Original Article - Kenyatta University
Original ArticleMolecular detection and phylogenetic analysis of Kenyan human bocavirusisolatesDennis Misigo1,2, Dufton Mwaengo3, David Mburu41Department of Microbiology and Parasitology, Kenya Methodist University, Meru, KenyaInstitute of Tropical and Infectious Diseases, University of Nairobi, Nairobi, Kenya3Department of Medical Microbiology, University of Nairobi, Nairobi, Kenya4Department of Biochemistry and Biotechnology, Kenyatta University, Nairobi, Kenya2AbstractIntroduction: The commonly expected causative agents associated with flu-like symptoms in Kenya are the classical viral pathogensidentifiable as influenza virus, adenovirus, parainfluenza virus, enteroviruses, respiratory syncytial virus (RSV) and rhinovirus. However,newer agents have been identified globally that present with illnesses clinically indistinguishable from those caused by the classicalpathogens; one of them is human bocavirus.Methodology: A total of 384 specimens were analyzed, primarily to determine if the emerging human bocavirus (HBoV) infections exist inKenya as coinfections with other respiratory viruses and to describe the genotype of the virus in circulation. In brief, viral nucleic acids wereextracted from culture supernatants, amplified by PCR, and sequenced.Results: HBoV DNA was amplified from 1.8% of screened specimens. Coinfection with parainfluenza virus, adenovirus, and enterovirus was2.5%, 2%, and 1.4%, respectively. Multiple coinfections consisting of HBoV plus two other viruses were found in 3% of specimens.Isolation occurred in the months of January, March, April, August, and November. Retrospective review of clinical parameters indicated thatall the individuals complained of non-specific symptoms, mainly fever, coughs, nasal stuffiness, runny noses, and vomiting.Phylogenetically, the GenBank deposited sequences of this study’s isolates cluster closely to the reference strain NC 07455 (HBoV1).Conclusion: Coinfections with human bocavirus (HBoV1) occur in Kenya, and high incidence might primarily be during the early stages ofchildren’s lives.Key words: human bocavirus; Kenya isolates; respiratory viral infectionsJ Infect Dev Ctries 2014; 8(2):221-227. doi:10.3855/jidc.3050(Received 27 September 2012– Accepted 15 August 2013)Copyright 2014 Misigo et al. This is an open-access article distributed under the Creative Commons Attribution License, which permits unrestricted use,distribution, and reproduction in any medium, provided the original work is properly cited.IntroductionViral upper respiratory infections (URTIs) arefrequent and have a significant global health impact,causing between six and nine infection episodes peryear in children and between two and four infectionsper year in adults[1]. Most of these infections arecaused by the influenza virus, adenovirus, rhinovirusand parainfluenza viruses. However, new evidencesuggests that, occasionally, URTIs are also caused byor may be associated with newer emerging pathogensthat include human bocavirus, metapneumovirus, andhuman coronaviruses (HCoV-229E, HCoV-OC43,SARs-CoV, HCoV-KU1, and HCoV-NL63) [2].Human bocavirus(HBoV) is a novel viral agentthat was discovered about seven years ago by Allanderet al. [3]. It belongs to the family Parvoviridae and isstructurally similar to the other parvoviruses [4], butmolecularly differentiable by the encoded nuclearphosphoprotein (NP1), whose function(s) is unknown[5]. The virus contains a single linear positive-senseand negative-sense single-stranded deoxyribonucleicacid (DNA). Currently, bocaviruses can be classifiedbased on their genetic variability into species 1 to 4[6]. Initially, only two genotypes were described in theliterature as ST1 and ST2 [7]. Accumulating datasuggests that the formation of other HBoV variants islikely through recombination events among the species[8,9]. The species HBoV1, which is the focus of thisstudy, is predominantly a respiratory virus, while therest of the species seem to be more enteric pathogens[10].A historical review of most survey activities onviral respiratory tract infections in Kenya [11,12]indicates that the focus is always on identifying theclassical common viral pathogens (influenza virus,adenovirus, rhinovirus, RSV,andparainfluenza virus).
