key: cord-354143-p2ofapbd
authors: Hellferscee, Orienka; Tempia, Stefano; Walaza, Sibongile; Variava, Ebrahim; Dawood, Halima; Wolter, Nicole; Madhi, Shabir A.; du Plessis, Mignon; Cohen, Cheryl; Treurnicht, Florette K.
title: Enterovirus genotypes among patients with severe acute respiratory illness, influenza‐like illness, and asymptomatic individuals in South Africa, 2012‐2014
date: 2017-07-06
journal: J Med Virol
DOI: 10.1002/jmv.24869
sha: 
doc_id: 354143
cord_uid: p2ofapbd

Enteroviruses can cause outbreaks of severe acute respiratory illness (SARI) and EV‐A, ‐B, ‐C, and ‐D species have different pathogenic profiles and circulation patterns. We aimed to characterize and determine the prevalence of enterovirus genotypes among South African patients with respiratory illness and controls during June 2012 to July 2014. Syndromic SARI and influenza‐like illness (ILI) surveillance was performed at two sentinel sites. At each site nasopharyngeal/oropharyngeal specimens were collected from SARI and ILI patients as well as controls. Specimens were tested for enterovirus by real‐time PCR. Positive specimens were further genotyped by sequencing a region of the VP1 gene. The prevalence of enterovirus was 5.8% (87/1494), 3.4% (103/3079), and 3.4% (46/1367) among SARI, ILI, and controls, respectively (SARI/controls, P = 0.002 and ILI/control, P = 0.973). Among the 101/236 (42.8%) enterovirus‐positive specimens that could be genotyped, we observed a high diversity of circulating enterovirus genotypes (a total of 33 genotypes) from all four human enterovirus species with high prevalence of Enterovirus‐B (60.4%; 61/101) and Enterovirus‐A (21.8%; 22/101) compared to Enterovirus‐C (10.9%; 11/101) and Enterovirus‐D (6.9%; 7/101) (P = 0.477). Of the enterovirus genotypes identified, Echovirus 30 (9.9%, 10/101), Coxsackie virus B5 (7.9%, 8/101) and Enterovirus‐D68 (6.9%, 7/101) were most prevalent. There was no difference in disease severity (SARI or ILI compared to controls) between the different enterovirus species (P = 0.167). We observed a high number of enterovirus genotypes in patients with respiratory illness and in controls from South Africa with no disease association of EV species with disease severity.

pneumonia in children also include enteroviruses, rhinovirus, human bocavirus, and human coronaviruses (229E, NL63, OC43, HKU1), although this remains controversial. 3, 4 Although influenza and respiratory syncytial viruses have been well described as causes of pneumonia in South Africa, little is known about enterovirus prevalence and circulating genotypes. 5, 6 Enteroviruses are members of the enterovirus genus in the family Picornaviridae. 7 The capsid protein VP1, the most variable protein containing the majority of neutralization epitopes, is commonly used to characterize enteroviruses. [7] [8] [9] More than 100 enterovirus genotypes are currently classified into four species, HEV-A, HEV-B, HEV-C, HEV-D, which have different pathogenic profiles and circulation patterns. 7, 10 Enteroviruses (EV) can cause symptoms similar to a mild cold, but have also been associated with severe respiratory infection requiring hospitalisation and may be fatal. 10 An outbreak of a re-emerging EV-D68 lineage causing severe acute respiratory illness (SARI) occurred in the USA and Canada in 2014, and these lineages were also detected in Europe, Asia, and South America from 2012 to 2013 samples. [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] Though EVs have been associated with SARI and influenza-like illness (ILI), 5 these viruses have been less well characterized in South Africa. We identified high diversity among EVs circulating in hospitalized South African patients, 25 however it is unknown whether different EV genotypes are associated with mild or severe respiratory illness in South Africa.

We aimed to characterize the EV genotypes circulating among South African patients with SARI, ILI, and asymptomatic individuals from June 2012 to July 2014. In addition we assessed the association of different EV species with mild (ILI) or severe (SARI) illness compared to asymptomatic individuals. 

An asymptomatic individual or control was defined as a person presenting at the same outpatient clinics with no history of fever, respiratory, or gastrointestinal symptoms during the 14 days preceding the visit. These individuals commonly presented to the clinics for visits such as dental procedures, family planning, well baby visits, voluntary HIV counseling and testing, or acute care for non-febrile illnesses. We aimed to enroll one HIV-infected and one HIV-uninfected control every week in each clinic within each of the following age categories: 0-1, 2-4, 5-14, 15-54, and ≥55 years. Individuals were not followed-up to ensure they remained asymptomatic after enrollment.

