research article novel gyroviruses, including chicken

Research ArticleNovel Gyroviruses including Chicken AnaemiaVirus in Clinical and Chicken Samples from South Africa

Heidi E M Smuts

Division Medical VirologyNational Health Laboratory Service Department of Clinical Sciences Faculty of Health SciencesUniversity of Cape Town Observatory 7925 South Africa

Correspondence should be addressed to Heidi E M Smuts heidismutsuctacza

Received 17 December 2013 Accepted 17 March 2014 Published 29 April 2014

Academic Editor Finn S Pedersen

Copyright copy 2014 Heidi E M Smuts This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Introduction Chicken anaemia virus CAV was until recently the only member of theGyrovirus genus 6 novel gyroviruses AGV2HGyV1 and GyV3-6 have since been discovered in human and chicken samplesMethods PCR amplification of the VP2 gene wasused to detect AGV2HGyV1 GyV3 and CAV in a range of clinical samples including stool respiratory CSF and HIV-positiveplasma Screening of fresh local chicken meat was also performed Results AGV2HGyV1 or GyV3 was detected in stools fromhealthy children (1749 347) and patients with diarrhoea (22149 148) 12 (3246) nasopharyngeal respiratory samples werepositive No AGV2HGyV1 or GyV3 was detected in nasal swabs from wheezing patients in CSF from patients with meningitisand in HIVpositive plasma CAV was found in 51 (2549) of stools from healthy children and 16 (24149) in diarrhoea samplesScreening of 28 chicken samples showed a higher prevalence of gyrovirus (2028 71) compared to CAV (128 36) Phylogeneticanalysis of the CAV VP1 gene showed South African sequences clustering with Brazilian isolates from genotypes D2 and A2Conclusion Novel gyroviruses including CAV are present in the South African population with diarrhoea and respiratory illnessas well as in healthy children Their presence suggests an origin from chicken meat consumption

1 Introduction

Until recently chicken anaemia virus (CAV) was the onlymember of the genus Gyrovirus in the Circoviridae familyThis genus is characterized by small nonenveloped DNAviruses with a negative sense single-stranded circular DNA ofabout 23 kb [1] Circoviruses in contrast have an ambisensegenomeThe similarity of the gyrovirus genome organizationto annelloviruses with 3 overlapping open reading frames(ORFs) has led to the recommendation that gyrovirusesbecome a subfamily Gyrovirinae within the Anelloviridae-family [2]

In early 2011 Rijsewijk et al [3] reported the discoveryof a distant relative to CAV avian gyrovirus 2 (AGV2) indiseased chicken from Brazil with only 40 homology toCAV Later that year Sauvage et al [4] identified a very closelyrelated gyrovirus on human skin (HGyV1) Subsequently4 other novel gyroviruses have been described Gyrovirus3 (GyV3) was identified by viral metagenomics in faecesfrom Chilean children with acute gastroenteritis and also

in chicken meat [5] A phylogenetically distinct gyrovirus(GyV4) was also discovered in both human stool samples andchicken meat by 454 pyrosequencing [6] Further 2 divergentgyroviruses GyV5 and GyV6 were found in the stools ofTunisian children with diarrhoea [7]

CAV is an economically important pathogen in the poul-try industry causing severe anaemia and immunosuppressionin young 2-3-week-old chicken that lack protective maternalantibodies [8] In adult chickens CAV infection is subclinicalbut financial lossesmay be incurred by poultry farmers due tolack of weight gain and susceptibility to secondary infections

The role of CAV in the African poultry context has beenaddressed by a number of researchers [9ndash16] Seroprevalencein Nigeria Egypt Central African Republic and Cameroonranged from 37 to 89 depending on age of chickenFurther CAV DNA could be detected in the majority (77)of seropositive chickens indicating persistent virus sheddingin the presence of antibodies To date there has only been onebrief report on CAV in South African chickens [17] where 3broiler chickens were seropositive

Hindawi Publishing CorporationAdvances in VirologyVolume 2014 Article ID 321284 7 pageshttpdxdoiorg1011552014321284

2 Advances in Virology

The aim of this study was to determine if some of thenovel gyroviruses and CAV are present in the South Africanpopulation and in chickens from this region

2 Materials and Methods

21 Clinical Samples Anonymized stool samples from 49healthy children aged 6ndash36 months were obtained withparentalguardian consent from a creche over 2 periodssummer (FebruaryMarch 2006) and winter (JulyAugust2006) Stool samples from patients with diarrhoea submittedto the virology and microbiology diagnostic laboratory overthe same time periods in 2006 were also examined (119899 = 149)