Misigo et al. –Kenyan isolates of human bocavirusFrom these surveys, influenza viruses account for 2%of the cases, adenovirus for 3.6%, RSV for 22.1%,rhinoviruses for 12.2%, herpes simplex virus (HSV)for 13.3%, and parainfluenza virus for 4.3%. Hence,there are few or no studies (especially surveillanceprograms) specifically examining the presence ofnewer pathogens associated with upper apneumovirus, and human coronaviruses) in EastAfrica.The objective of this investigation was to fill in thegap of information about such emerging viralpathogens by isolating and determining the genotypeof Kenyan isolates of human bocavirus (HBoV) basedon the viral capsid protein gene (VP 1/2).Our focuswas on archived supernatants collected from cellcultures that had been exposed to clinical specimens ofsubjects suspected of having viral acute respiratoryinfections.MethodologyEthics and study designThis study was within the limits of a descriptiveretrospective cross-sectional design and was approvedby the Kenyatta National Hospital/University ofNairobi Ethical Review Committee (KNH/UON ERC)and assigned project number P85/3/2010.SpecimensA total of 384 samples collected from six regionsof Kenya (Kisumu, Busia, Nairobi, ngeal swab specimens were collected fromindividuals presenting with influenza-like illnessesbetween January 2007 and June 2009. Thesespecimens were confirmed by immunofluorescenceassay to be positive for HSV type 1, RSV,parainfluenza virus, adenovirus, and enterovirus. Theclinical specimens were inoculated into the LLCMK2, HEp-2, rhabdomyosarcoma (RD), and MDCKcell lines and the supernatant was collected bycentrifugation within 48 hours after primaryJ Infect DevCtries 2014; 8(2):221-227.inoculation. The processed supernatant was stored at 80oC in 1 mL aliquots containing a mixture of viraltransport media and culture media containingDubelcco’s modified eagle medium supplementedwith 2% fetal bovine serum and 1% antibiotic.Clinical parametersDemographic data, history of illness, andlaboratory studies were examined retrospectively fromthe clinical parameters documented in the database.Cross-tabulation of proportions and frequencies wereanalyzed by SPSS version 13.0 (SPSS Inc., USA) andEPI info (CDC, Atlanta, USA).DNA extraction and polymerase chain reaction (PCR)DNA was extracted from 100-150 µL of thesamples using Qiagen QIAmp DNA blood mini kit(Qiagen, Hilden, Germany). Primers used in a seminested PCR for HBoV screening originated from apublished report by Smuts and Hardie [7], and weredesigned for the NP1andVP1/VP2 genes (Table 1).These primers are expected to amplify a 368 bpproduct of NP1 gene, which is confirmed byamplifying a 980 bp product of the VP1/VP2 gene. Inbrief, a 50 µL PCR reaction was set up containing 1XPCR buffer, 0.2 mM of each primer, 0.2 mM ofdNTPs mix, 1.5 mM MgCl2, 2.0 units of Taqpolymerase, and 10 µL of DNA. Cycling profileincluded an initial denaturing at 94 C (10 minutes),followed by 40 cycles of denaturing at 94 C (30seconds), annealing at 55 C (30 seconds), 72 C (1minute), a final extension at 72 C (5 minutes), and ahold at 4 C. Two microliters of the primary PCRproduct were used as a template in separate reactiontubes in the secondary PCR under the same cyclingconditions. Ten-µL aliquots of amplicons were loadedon gels and electrophoresis performed in1.5 % (w/v)agarose containing 2 µL of ethidium bromide. Thegels were run at 100 mV-150 mV in 1X Tris BorateEDTA buffer pH 8.0. Two markers, a standard 100 bpand 1 Kb DNA ladder (Fermentas, Burlington,Canada) were used as size references, and gels wereTable 1. Oligonucleotide primers for PCR amplification and sequencing of HBoV targeting the viral capsid protein (VP1/2)and nucleoprotein (NP1)GeneForwardVP1/2 outerVP-A (5’GCA CTT CTG TAT CAG ATG CCT T-3)VP1/2 innerVP-C (5’CTT AGA ACT GGT GAG AGC ACT G-3’)NP-1 outerNP-A (5’TAA CTG CTC CAG CAA GTC CTC CA-3’)NP-1 innerNP-C (5’CTC ACC TGC GAG CTC TGT AAG TA-3’)Adapted from Smuts and Hardie (2006)ReverseVP-B (5’CGT GGT ATG TAG GCG TGT AG-3’)VP-B (5’CGT GGT ATG TAG GCG TGT AG-3’)NP-B (5’-GAA GCT CTG TGT TGA CTG AAT-3’)NP-B (5’-GAA GCT CTG TGT TGAC TGA AT-3’)222
