Background: Norovirus is a leading cause of viral gastroenteritis worldwide with a peak of disease seen in children. The epidemiological analysis regarding the virus strains in Africa is limited. The first report of norovirus in Botswana was in 2010 and currently, the prevalence and circulating genotypes of norovirus are unknown, as the country has no systems to report the norovirus cases. This study investigated the prevalence, patterns and molecular characteristics of norovirus infections among children ≤5 years of age admitted with acute gastroenteritis at four hospitals in Botswana. Methods: A total of 484 faecal samples were collected from children who were admitted with acute gastroenteritis during the rotavirus vaccine impact survey between July 2013 and December 2015. Norovirus was detected using real- time RT-PCR. Positive samples were genotyped using conventional RT-PCR followed by partial sequencing of the capsid and RdRp genes. Norovirus strains were determined by nucleotide sequence analysis using the online Norovirus Genotyping Tool Version 1.0, and confirmed using maximum likelihood tree construction as implemented in MEGA 6.0. Results: The prevalence of norovirus was 9.3% (95% CI 6.7–11.9). The genotype diversity was dominated by the GII.4 strain at 69.7%. This was followed by GII.2, GII.12 each at 9.1%, GI.9 at 6.6% and GII.6, GII.10 each at 3.0%. The most common combined RdRp/Capsid genotype was the GII.Pe/GII.4 Sydney 2012. Norovirus was detected during most part of the year; however, there was a preponderance of cases in the wet season (December to March). Conclusion: The study showed a possible decline of norovirus infections in the last 10 years since the first report. An upward trend seen between 2013 and 2015 may be attributable to the success of rotavirus vaccine introductions in 2012. Knowledge of circulating genotypes, seasonal trends and overall prevalence is critical for prevention programming and possible future vaccine design implications. Keywords: Norovirus, GII.4 variants, GII.Pe-GII.4, Botswana, Genotyping Background or water sources , person to person contact or Globally, noroviruses are a leading cause of acute gastro- through aerosols . Additionally, norovirus has been enteritis . Its symptoms include nausea, diarrhoea, shown to have low infectious dose . Currently, there vomiting, stomach cramps and fever . While these is no antiviral treatment for norovirus infections, and symptoms can be self-limiting in healthy individuals, vaccines are in late development stages [9, 10]. Noro- children, elderly and immune-compromised individuals viruses are single stranded, small non-enveloped RNA often experience prolonged symptoms [3, 4]. The shed- viruses that belong to the Caliciviridae family [11, 12]. ding of norovirus has also been shown in asymptomatic Their genome is approximately 7.5 kb long and com- individuals , and its transmission occurs mainly by prises three to four open reading frames (ORF), [12, 13]. the faecal-oral route, through either contaminated food ORF-1 encodes a large polyprotein, which is cleaved by virus-encoded protease, into six non-structural proteins * Correspondence: firstname.lastname@example.org that include the RNA-dependent RNA polymerase Department of Biological Sciences and Biotechnology, Botswana (RdRp) . ORF-2, ORF-3 and ORF-4 encode the International University of Science and Technology, Private Bag, 16 Palapye, major capsid protein, VP1, minor structural protein, VP2 Botswana Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Makhaola et al. BMC Infectious Diseases (2018) 18:246 Page 2 of 8 and virulence factor, respectively. The ORF-4 is unique 2015 . The facilities represent geographical regions to the murine norovirus cluster [6, 8, 14]. When using where more than 80% of Botswana’s population resides. complete amino acid sequences of the capsid protein, noroviruses cluster into seven genogroups [10, 15], and Sample collection and preparation each genogroup is subdivided into several genotypes. Bulk stool (~ 5 ml) or flocked rectal swab was obtained While genogroups I, II and IV have been shown to infect from each participant within 48 h of hospitalization to humans [5, 8], GII.4 is the predominant genotype glo- avoid detection of hospital-acquired infections. All sam- bally, and every few years new GII.4 variants surface