norovirus and Sapovirus 5 ; human noroviruses are divided into 3 distinct genogroups, GI, GII, and GIV, each including genotypes. The classification is constantly evolving with the discovery of new strains. Recently Kageyama and others 6 proposed a classification with 31 genotypes (14 for GI and 17 for GII). At the same time, Vinje and others 7 identified 15 genotypes in the GII. Also, Zheng and others 8 defined 8 and 17 genotypes in the GI and II, respectively. In Africa where acute diarrhea causes high morbidity and mortality in children under 5 years of age, 9 the role and diversity of viruses other than RV has been sparsely documented, particularly among adults. Studies in Ghana, Malawi, and South Africa have reported a frequency of % for HuCVs in hospitalized children with acute diarrhea. A rather large diversity with a predominance of GII strains and new described genotypes was reported in Ghana and Malawi. 10,11 We report here a study that we conducted in Djibouti (Horn of Africa) in patients over 15 years old, most of them were European soldiers, during an 18-month period ( ) with the aim to investigate the prevalence of enteric * Address correspondence to Jérôme Maslin, Laboratoire de Biologie Clinique, Groupement Médico Chirurgical Bouffard, SP Armées-Djibouti. maslin_j@yahoo.com 522 viruses among adults presenting with sporadic acute diarrhea and to determine the role and diversity of HuCVs in this African tropical area. Compared with pediatric data, data concerning sporadic acute diarrhea in adults are relatively rare. Hence the data we present in this study of sporadic diarrhea in adults are of particular interest, especially where military forces deployed overseas are concerned. MATERIALS AND METHODS Patients and samples. Djibouti, situated in the Horn of Africa, is surrounded by Eritrea, Ethiopia, and Somalia; it is an important regional crossroads for trade and exchange. The total population of Djibouti is estimated about 600,000, twothirds of which live in the city of Djibouti. The French Military Hospital Bouffard (Djibouti city) is open to expatriates (French, American, and other European soldiers are the majority), Djiboutians (especially soldiers and their families), and more rarely patients from other countries of the Horn of Africa living in Djibouti. Patients were enrolled in the study from September 2002 to February They were all over 15 years old, largely urban, seen for acute diarrhea (more than 3 stools per day for a duration of less than 7 days) at Bouffard Hospital. For each patient, the physician filled out an epidemiologic and clinical questionnaire, and a stool sample was immediately sent to the laboratory for bacteriological and parasitological analysis. Only negative samples were further tested for viruses. Virus detection. After shipment of frozen samples (stored at 80 C in dry ice) to the Teaching Military Hospital Val de Grâce in Paris, France, specimens were screened by enzyme immunoassay (EIA) for the presence of group A RV (IDEIA Rotavirus, Dako Ltd.), AdV (IDEIA Adenovirus, Dako Ltd.), and AstV (Amplified IDEIA Astrovirus, Dako Ltd). Cell cultures (Vero, MRC-5) were inoculated for AdV detection by immunofluorescence with monoclonal antibodies (11-020, Argene, France). HuCVs and also AsV were detected by a commercial reverse transcription polymerase chain reaction test (RT-PCR) (Calici/Astrovirus Consensus, Argene, France). 13

2 HUMAN CALICIVIRUSES IN THE HORN OF AFRICA 523 TABLE 1 Characteristics of the population Sex Male 86 (80%) Female 22 (20%) Ratio (M/F) 3.9 Origin European 82 (76%) Djiboutian 17 (16%) Others 9 (8%) Age (y) Mean 29.5 Median 29 Range [15 49] Status Military* 77 (71%) Civilian 31 (29%) * French soldiers posted in Djibouti (2 3 years). HuCVs characterization. HuCVs were further characterized by genotyping by the National Reference Center for enteric viruses in Dijon, France. Strains were sequenced in the polymerase gene using the following primers: JV12-JV13 14 for noroviruses and SR80/p110 15,16 for sapoviruses. Some strains were sequenced in the capsid gene using primers G1SKF/G1SKR for genogroup I and G2SKF/G2SKR 17 for genogroup II noroviruses, respectively, and 3 of them were also sequenced in the region spanning ORF1/2 junction as previously reported. 18 Briefly, a RT-PCR was performed with primers JV12 and G1SKR or NI 19 and Mon depending on genogroup I or II, and the PCR products were cloned into the pgem-t Easy Vector System (Promega Corporation, Madison, WI). The cycling conditions were as follows: one cycle of reverse transcription at 42 C for 15 min; PCR: denaturation for 2 min at 94 C; 40 amplification cycles with denaturation for 30 sec at 94 C, annealing for 1 min at 50 C, and extension for 1 min at 72 C; and a final cycle of incubation at 72 C for 15 min. Genotyping was realized by direct sequencing of the PCR products or plasmids using the ABI Prism Big Dye Terminator Cycle Sequencing Ready Reaction Kit on an automated sequencer (model 3100 DNA Sequencing System), both from Applera Corporation, Foster City, California, United States. Sequence alignments with the GenBank library were carried out by using Fasta Version 3.3t06. For the RNA polymerase, alignments were also performed with reference strains available in the database of the European Foodborne Viruses network. 