Study staff completed case report forms for all enrolled SARI and ILI cases as well as controls. In addition, for SARI cases, hospital records were reviewed to assess disease progression and outcome (ie, discharge, transfer, or in-hospital death). Referral to hospital was recorded for all enrolled ILI cases. ILI cases that were referred to hospital were excluded from the analysis, as these patients did not adhere to the case definition of ILI anymore.

HIV results were obtained from a combination of two sources: (i) patient clinical records when available and (ii) for consenting patients, an anonymized linked dried blood spot was tested at the National Institute for Communicable Diseases (NICD). When both results were available, the NICD result was used.

Nasopharyngeal aspirates for children <5 years of age and nasopharyngeal and oropharyngeal swabs from persons ≥5 years of age were collected from all enrolled participants and placed in universal transport medium (Copan, Murruieta, CA).

All respiratory tract specimens were tested for the presence of 10 respiratory viruses (influenza virus A and B, respiratory syncytial virus, parainfluenza virus types 1, 2, and 3, adenovirus, human metapneumovirus, and rhinovirus) including EV, using a multiplex real-time reverse transcriptase polymerase chain reaction assay. 26 The real-time reverse transpriptase polymerase chain reaction used for EV testing, is a sensitive method for the routine detection of all members of the enterovirus genus. This method has a weak cross-reactivity to hightitre rhinovirus stocks, but not with rhinovirus-positive clinical samples. 27 

All specimens testing positive for enterovirus were selected for molecular characterization. A 400 base pair region of the VP1 gene was amplified and sequenced as previously described using primer set 224/222 during the first round of amplification and primer set AN89/AN88 for the nested amplification. 8 This assay was originally designed to detect and identify EV, but also detects and identifies a subset of rhinoviruses.

Amplicons were purified using the ExoSAP enzyme system 

The genetic diversity of each VP1 sequence was first determined by comparison with the reference strains in GenBank (US National Center for Biotechnology Information, NCBI, http://www.ncbi.nlm.nih.gov/, accessed 01 July 2016) using BLAST (Basic Local Alignment Search Tool) and confirmed by phylogenetic analysis of the partial VP1 sequences. Multiple sequence alignments were generated in the MAFFT (Multiple Alignment using Fast Fourier Transform) multiple sequence alignment program 28 and analyzed in the BioEdit Sequence Alignment Editor Software. 29 Phylogenetic trees were generated using the neighbor-joining method and genetic distances were calculated with the Kimura-2-parameter model using MEGA (Molecular Evolutionary Genetics Analysis) 6 software. 30 The statistical significance of the phylogenies was estimated by bootstrap analysis using 1000 pseudo replicates. Sub analysis was similarly done for the three dominant EV species in this study.

Sequences of enterovirus partial VP1 genes generated in this study have been deposited in GenBank with the following accession numbers: KX940982-KX941096.

The Chi-squared or Fisher's exact test were used for comparison of categorical variables. Unconditional exact logistic regression was used to assess the association of EV species with disease severity among patients with mild (ILI) or severe illness (SARI) using asymptomatic individuals as control group. Exact logistic regression was used to account for the fact that no EV-D species was detected among controls. EV-B was used as the comparison group as it was the species most frequently detected. Significance was assessed for P < 0.05. All models were adjusted for age, HIV serostatus and underlying medical conditions. The analysis was performed using STATA 14 On multivariable logistic regression analysis using EV species B as the reference group and controlling for age, HIV status and underlying illness, no association with disease severity (SARI or ILI compared to controls) was identified for any of the EV species (Supplementary   Table S1 ).

Enterovirus genotypes could be identified in 59% (51/87), 33% (34/103), and 35% (16/46) of enterovirus-positive samples among SARI and ILI cases and controls, respectively. We identified a total of 33 genotypes, distributed among all four EV species. The most prevalent genotype was E30 (9.9%, 10/101), followed by CVB5 (7.9%, 8/101) and EV-D68 (6.9%, 7/101). All other genotypes identified in the study were detected in <5% of genotyped samples (Supplementary Fig. S2 ). All E30 strains identified in this study displayed >98% nucleotide (nt) homology and formed a distinct bi-phyletic cluster with 79% bootstrap support (Fig. 3) . In the bi-phyletic cluster, strains from Edendale SARI cases and Klerksdorp SARI and ILI cases are distinctly located in each of the sub clusters with 92% bootstrap support, respectively.

The E30 strains in our study differ from other E30 genotypes by mean nucleotide pairwise distance of 20.2% (15.6-27.5 ) and mean amino acid sequence distance of 6% (2.7-11.6) in amino acid sequence.