that ples were transported to the National Health Laboratory rapidly replace the dominant circulating strain . Sev- on ice and kept at -80 °C until testing. A 10% stool sus- eral factors have been suggested that enable this strain pension was prepared using Precellys® Lysing kit (Bertin to dominate infections and these are: high mutation Corp, Rockville, MD), according to the manufacturer’s rates, wide receptor specificity, and short-term immunity instructions. The supernatant was then stored at -80 °C compared to other strains [2, 6]. Routinely, the norovirus prior to nucleic acid extraction. genotype determination is by either the partial capsid or RdRp gene sequences. However, noroviruses being RNA RNA extraction and viral detection viruses undergo recombination at ORF1/ORF2 that leads Norovirus was detected using multiplex real-time PCR to a single virus strain clustering under different geno- as described previously . Briefly, total nucleic acid types when different genomic regions are used for extraction was performed using easyMag (bioMerieux, sequencing [8, 17, 18]. Based on these, there is need to Lyon, France). 200 μl of the supernatant was used to determine both the capsid and RdRp genotypes. yield a nucleic acid elution volume of 70 μl. Norovirus In most of Sub-Saharan Africa, routine norovirus GI/GII was detected using the viral multiplex panel and surveillance is lacking or limited. Prevalence of noro- qualitative real-time PCR with the ABI 7500 Fast virus in Africa among symptomatic children is esti- (Applied Biosystems, Carlsbad, CA, USA). The samples mated at 13.5% (95% CI 0.8–25.5) and among the were taken through 45 PCR cycles. Targets with Cycle asymptomatic at 9.7% (95% CI 7–31%) . A previ- threshold (Ct) values below 40 were considered positive ousstudy in Botswana hasestimated noroviruspreva- for that particular sample. For quality check, there were lence at 24%  among symptomatic children. three controls included in every batch of testing in the Following multiple gastroenteritis outbreaks that assay: extraction negative control, in-house positive con- claimed many lives, the rotavirus vaccine was intro- trol, and master mix/reagent negative control. The duced in Botswana in July 2012 [20, 21]. While this extraction negative control was used to ensure adequate move has significantly reduced cases and deaths extraction process; the in-house positive control was related to gastroenteritis , understanding the used to ensure detection of all available targets in the causative agents of the remaining gastroenteritis cases assay; master mix/reagent negative control was used to is critical for prevention and management. Studies ensure there was no contamination in master mix report that where rotavirus vaccine is used on a large preparation. scale, norovirus infections become significant . It is therefore critical to understand norovirus infections Capsid and RdRp gene amplification in Botswana after a large scale-up of the rotavirus Following norovirus detection, positive samples were vaccine. In this study, we determined the prevalence, tested by conventional RT-PCR using the Transcriptor genetic diversity and trends of norovirus infections One-Step RT PCR Kit (Roche Diagnostics, Mannheim, among children 5 years and younger admitted with Germany). The capsid gene (5′ end, region C) was amp- acute gastroenteritis in Botswana between 2013 and lified with oligonucleotide primer sets G1SKF/R and 2015. G2SKF/R to amplify the 329 bp of GI and 342 bp GII capsid genes respectively, as previously described . Each 50 μl reaction mixture contained 1 μl of each pri- Methods mer, 1 μl Taq DNA polymerase and 5 μl of cDNA. The Study population and setting PCR was performed under these conditions: 50 °C for Between July 2013 and December 2015, faecal samples 30 min, 94 °C for 10 min followed by 40 cycles, 94 °C were collected from children under 5 years admitted for 30 s, 50 °C for 30 s, 72 °C for 1 min and final exten- with acute gastroenteritis at four health facilities in sion at 72 °C for 5 min. The RdRp gene was amplified Botswana (Princess Marina Hospital, Nyangabgwe using published oligonucleotide primer set, JV12/JV13 Referral Hospital, Letsholathebe II Memorial Hospital . The reactions conditions were as follows: Each and Bobonong Hospital), as part of the National Sur- 50 μl