21 For the phylogenetic analyses, sequences were first aligned using CLUSTAL-W program in the PHYLIP format. Replicate data sets (N 100) were generated by bootstrap resampling and were analyzed by the neighbor-joining method. Nucleotide sequence accession numbers. The nucleotide sequences determined in this study have been deposited in Genbank under accession numbers EF190918, EF190919, and EF for samples VDG5, VDG50, and VDG66, respectively. RESULTS Virological testing. A total of 108 fecal samples were collected; samples were first analyzed with an immunologic test, and 75 were analyzed by RT-PCR (the difference between the total fecal samples collected and the samples analyzed was due to the amount of stool collected). Ninetyfive questionnaires were usable. The characteristics of the population are presented in Table 1. In expatriates, the majority of diarrheas occurred during the first 2 months after arriving in Djibouti (Table 2). The average number of stools was 8 per day (predominately watery), and 33 cases needed treatment (30.5%) with anti-secretory agents (19/33) and intestinal coating agents (15/33). Only 6 patients (all with fever) received antibiotics. Patients presenting nausea were the most often treated (19/ 44, 43%) in comparison with those without nausea (12/51, 23.5%), P The results of virological testing are presented in Table 3. Nineteen samples (25.3%) were positive for HuCVs. The other enteric viruses were less prevalent: AstV: 4.8% and 4% by EIA and RT-PCR, respectively (the AstV were not phylogenetically investigated); AdV: 3.8% and 4.8% by EIA and IFI respectively; RV: 1.9%. No co-infection was detected. The ELISA technique allowed us to identify 5 AstV strains; the PCR only 3. However, the numbers analyzed were different for the 2 techniques (105 and 75 stool samples, respectively). For the AdVs, the difference can be explained by the higher expected sensitivity of the IFI. There was no significant difference (P > 0.05) between the patients with HuCV infection and patients with infection caused by another virus with regards to sex, origin, age, status, and number of stools per day (mean 8/day). The frequency of asthenia was higher among patients infected by HuCVs (P 0.03). There was no significant difference regarding the other symptoms (abdominal pain, vomiting, nausea, fever, myalgia), but this may arise from the low number of comparable cases of each virus in each age group. HuCV characterization. Eleven strains of HuCVs could be genotyped in the pol and/or the capsid region. A diversity of strains was observed; among them, 8 (73%) were noroviruses and 3 (27%) were sapoviruses. Two of the sapoviruses were related to Parkville virus (Accession number: U73124, 90.8% and 92% nucleotide identity in a 285 bp region of the pol gene, respectively) and one showed 85.8% nucleotide identity with Mexico/339/1991 virus (Accession number: AY157865). 22 Among the noroviruses (Figure 1), 1 belonged to genogroup I (VDG50) and was close to Saitama-KU8G1/ 99/JP virus, which has been proposed recently as a new GI genotype (GI-11), 6 6 belonged to genogroup II and 1, VDG63, to genogroup IV (Alphatron). The 6 GII strains were the following: 2 M7/99/US-like strains, VDG68 and VDG82 (new proposed genotype, GII-14), 8 1 Idaho Falls, VDG66 (new proposed genotype GII-9), 8 and 1 CS-E1-like strain, VDG39 (new proposed genotype GII-17) 8 and 2 putative recombinants showing a pol gene that could not be TABLE 2 Time after arrival in Djibouti and characteristics of the diarrhea Delay arrival date/first clinical signs Mean 1 month Median 2 months Range [1 day 2 years] Number of stools/day Mean 8 Median 8 Range [1 24] Stool aspect (N 65) Watery 48 (74%) Mucous 4 (6%) Bloody 2 (3%) Other 11 (17%)

3 524 MASLIN AND OTHERS TABLE 3 Results of the Virological testing Stool samples (N) Virus Method Positive samples (N) % 105 Adenovirus EIA Astrovirus EIA Rotavirus EIA Adenovirus IFI Calicivirus RT-PCR Astrovirus RT-PCR 3 4 assigned to any known genotype but was close to GGIIb recombinant strains. 