Misigo et al. –Kenyan isolates of human bocavirusphotographed on a UVP trans-illuminator. For the NP1and VP1/2 genes, a positive PCR was one thatdisplayed amplicon of just under 1000 bp and 400 bp,respectively. Figure 1 shows the PCR productrepresenting both the NP1 and the VP1/2 genes. Thelater fragment was subjected to sequencing asindicated below; this is the most variable region, so itis highly informative for diversity studies.Amplicons concentration and purityThe 960 base amplicons for sequencing werepurified using the Qiagen QIAquick PCR (Qiagen,Hilden, Germany) purification kit from gels orsolutions depending on the level of purity. ically using a Thermo ScientificNanoDrop 2000/2000c spectrophotometer (NanoDropTechnologies, Wilmington, USA) on 0.5-2.0 µL of thesamples against blanked water controls. The ratio ofabsorbance at 260 and 280 nm was used to assess thepurity of DNA. A ratio of 1.8 and above wasgenerally accepted as pure for DNA. The eluted DNAwas stored at -20 C until analyzed.SequencingCycle sequence was performed according to theprotocol specified for the Gene Amp ). The primers used for sequencing weresimilar to those used for the second round ofamplification of the VP gene. Excess deoxy terminatorwas removed from the DNA sequencing products withCentri–SepTM columns. The purified products weredried in a vacuum centrifuge for 10-15 minutes at 60 C and stored at -20 C. In brief, automatedsequencing was performed on the sense and anti-sensestrands of HBoV DNA on an ABI Prism 3100 Geneticanalyzer version 2 (Applied Biosytems, Darmstadt,Germany) using the ABI Prism BigDyeTerminatorcycle ready reaction kit version 2.0 as recommendedby the manufacturer. The raw data sequence wasanalyzed with sequencer software version 4.0.5.Phylogenetic analysisThe raw ABI sequences of the VP1/2 region werecleaned by trimming off the low quality bases at theend of the chromatogram and assembled into a contig.Base pair errors were corrected using the DNA baserversion 2.91.5 sequence assembly software (HeracleSoftware, Bremen, Germany). The Kenyan isolateswere preliminarily compared to known referencesequences of bocavirus HBoV1, HBoV2, HBoV3,J Infect DevCtries 2014; 8(2):221-227.Figure 1. A representative agarose gel (1.5 % [w/v]) showingPCR results of NP1 and VP1.Lane M (100 bp ladder markers). Lanes 1 and 2 are VP gene PCRproducts of 980bp, while lanes 3 and 4 show NP1 gene PCR products of398 bp from two specimens (KE-4S9/04) and (KE-10N8/03). 5 negative control. PCR-amplified products of 980 bp and 398 bp areshown with arrows.HBoV4, parvovirus isolates (canine minute virus andbovine parvovirus), and a few other selected sequencesof the virus isolates from South Africa (ZA190/04,ZA2591/04), China (HK5), Europe (Bonn-1), andAmerica (CRD2) via BLASTn [13]. The comparisonsequences had accession numbers DQ317556.1 (ZA2591/04), bovine parvovirus (NC 001540.1), canineminute virus (NC 004442.1), EF450721.1 HK5,NC 014358.1 (bocavirus gorilla), NC 007455.1(human bocavirus 1), NC 012042.1 (human bocavirus2), NC 012564.1 (human bocavirus 3), NC 012729.2(human bocavirus 4), FJ858259.1 (Bonn-1) and(DQ340570.1) (CRD2). The sequences weredownloaded as Fasta files from GenBank database andwere aligned together with five of this study’ssequences labeled KE-4S9/04, KE-9N7/11, KE10N8/3, KE-48G7/8 and KE-23A8/11 in ClustalWusing multiple alignment algorithms. Phylograms wereconstructed with Mega version 4 [14] using Kimuratwo-distance-based parameters via neighbor-joininganalysis of the amino acid arrangement generated from1000 bootstrap replicates.ResultsNumber of isolates and demographicsHalf of all the specimens assayed in this study(51.3%) were from children between one and twoyears of age, 29% were from children below one yearof age, and 18.5% were from subjects older than threebut younger than seven years, while 0.9% were fromadults.223