reaction mixture, contained 2 μl each primer, 1 μl veillance on Rotavirus Vaccine Impact Survey 2012– Taq polymerase and 15 μl of cDNA. PCR conditions Makhaola et al. BMC Infectious Diseases (2018) 18:246 Page 3 of 8 were 50 °C for 30 min, 94 °C for 10 min followed by Table 1 Demographic and clinical characteristics of a study conducted in children with gastroenteritis in Botswana, 40 cycles 95 °C for 30 s, 37 °C for 90 s, and 72 °C for 2013–2015 2 min and final extension of 5 min. The PCR products Characteristic Total No. of norovirus No. of norovirus were visualised on agarose gel. Tested Positive (%) negative (%) Gender, n = 484 Sequencing and phylogenetic analysis Males 269 26 (9.7) 243 (90.3) The amplicons from the partial gene regions for the RdRp Females 215 19 (8.8) 196 (91.2) and capsid were purified using QIAquick PCR Purification Age (months), n = 484 (43 missing age) Kit (Qiagen Inc., Valencia, CA), nucleotide sequencing 0 to 6 129 14 (10.9) 115 (89.1) was done using BigDye® Terminator v3.1 Cycle Sequen- 7 to 12 166 24 (14.5) 142 (85.5) cing Kit (Applied Biosystems, Carlsbad, CA) on 3130 DNA genetic analyser (Applied Biosystems, Carlsbad, 13 to 18 80 5 (6.2) 75 (93.8) CA). Sequencing was done in both directions using same > 19 66 0 66 (100) specific primers used for RT-PCR. Sequences were edited Facility, n = 484 (2 unknown facility) using Sequencher® version 5.4.6 DNA sequence analysis Princess Marina Hospital 255 28 (11) 227 (89) software, Gene Codes Corporation, Ann Arbor, MI USA Nyangabgwe Referral 112 11 (9.8) 101 (90.2) http://www.genecodes.com and norovirus strains were Hospital determined by nucleotide sequence analysis using the Letsholathebe II Memorial 83 3 (3.6) 80 (96.4) online Norovirus Genotyping Tool Version 1.0  avail- Hospital able at www.rivm.nl/mpf/norovirus/typingtool and con- Bobonong Hospital 32 3 (9.4) 29 (90.6) firmed using maximum likelihood method based on the Year, n = 484 Kimura 2-parameter model as implemented in Molecular 2013 243 22 (9.1) 221 (90.9) Evolutionary Genetic Analysis (MEGA) software version 6.0 . Robustness of the trees was assessed using boot- 2014 141 10 (7.1) 131 (92.9) strap analysis of 1000 replicates. The sequences generated 2015 100 13 (13.0) 87 (87) have been submitted to GenBank. Accession numbers Symptoms, n = 484 (14 missing symptoms) for RdRp gene sequences are MF817977 to MF818009 Diarrhoea and vomiting 360 30 (8.3) 330 (91.7) and for capsid gene sequences the accessions num- Diarrhoea only 62 6 (9.7) 56 (90.3) bers are MF920449.1 to MF920478.1. The GII.4 Vomiting only 48 6 (12.5) 42 (87.5) strains that were unidentified using the Norovirus Genotyping tool were identified using the online NCBI databases (https://blast.ncbi.nlm.nih.gov). Norovirus prevalence The results for the norovirus prevalence are given in Table 1. Of the 484 participants, 45 were positive, Statistical analysis 9.3% (95% CI 6.7–11.9). The prevalence for 2013, Categorical variables and proportions were compared 2014 and 2015 were 9.1% (22/243), 7.1% (10/141) and using a two-sided Chi-square or Fisher’sexact test 13.0% (13/100), respectively. The prevalence rates for where appropriate. Ninety-five confidence intervals 0–6months, 7–12 months and 13 to 18 months were (95% CI) were estimated using the binomial exact 10.9% (14/129), 14.5% (24/166) and 6.3% (5/80), re- method and P-value of < 0.05 was considered statisti- spectively and these were not statistically different. cally significant. There were no norovirus infections detected in the months of April, June and November for all the three Results years of the study. However, the infections were more Clinical and epidemiological data likely to occur in the wet than in the dry months, Clinical and epidemiological data are given in Table 1. (OR = 3.4; 95% CI 1.31–8.81). Diarrhoea was associ- From July 2013 to December 2015 a total of 484 partici- ated with vomiting symptoms, 360/470 (83%) (Fisher’s pants were enrolled in the study, 269 (55.6%) males and exact p = 0.001) and the majority of norovirus positive 215 (44.6%) females. The median age was 9 months (Q1, children presented with vomiting and diarrhoea, 30/ Q3: 6, 14; range 1 to 56 months). Participants were 45 (67%). enrolled from the following hospitals as follows Princess Marina, 255 (53%); Nyangabgwe Referral, 112 (23%); Norovirus genotyping Letsholathebe