246 nasopharyngeal respiratory samples from childrenaged 1ndash60 months previously screened for the 7 commonrespiratory viruses (adenovirus human respiratory syncytialvirus human metapneumovirus influenza A and B parain-fluenza virus 1ndash3 and human rhinovirus A) were tested ofwhich 152 were negative and the remainder (119899 = 94) werepositive for one of the above viruses

Nasal swabs (119899 = 48) collected in May 2004 from chil-dren aged 6ndash25 months with acute wheezing were alsoscreened Informed consent from a parent or guardian hadbeen obtained

The presence of gyrovirus in plasma from HIV-infectedpatients (119899 = 48) was evaluated as Maggi et al [18] hadidentified HGyV1 in one Italian patient The CD4120583L countranged from 13 to 1065 with a mean of 397

94 cerebrospinal fluid (CSF) samples from patients withsuspected meningitis were also screened

22 Chicken Meat Fresh chicken meat for human consump-tionwas purchased inAugust 2012 from4 retail stores inCapeTown South Africa These included 4 thighs and 4 drumsticks from3of 4 stores and 4 thighs from the remaining store

A sterile scalpel was used to remove a small piece of theskin and underlying meat from each sample This was placedin a sterile 15mL Eppendorf tube and stored at minus20∘C untilextracted

23 DNA Extraction Total nucleic acid was extracted fromnasopharyngeal respiratory samples using the MagNA PureLC automated extraction method as per manufacturerrsquosinstructions (Roche Diagnostics GmbH Penzberg Ger-many) Nucleic acid was extracted from stool nasal swabCSF plasma and chicken meat samples using the QIAampDNA stool mini kit and QIAamp DNA mini kit with proto-cols appropriate for body fluid or tissue as per manufacturerrsquosinstructions (Qiagen Hilden Germany)

24 PCR for Detection of Gyrovirus or CAV Consensus prim-ers targeting the conserved VP2 region of the gyrovirusesCAV AGV2HGyV1 GyV3 and GyV6 were used as outerprimers as described by Phan et al [5] Nested primersspecific for either CAV (CAV F1n 51015840 GGCAGTGAATCG-GCGCTTAGCCG and CAV R1n 51015840 AGTCGCTTGAGGTG-GTGCCACCG) or AGV2HGyV1 and GyV3 (ConGy F1n51015840 GGCAGTGAATTGCCGCTTAGGC and ConGy R1n 51015840

CGCAGTCTGTGTCTCCAGTGC) were used to improvesensitivity The inner PCR product was sim550 bp in size ThePCR conditions for the first round were as follows denatura-tion at 95∘C for 3minutes 40 cycles of 95∘C for 15 sec 45∘C for25 sec and 72∘C for 35 sec followed by a final 72∘C elongationstep for 7min The second round of amplification was thesame as above with an increase in the annealing temperatureto 55∘C PCR products were visualised by electrophoresisthrough a 2agarose gel ethidiumbromide staining andUVillumination GyV4 GyV5 and GyV6 are not amplified withthe nested primers and therefore this study did not measuretheir prevalence

25 Sanger Sequencing The VP2 amplicons were sequenceddirectly in both directions using either CAV or GyV nestedPCR primers The BigDye terminator cycle sequencing kitwas used (Applied Biosystems Foster City CA USA) TheVP2 genes were aligned with reference sequences from Gen-Bank using ClustalW [19] and phylogenetic trees constructedusing MEGA 505 [20] with 1000 bootstrap resamplingsBLAST analysis was also undertaken

Improved phylogenetic resolution of CAV positive sam-ples was investigated by amplification of the 1349 bp of theVP1 and overlapping VP2 and VP3 genes using primers pre-viously described by Natesan et al [21] and Eltahir et al [22]Increased sensitivity was achieved by the design of a nestedVP1 forward primer (51015840CCGCAAGAAGTATAAGAC)

3 Results

31 Gyrovirus in Clinical Samples AGV2HGyV1 or GyV3was detected in stools from both healthy children (1749347) and patients with diarrhoea (22149 148) (Table 1)The detection rate in respiratory samples was considerablylower at 13 (2152) in samples negative for the com-mon respiratory viruses and 11 (194) in positive samples(Table 1) In the latter sample parainfluenza 1ndash3 was previ-ously detected using the Seeplex RV7 assay (Seegene SeoulSouth Korea) NoAGV2HGyV1 or GyV3was detected in thenasal swabs from children with acute wheezing in plasmafrom HIV-infected individuals or in the CSF from patientswith suspected meningitis

32 CAV in Stool Samples CAV was detected in 51 (2549)stool samples from healthy children and 161 (24149)in diarrhoea samples (Table 1) Nasopharyngeal respiratorysamples nasal swabs HIV-positive plasma andCSFwere notscreened for the presence of CAV