Misigo et al. –Kenyan isolates of human bocavirusJ Infect DevCtries 2014; 8(2):221-227.Table 2. Summary of clinical and demographic data on seven individuals positive for HBoV DNAHBOV-POSITIVE SUBJECTS (n 7)Subject no.Age in monthsSexGeo-locationPeriod of isolation122MBusiaNov 08Temp 38 CCough, nasal stuffiness, runny noseBreathing difficultyDiarrheaVomitingFatigue, malaise, poor appetiteNeurological - restlessnessCoinfections Para 3Demographics232213MFNairobiKisumuAug 07Jan 07Clinical Presentation hAdvEnt48FKisumuOct 07513FKisumuNov 07614FKisumuMar 0878FKisiiApr 09 hAdv Ent/hAdv HSV/Ent Para 3Para: parainfluenza 3; hAdv: human adenovirus; Ent: enteroviruses; HSV: herpes simplex virus; : symptom present; -:symptomabsent; M: male;F:female; , 1: KE-23A8/11; 2: KE-48G7/8; 3:KE-38N7/01; 4: KE-9N7/10; 5: 9N7/11; 6: KE-10N8/3; 7: KE-4S9/04; Bus: Busia; Nai: Nairobi; Kis:Kisumu; Kss: KisiiThere was a slight predominance of males (56%) incomparison to the females (44%). HBoV DNA wasamplified from seven samples out of 384 screenedspecimens (1.8%); this was from five females and twomales.A retrospective review of records for age revealed thattwo (28.5%) of the seven positive subjects werechildren less than 12 months of age (8 monthsprecisely) and the other five were children betweenone and two years of age (71.5%).Co-pathogensAmong the HBoV-positive samples, coinfectionswith a single virus were observed in two withparainfluenza 3 only (2.5%), two with adenovirus only(2%), and one with enterovirus only (1.4%). Multipleinfections consisting of HBoV plus two other viruses,which collectively accounted for 3%, were observedwhen enteroviruswas isolated together withadenovirus, and also separately with HSV-1. HBoVwas isolated in all the three years represented by thesamples screened.HBoV was isolated twice, consistently in Novemberand March, in two different years and from twodifferent regions. Clinical characteristics of subjectswith HBoV DNA positive specimen alongside themonth and place of isolation are outlined in Table 2.Most of the isolates were from the samples from thewestern and Nyanza regions of Kenya.Sequence analysisThe VP1/2 (980 bp) gene region amplifiedcorresponded to nucleotide positions 4102-5002 of thepublished sequence of the prototype strain. A searchfor homologies between this sequence and sequencesin the public database indicated that the Kenyansequences had 99% homology to human bocaviruses.The phylogenetic tree indicated two levels ofseparation. At the first level, HBoV2, 3, and 4 werefound to cluster together and were separated fromother HBoV isolates and the GBV (Figure 2).Interestingly, the data suggests that HBoV1 is moreclosely related to the gorilla bocavirus than it is toHBoV2, 3, and 4.Figure 2. Phylogenetic analysis of five isolates of humanbocavirus originating from KenyaSequences retrieved from GenBank for comparison are labeled as HK5,GBV, CRD2, ZA, and Bonn-1. The reference DNA sequences forbovine parvovirus and canine minute virus served as the out-groupsequences.224
Misigo et al. –Kenyan isolates of human bocavirusAt the second level of separation, the gorillabocavirus was separated from the rest of the isolates,which were found to cluster together. The lattercomprised the five isolates from Kenya and CRD2,Bonn-1, HK5, CRD2, ZA 190-05, ZA2591/04, andhuman bocavirus 1. The clustering of the tree wassupported by high bootstrap values of 61%-100%.Accession numbersFive Kenyan sequences of the HBoV-VP1/VP2gene representing DNA of five samples weredeposited in GenBank and have been 42 (KE-23A8/11), HQ288043 (KE-10N8/3),HQ326235 (KE- 48G7/8) and HQ326236 (KE4S9/04).DiscussionThe first comprehensive investigation of the roleof viruses in causing acute respiratory infections inKenya dates back to the mid-1980s, when Hazlett etal. [12] isolated a large proportion of