23 For one of them, a capsid gene belonging to GII-2 (Melksham genotype), the second capsid gene could not be sequenced. DISCUSSION Viral gastroenteritis is now well documented in industrialized countries, in contrast to Africa where epidemiologic data concerning viruses other than RV are still lacking. This may be attributed to the fact that methods for the detection of these viruses, especially HuCVs, are costly and require specialized laboratories. Moreover, local adaptation of surveys remains difficult because of preservation and shipment of samples. In this survey, which was focused on the detection and characterization of Human Caliciviruses associated to acute diarrhea among adults (outside of the context of an outbreak) the results of virological testing confirm the relative FIGURE 1. Relationships among pol (A) and capsid (B) nucleotide sequences of 8 norovirus strains isolated in Djibouti from 2002 to 2003 and prototype viruses using a neighbor-joining tree (Phylip version 3.6). The input alignment file was generated using Clustal W version 1.8. The Genbank accession numbers are as follows: Norwalk: M87661; Southampton: L07418; Desert Shield: U04469; Chiba: AB022679; Musgrove: AJ277614; Hesse: AF093797; Winchester: AJ277609; Boxer: AF538679; SaitamaKU8GI/99/JP: AB058547; Hawaii: U07611; Melksham: X81879; Toronto: U2030; Bristol: X76716; Hillingdon: AJ277607; Seacroft: AJ277620; Leeds: AJ277608; Amsterdam: AF195848; Idaho-Falls: AY054299: Erfurt-546: AF427118; Limburg: AY077644; SW918: AB074893; Wortley: AJ277618; Fayetteville: AY113106; M7: AY130761; J23: AY130762; Tiffin: AY502010; CS-E1: AY502009; SaitamaT29: AB112221; Alphatron: AF

4 HUMAN CALICIVIRUSES IN THE HORN OF AFRICA 525 frequencies generally observed in industrialized countries for HuCVs (and also RV, AstV, AdV) in this age group. 24 RV, which remains the main agents of gastroenteritis among children is more rare in adults, HuCVs being the most prevalent in this age group. It is important to recognize the diversity of HuCVs strains that were probably contracted in Djibouti. We sequenced 11 of the HuCV detected and observed a large diversity of strains with 3 sapoviruses and 8 noroviruses in this area. Among noroviruses, GII strains were predominant as previously shown in industrialized countries but also more recently in African countries Five of the 8 noroviruses were newly described genotypes, GI-11, 6 GII-9, GII-14, and GII-17 8 and 2 of them were recombinant strains showing a pol gene related to GGIIb strains. These strains, which were detected first in 2000 in France 23 have been predominant in Europe since 2004; they have the particularity to associate a unique pol gene with different capsid genes, here belonging to GII-2 genotype. Of note, a strain (E673) showing a great similarity with strain Djibouti VDG5 (97.7% identity in the region spanning ORF1/2, Acc. N AY682549) has been previously reported in France in Such a diversity with new described genotypes and recombinant strains has also been reported in Ghana (rgii-8/gii14) 10 and in Malawi (GII/ 11, GII/13, GII/16, GII/20). 11 In conclusion, this study shows the occurrence and diversity of HuCVs among adults presenting acute diarrheas in a tropical area (Djibouti, Horn of Africa). Considering a virus as readily transmissible as norovirus, we cannot judge if the strains are really endemic and if the traffic in and out of Djibouti has played a role in the introduction of new strains from elsewhere. The majority of our patients were European soldiers at the beginning of their deployment in Djibouti. Because their deployment in other countries before coming to Djibouti were for months at a time, it is likely that they contracted the diarrhea in Djibouti. Moreover they did not present any gastroenteritis before their arrival. Still, it is difficult to extrapolate our findings to the Djiboutian population in general. Surely, the development of RT-PCR assays among some references laboratories in tropical Africa should be considered to survey those viruses. Received November 13, Accepted for publication December 4, Acknowledgments: The authors thank Dr. R. Thiel for the proofreading of this article. We also gratefully acknowledge the contributions by Drs. J. J. Depina, P. Mathecowitsch, and H. Dampierre. Authors addresses: Jérôme Maslin, Laboratoire de Biologie Clinique, Groupement Médico Chirurgical Bouffard, SP Armées Djibouti, maslin_j@yahoo.com. Elisabeth Nicand, Laboratoire de Biologie Clinique, Hôpital d Instruction des Armées du Val de Grâce, 74 Bd de Port Royal, Paris cedex 05, France. Katia Ambert-Balay, Laboratoire de Virologie-Reference Laboratory for Enteric Viruses-Hôpital du Bocage, Service de Microbiologie Medicale, Dijon cedex, France. Christine Fouet, Laboratoire de Biologie Clinique, Hôpital d Instruction des Armées du Val de Grâce, 74 Bd de Port Royal, Paris cedex 05, France. Jérôme Kaplon, Laboratoire de Virologie-Reference Laboratory for Enteric Viruses-Hôpital du Bocage, Service de Microbiologie Medicale, Dijon cedex, France. Rachel Haus, Département Epidémiologie et Santé Publique Nord-Ecole d Application du Service de Santé des Armées Hôpital d Instruction des Armées Bégin, 69 Av de Paris, Armées, St. Mandé, France. Pierre Pothier, Laboratoire de Virologie-Reference Laboratory for Enteric Viruses-Hôpital du Bocage, Service de Microbiologie Medicale, Dijon cedex, France. 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