Dual infection with both CAV and AGV2HGyV1GyV3was found in 1049 (204) and 8149 (54) stools fromhealthy children and patients with diarrhoea respectivelyCodetection of CAV and AGV2HGyV1GyV3 was not sta-tistically linked (119875 = 085)

33 CAVGyrovirus in Chicken Meat Screening of 28chicken samples for the presence of CAV DNA or AGV2HGyV1GyV3DNA showed a considerably higher prevalenceof gyrovirus (2028 714) compared to CAV (128 36)

Advances in Virology 3

Table 1 PCR screening of clinical and chicken samples for thepresence of AGV2HGyV1 and CAV

Samples AGV2HGyV1Number ()

CAVNumber ()

Control stool 1749 (347) 2549 (51)Diarrhoea stool 22149 (148) 24149 (161)Negative respiratory virusNPA 2152 (13) ND

Positive respiratory virusNPA 194 (11) ND

HIV-positive plasma 048 NDAcute wheezing nasalswabs 048 ND

Meningitis CSF 094 NDChicken meat store P 78 (875) 08Chicken meat store W 88 (100) 08Chicken meat store C 18 (125) 08Chicken meat store S 44 (100) 14 (25)NPA nasopharyngeal aspirates ND not done

From one store C 78 samples were negative for bothgyrovirus and CAV while chicken meat obtained from theremaining 3 stores was positive for AGV2HGyV1 with onlyone sample coinfected with CAV (Table 1)

34 Phylogenetic and Amino Acid Analysis Phylogeneticanalysis of the VP2 region of samples successfully sequencedshowed the presence of only AGV2HGyV1 with 563(1832) grouping with AGV2 sequences and 435 (1432)with HGyV1 sequences (Figure 1(a)) No GyV3 was detectedAll 3 respiratory samples clustered with AGV2 sequencesOf the chicken samples successfully sequenced 69 hadsequence homology to HGyV1 and the remaining 3 sampleshomology with AGV2 sequences

The VP1 region of CAV provided phylogenetic informa-tion for the classification of isolates into different genotypes(Figure 1(b)) Analysis of the 1349 bp region showed that67 CAV-positive samples grouped together with genotypeD particularly D2 isolates from USA Japan Malaysia andAustralia The remaining sample clustered within genotypeA but formed a separate lineage with a high bootstrap value(data not shown) Phylogenetic analysis of a truncated VP1region (481 bp) to allow inclusion of VP1 sequences fromBrazil (AY855079-88) retained the phylogeny as determinedusing the larger VP1 region but now showed that all SouthAfrica isolates clusteredwith the Brazilian sequences of eithergenotype A or D (Figure 1(b))

Analysis of the deduced 410 amino acids of CAV VP1protein revealed that no amino acids were characteristic ofthe South African isolates although a proline (P) at position286was found in all sequences and in commonwith 2 isolatesfrom Japan and 1 each from Australia and Malaysia Theregion responsible for virulence [23] showed a glutamine (Q)at position 394 instead of histidine indicative of a highlyvirulent strain (data not shown)

4 Discussion

The recent identification of novel gyroviruses in the humanpopulation led to the screening of a range of clinical sam-ples from patients and healthy children from South AfricaThe present study confirms the presence of AGV2HGyV1and CAV in this region GyV3 was not detected and thisstudy did not test for GyV4 GyV5 and GyV6 As limitedstudies on gyroviruses in healthy and sick individuals areavailable the pathogenesis and clinical importance are notwell understoodHGyV1may be amember of the skin virome[4] although Chu et al [6] were unable to detect gyrovirusesin skin swabs from healthy individuals Novel gyroviruseshave been detected in stools from patients with diarrhoeawith a prevalence of 1ndash93 while no gyrovirus was detectedin stools from healthy individuals [5 6] The present studyshowed a higher prevalence of gyroviruses (AGV2HGyV1)in stools from patients with diarrhoea (15) while 35 ofhealthy children also had evidence of AGV2HGyV1 DNAChu et al [6] demonstrated that gyroviruses were morecommonly detected in patients younger than 20 years of age

Viruses found in the stool samples may also play arole in respiratory and systemic diseases This was investi-gated by screening respiratory samples from young patientshospitalized with respiratory illness and children attendingan outpatient hospital clinic with acute wheezing AGV2was detected in 3 hospitalized patients with one patientcoinfected with parainfluenza virus 1ndash3 Further studies arerequired to determine if gyroviruses can cause respiratoryinfections requiring hospitalization