viral agents,many of which were considered less important then.Currently, that trend has greatly changed due to acomplex interaction of viruses with other factors in thehuman population and environment resulting in theresurgence and emergence of new viral pathogens. Inthe Kenya Demographic Health Survey (KDHS) reportof 2009, it was estimated that countrywide, 56% of allcases of outpatient clinic consultations in childrenbelow five years of age were due to repeated episodesof acute respiratory tract infections. These childrengenerally present with complaints of coughaccompanied by congestion and difficulty in breathingor fever. These signs perhaps are often erroneouslyattributed to influenza virus infections, butnevertheless rarely warrant initiation of aninvestigation of the etiological agent responsible. It ismore likely that some of these respiratory infectionsare caused by the newer emerging pathogens thatinclude human bocavirus, metapneumovirus, andhuman coronaviruses. Globally, as reviewed byAllander [15], human bocavirus has variably beendetected in 1.5%-19% of individuals with acuterespiratory illness, with a noted frequency of detectionof between 5.0%-5.5% in most of the studies.A review of the literature indicates that there hasbeen no study specifically investigating the presenceof HBoV in respiratory infections in Kenya. The mainobjective of this report was to demonstrate thepresence of HBoV in Kenya as a co-infecting agent, todescribe the genotype in circulation, and to establishJ Infect DevCtries 2014; 8(2):221-227.its phylogenetic relationship with isolates from otherregions. Here we report the presence of HBoV inKenya among patients with upper respiratoryinfections. Our findings suggest that HBoV1-likelineage (St2) is the main strain circulating in Kenya. Inthis study, the total number of HBoV isolated was1.8%, and it is unclear as to whether or not thisstatistic implies that HBoV plays a relativelyunimportant role in acute respiratory infections inKenya. However, the findings show that the virus istically, all of the seven individuals inwhom the virus DNA was isolated ranged in age froma few months to less than two years, and were alsoinfected with one of the other regular respiratoryviruses. This observation is consistent with theobservations of many other authors [16-18]. Oneexplanation that is consistently put forward to accountfor these observations is that maternal antibodiesprovide protection to children younger than ninemonths of age, meaning that, as the concentration ofmaternal IgG declines, the incidence will increase asthe children grow older [19-21].In regards to the clinical presentation of infectionsinvolving HBoV, it seems that the general symptomsthat usher in influenza-like illnesses are common to awide variety of viral infections, and thus there is noone single sign that can be said to be pathognomonicfor HBoV infections. The presence of vomiting insome of the individuals suggested gastric discomfort,even though there was no record of diarrhea episodes.These symptoms of gastroenteritis have beenhighlighted in other studies. For example, one of thereasons for seeking health care services for acuterespiratory infections in 10% of the children in whomHBoV was isolated in Italy was gastroenteritis [22].Perhaps HBoV is a pneumoenteric virus, meaning thatunder certain settings, HBoV infection might beinitiated at the respiratory site before it extends toinvolve the intestinal sites. However, examiningwhether HBoV was the c
Original Article Molecular detection and phylogenetic analysis of Kenyan human bocavirus isolates Dennis Misigo1,2, Dufton Mwaengo3, David Mburu4 1 Department of Microbiology and Parasitology, Kenya Methodist University, Meru, Kenya 2 Institute of Tropical and Infectious Diseases, University of Nairobi, Nairobi, Kenya 3 Department of Medical Microbiology, University of Nairobi, Nairobi, Kenya
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