All CVB5 strains identified in our study (six SARI samples and two ILI samples from Edendale) were categorized as genogroup C and formed a distinct sub cluster with a bootstrap value of 91% (Fig. 4) . The nucleotide 

The majority (71.4%, 5/7) of EV-D68 strains identified in 2013/2014 distinctly clustered in lineage B2 (89% bootstrap support) of which three were ILI cases and two were SARI cases, together with strains from one of the two co-circulating EV-D68 lineages that caused the large 2014 USA outbreak (Fig. 5) . The nucleotide sequences for EVD-68 strains were downloaded from GenBank and sequences depicted in Fig. 5 represent all known lineages.

The lineage B EV-D68 strains in our study differed from other lineage B EV-D68 strains by 1.5% nucleotide and 1.4% amino acid sequence. These included two SARI cases (one from Klerksdorp and one from Edendale) and three ILI cases (one from Klerksdorp and two from Edendale). 

We describe the EV species circulating among SARI and ILI cases and asymptomatic controls in South Africa. We observed a high diversity of circulating EV genotypes (a total of 33 genotypes) from EV species A-D with high circulation rates for EV-B and EV-A compared to EV-C and EV-D.

From our systematic surveillance across all age groups, the majority of patients testing positive for EV were <5 years of age.

We did not observe any difference in disease association due to different EV species since EV disease association cannot be conclusively ascribed since the analysis did not include coinfections with other pathogens. Epidemics of human EV disease display a seasonal pattern, with infections more common in summer and early autumn in geographical regions with a temperate climate. 7, 33 Most clusters of EV-D68 showed an atypical late seasonality compared to other EVs, with a peak in autumn, instead of summer. 7 A common feature of E30 molecular epidemiology is the progression of circulating lineages within one prevalent genotype. 9, 31 The E30 strains identified in this study clustered together (designated genotype k) although no differences were observed between viruses from SARI compared to ILI cases. There is no report of a concurrent E30 aseptic meningitis outbreak at the same time the E30 NP/OP positives were detected. It has been reported that genotypes of E30 can become dominant for 3-4-year periods before they disappear from circulation, 34 and they are therefore known as epidemic strains. In our study, it seems that E30 genotype k became dominant and circulated in South Africa during 2012-2014. Phylogenetic studies with VP1 sequences indicate that E30 variants are continuously emerging and replacing the circulating E30 strains, largely due to the error prone EV RNA polymerase 31 .

CVB5 has been detected for over 50 years and, similar to E30, sporadic cases of aseptic meningitis, as well as outbreaks, have been reported, remaining one of the most predominant reported EV genotypes in a number of countries. 35 All of the CVB5 strains identified in this study formed a sub cluster in genogroup C.

Phylogenetic clustering by year of study enrolment was observed for the South African strains. Different co-circulating strains of EV are often observed in EV disease outbreaks as EV genomes vary with respect to time but may also vary by geographical distribution. 36 In 2014, the largest outbreak to date of severe respiratory illness associated with EV-D68 occurred in the USA, and almost all of the case patients were children. 37 Coinciding with the 2014 USA outbreak The mechanisms for emergence of EV-associated outbreaks are not known; however, a combination of virus-specific, population-level, and other external factors are likely to be involved. 7 Our study has some limitations. Previous studies observed a difference in the predominant EV genotypes among neonates and older children 39 but we were not powered for this analysis. Some rhinovirus sequences were detected in samples that tested positive for EV on our in-house assay indicating that our EV detection assay is not 100% specific to detect only EVs. This study was not designed to describe the role of co-infections with other respiratory pathogens. In addition the association with EV detection with mild and severe illness was not investigated.

In conclusion, we showed that there was a high diversity in the VP1 sequences of the EV species circulating in South Africa during 2012-2014 and most of these genotypes are associated with meningitis worldwide. EV was detected in outpatient and hospitalized patients with SARI but was also detected in controls. We determined no disease association of EV species with disease severity; however, some genotypes (E30, CVB5, EV-D68) were more prevalent in symptomatic cases. Further studies are needed to determine if other factors such as viral load or host interactions play a role in EV-associated disease as well as a robust spatiotemporal phylogenetic analysis based on complete VP1 sequences.

We thank all members involved in SARI and ILI surveillance for the collection of specimens and data management. This work was supported by the National Health Laboratory Service, South Africa, and the United States Centers for Disease Control and Prevention, Atlanta, Georgia, USA (co-operative agreement number: 5U51IP000155).

The findings and conclusions in this paper are those of the authors and do not necessarily represent the views of their affiliated institutions or the agencies funding the study.

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