II Memorial, 83 (17%) and Bobonong, 32 From 45 positive norovirus samples, 33 (73.3%) were (7%). genotyped. Norovirus geno-grouping revealed that GII Makhaola et al. BMC Infectious Diseases (2018) 18:246 Page 4 of 8 was most prevalent 93.9% (31/33) followed by GI at characterized by GII.12, GII.6 and GII.2 strains, how- 6.1%, (2/33). In total 6 RdRp-based (33 samples) and 5 ever based on the RdRp; GII.Pe was detected in com- capsid-based (30 samples) genotypes were identified bination with GII.2. in GII geno-group as represented in Fig. 1a and b. Of the 23 identified GII.4 strains, 13 were identified Based on the capsid gene, the GII.4 strain was pre- on the Norovirus Genotyping Tool as phylogenetically dominant with 69.7% (23/33) prevalence, other strains related to the Sydney 2012 variant, and 10 were un- identified were GII.2 and GII.12 each at 9.1% (3/33); assigned. The unassigned samples were queried in the GI.9 strain at 6.1% (2/33); GII.6 and GII.10 strains NCBI database (Additional file 1: Table S1) and 9 sam- each at (1/33), 3.0% (Fig. 1a). Regarding classification ples had close identity to the Sydney 2012 variant by the RdRp gene, GII.Pe was most prevalent at 50% isolated in South Africa while one had close identity to a (17/34), followed by GII.P4 at 29% (10/34). The other variant isolated in Japan. Five of these nine GII.4 strains identified genotypes were GII.Pg at 9% (3/34), GII.P7 were closely related to GII/Hu/ZA/2012/GII.Pe-GII.4 at 6% (2/34), GII.P2 and GII.P17 each at (1/34), 3% Sydney 2012/Johannesburg 9814 (KJ710247.1). Their (Fig. 1b). As shown in Fig. 2a, in 2013 and 2015 query coverage ranged from 95 to 97% and nucleotide based on capsid gene, the predominant strain was identity was 98–99%. The other 4 strains were closely GII.4 but interestingly this strain was not detected in related to the GII/Hu/ZAF/2013/ GII.Pe-GII.4 Sydney the year 2014. Norovirus infection in 2014 was 2012/Cape Town (KR904238.1) with query coverage of ab Fig. 1 Phylogenetic tree of noroviruses based on (a) partial capsid and (b) RdRp genes of norovirus strains detected in children with acute gastroenteritis in Botswana, 2013–2015. The study samples are in shaded triangles and reference strains (unshaded triangles) are indicated by GenBank accession numbers. The scale bar represents nucleotide substitutions per site and the number above each branch corresponds to the bootstrap value. Scale bar is proportional to genetic distance Makhaola et al. BMC Infectious Diseases (2018) 18:246 Page 5 of 8 Fig. 2 Distribution of norovirus genotypes by year from Botswana, 2013–2015. a Capsid and b RNA dependent RNA polymerase 99–100% and 95–99% nucleotide identity. The last sam- the southern and northern regions of the country where ple was similar to NV/Saitama T37Dgii/01/JP more than 80% of the population resides. The prevalence (AB112236.1) with 96% query coverage and 95% nucleo- estimate falls close to the regional average of 13.5% tide identity, Additional file 1: Table S1. Based on the (range 0.8 to 25.5)  but lower than that of the previ- RdRp genotype sequences, in 2013 the GII.Pe strain was ous study in Botswana at 24% . This may suggest a the most prevalent whereas in 2015, the GII.P4 was possible decline in the NoV infections in the last 10 years dominant, Fig. 2b. The GII.P4 strains were mainly New but not necessarily a reduction in the number of gastro- Orleans 2009 variants. Combined RdRp and capsid enteritis cases. The prior study had a smaller sample size genotypes of 31 GII strains were determined resulting in (collected over a 7 year period, ~ 10 samples per year) 11 possible variants, of which 23/31 (67.7%) were con- and did not only enrol children admitted to hospital. In cordant between the two classification and 10/31 the previous study, there was a high prevalence on noro- (32.3%) were discrepant. These were GII.Pe/GII.4 at virus and GII.4 and it is possible that this could have (36%, n = 11/31); GII.P4/GII.4 at (26%, n = 8/31); been due to outbreak of new GII.4 variant. There is over followed by GII.Pe/GII.12; GII.Pg/GII.4; and GII.Pe/ Table 2 Norovirus polymerase and capsid genotypes obtained GII.2 each at (7%, n = 2/31) and lastly; GII.P4/GII.10; from faeces of children with gastroenteritis in Botswana, 2013– GII.Pg/GII.12; GII.Pg/P2; GII.P2/GII.4; GII.P7/GII.4; GII.P2/GII.6 at (3%, n = 1/31) each, Table 2. Polymerase (RdRp) genotype Capsid (VP1) genotype Number (%) GII.Pe GII.4 11 (36%) Discussion Viral gastroenteritis remains an important cause of mor- GII.P4 GII.4 8 (26%) bidity and mortality in the developing countries. GII.Pg GII.4 2 (7%) Furthermore, with the considerable decline of GII.Pe GII.12 2 (7%) rotavirus-associated diarrhoea in countries that have in- GII.Pe GII.2 2 (7%) troduced the rotavirus vaccine, it’s important to account GII.P4 GII.10 1 (3%) for the high numbers of diarrhoea cases that are still GII.Pg GII.12 1 (3%) seen. In this study, we report a possible shift in the vari- ant distribution of norovirus infections in Botswana. The GII.Pg GII.2 1 (3%) prevalence of norovirus was found to be at 9.3% among GII.P2 GII.4 1 (3%) children admitted with acute gastroenteritis at four GII.P7 GII.4 1 (3%) health facilities in Botswana between July 2013 and GII.P2 GII.6 1 (3%) December 2015. These facilities are distributed across Makhaola et al. BMC Infectious Diseases (2018) 18:246 Page 6 of 8 ten-year gap between sample collection of the study and summer . In some countries like Burkina Faso and our study. In this study, we have increased the sample Nigeria norovirus infections peaked in the dry season [31, size by prospectively enrolling each child admitted to 36], whereas in countries like Malawi and Morocco noro- hospital, determined the seasonality and included more virus infections peaked in the wet seasons [37–39]. A study geographic areas to understand the current burden of in Tunisia did not show any clear seasonal peaks . norovirus in the country. Additionally, it is possible that Several factors have been suggested to contribute to the this represents a shift in the aetiology of diarrhoea and seasonality of norovirus infection and they include rainfall, that the children could be exposed to wider causes of population density and human behaviour , and as these gastroenteritis. A study that characterized the aetiology are varied across African countries they could perhaps of infections in a diarrhoea outbreak that occurred in explain the discrepancy observed in the seasonality of nor- 2006 in Botswana, revealed diverse aetiology of infec- ovirus infection. Additionally, there is a disparity on the tions, mainly due to Cryptosporidium parvum, Crypto- prevalence of norovirus infections by health facility. A sporidium homins and Escherichia coli, while viral small proportion of norovirus cases were detected in Let- pathogens contributed a smaller proportion . We sholathebe II Memorial Hospital and one explanation is also found the high number of gastroenteritis cases in that the health facilities are in geographically distinct areas spring but with low norovirus prevalence, which may be with different livelihoods. While there is a lot of movement indicative of a different cause for the gastroenteritis. between the other three locations, Letsholathebe is situated While the prevalence is not statistically significant in a village with less interaction than the other three. across the years, there is an upward trend that was Consistent with other RNA viruses, noroviruses are observed between 2013 and 2015. Although causality genetically diverse even within genogroups. The results cannot be ascertained at this point, it is possible that this from this study showed that GII was the most com- significant increase could be attributable to the success monly detected strain. These findings are consistent with of the rotavirus vaccine, which was introduced in 2012 those of previous reports from Botswana and elsewhere . Studies from other regions have shown that where [5, 19]. We also found a dominance of the GII.4 strains. rotavirus vaccines are used on a large scale, there is an While this study detected GII.4 variants Sydney 2012 increase in the proportion of norovirus cases [10, 30]. By based on capsid gene and New Orleans 2009 based on 2014 rotavirus vaccine rollout or coverage in Botswana the RdRp gene, a previous study in Botswana reported among the under 2 years was reported to be over 70%, a more diverse GII.4 variants like Farmington Hills 2007, move that may have significantly reduced rotavirus Hunter 2004, Yerseke 2006a and New Orleans 2009 related gastroenteritis . The norovirus infection . A possible shift in GII.4 variant distribution could among the age categories was not statistically significant be attributed to the high mutation rate and evolution of though there was a trend of higher prevalence in the age norovirus strains leading to introduction of novel strains. category of 7 to 12 months. This trend is consistent with The analysis of combined capsid and RdRp genes also some of the published studies, which showed that noro- showed diverse norovirus infections. GII.Pe/GII.4 Sydney virus infections were more common in the 7 to 2012 was the most prevalent. These results suggest the 12 months age groups [16, 31, 32]. One explanation possibility of recombination between open reading given is that this is a period where kids begin to interact frames ORF1/ORF2 overlap region. We observed several more actively with their environment and can be easily GII.4 variants that were unassigned (10/23) by the Noro- exposed to norovirus [5, 32]. Furthermore, this is also a virus Genotyping tool, but were resolved by querying the period when solid foods are introduced and children are NCBI database (Additional file 1: Table S1). Most of often weaned from breastfeeding and in regions where these strains showed between 95 and 100% identity with clean water is not always available, there could be in- Sydney 2012 strains isolated in neighbouring South creased exposure to contaminated feeds . Norovirus Africa . The most common combined capsid and infections from this study were detected in most parts of RdRp was the GII.Pe/GII.4 Sydney 2012, these two the year with exception of April, June and November for genotypes were detected in major outbreaks in all the three years of the study. Nonetheless, a higher Australia , and thereafter became the predominant proportion of infections occurred in the wet months species. Some of these strains have been described than in the dry season. While studies from some regions before [8, 41] and some are possible novel recombi- show distinct patterns in norovirus seasonality [34, 35], nants. In this study the combined genotypes were de- results from African countries have not shown termined from separate RdRp and capsid amplicons, well-defined seasonal patterns. A study by Mans et al. in as such they could also represent coinfections and South Africa, which borders Botswana on the southern not recombinant strains. In 2014, GII.Pe was detected side, has shown a comparable norovirus seasonal pattern in combination with GII.2. To confirm recombinant with infections occurring in the spring and early strains the RdRp and capsid sequences will need to Makhaola et al. BMC Infectious Diseases (2018) 18:246 Page 7 of 8 be determined from a single amplicon overlapping the Publisher’sNote Springer Nature remains neutral with regard to jurisdictional claims in ORF1/ORF2 junction [8, 41], a subject for further published maps and institutional affiliations. study. Author details Department of Biological Sciences and Biotechnology, Botswana Conclusions International University of Science and Technology, Private Bag, 16 Palapye, In conclusion, this study has shown that norovirus strains Botswana. Botswana Harvard AIDS Institute Partnership, Gaborone, Botswana. 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Prevalence and genetic diversity of enteric viruses in approved the final manuscript. children with diarrhea in Ouagadougou, Burkina Faso. PLoS One. 2016;11(4): e0153652. Ethics approval and consent to participate 16. Mans J, de Villiers JC, du Plessis NM, Avenant T, Taylor MB. Emerging Written informed consent was obtained from all parents and guardians of norovirus GII.4 2008 variant detected in hospitalised paediatric patients in the children that were enrolled in this study. Ethical approval for the study, South Africa. J Clin Virol. 2010;49(4):258–64. to perform molecular analyses on the samples was obtained from Botswana’s 17. Mans J, Murray TY, Taylor MB. Novel norovirus recombinants detected in institutional review board, the Health Research and Development Council South Africa. Virol J. 2014;11(168):11–168. (HRDC). Furthermore, permission to collect data, samples from patients and 18. Hoa Tran TN, Trainor E, Nakagomi T, Cunliffe NA, Nakagomi O. 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BMC Infectious Diseases – Springer Journals
Published: May 30, 2018
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