Gyrovirus was not detected in blood from HIV-infectedindividuals although other studies have reported a lowprevalence in both blood healthy donors [24] and immuno-compromised HIV-positive and kidney transplant patients[18] Our results may reflect the small number of samplesscreened

Circoviruses are able to cross the blood-brain barrierand cause infections of the central nervous system Porcinecircovirus type 2 infection in piglets results in cerebellarvasculitis [25] while in humans TTV and novel cycloviruseswere found in the CSF of patients with subacute demen-tia unexplained paraplegia and acute CNS infections ofunknown aetiology [26ndash28] Screening of CSF samples frompatients with possible meningitis showed no evidence ofgyrovirus infection

CAVwas detected more frequently in stools from healthychildren (gt50) compared to those with diarrhoea (16)Prior studies by Phan et al [5] and Chu et al [6] also reporteda high CAV prevalence in faecal samples from healthydonors of 25 and 53 respectively This high detectionrate suggests that consumption of infected chicken meat maypossibly play a role However in this report screening offresh chicken meat purchased from a number of differentstores in the study area does not support this assumptionas only one positive CAV sample was found In contrasta significant proportion of chicken meat was positive forAGV2HGyV1This discrepancymay be explained by the factthat only fresh chicken meat purchased in 2012 was screenedwhile the clinical samples were collected from 2004ndash2006


JQ308212 HGyV1

STH-46-healthy

Chicken-P6L

STH-4-healthy

Chicken-C5L

Chicken-P1

STH-39-healthy

FR823283 HGyV1

Chicken-S1

NC015630 HGyV1

Chicken-P2

Chicken-W1

STH-53-healthy

STH-31-healthy

STH-42-healthy

STH-35-healthy

V06415-diarrhoea

JQ690763 AGV2

STH-47-healthy

SCH3165874-diarrhoea

RV4078-respiratory



RV5842-respiratory

STH-6-healthy

V083133-diarrhoea

STH-44-healthy

RV8347-respiratory

NC015396 AGV2

HM590588 AGV2

STH-33-healthy

Chicken-W5L

Chicken-W2

Chicken-W6L

STH-54-healthy

STH-41-healthy


V062119-diarrhoea

NC017091 GyV3

JQ308210 GyV399

73

81

0001

(a)

Figure 1 Continued


AY855081 Brazil

AF395114 Bangladesch A1

AY855080 Brazil

L14767USA A1

AY855084 Brazil

AF313470 USA A2

AY846844 China A3

AB046590 Japan A3

AY855086 Brazil

STH23-healthy

AF475908 China A2

AY855079 Brazil

D10068 Netherlands A2

AY040632 Malaysia A2

AB031296 Japan A2

AY855085 Brazil

AY843527 China A2

AF390038 Malaysia A2

M81223 Germany A2

M55918 Germany A2

D31965 Japan B

AF227982 Australia C

EF683159 Australia C

AF311900 USA D1

AY855083 Brazil

AB119448 Japan D2

AF285882 Malaysia D2

U65414 Australia D2

AB027470 Japan D2

STH44-healthy

STH33-healthy

STH19-healthy

AY855087 Brazil

STH28-healthy

AY855082 Brazil

STH47-healthy

AY855088 Brazil

STH27-healthy

100

99

99

98

95

88

86

78

87

84

93

0005

75

(b)

Figure 1 Phylogenetic trees generated by neighbour-joining analysis of the VP2 region of AGV2 HGyV1 and GyV3 (a) and CAV VP1 (b) ofSouth African isolates of human and chicken origin Reference sequences of AGV2 HGyV1 GyV3 and CAV were retrieved from the NCBIGenBank database and are indicated by accession numbers and country of origin Bootstrap values greater than 75 are indicated at thenodes of the tree The branch lengths are proportional to the evolutionary distances as shown on the scale


when imported frozen chicken products were more readilyavailable for consumption

A number of live attenuated CAV vaccines are availableand used in the poultry industry However evidence of thispractice occurring in the South African poultry industryis not readily available If undertaken breeder hens arevaccinated to obtain high levels of circulating antibodieswhich can be passed onto progeny preventing early infectionsand disease caused by CAV (personal communication DrNkuna South African Poultry Association)

From 2001 a significant amount of in-bone frozen chick-ens were imported from Brazil which in 2010 accounted forover 70 of the South African import market of chickens[29] This dropped substantially since 2012 with the intro-duction of stricter antidumping legislation These importedproducts were significantly cheaper than the locally sourcedchickens and may have been preferentially purchased andconsumed by a large proportion of the South African popu-lation Simionatto et al [30] reported that CAV was detectedin 90 of field samples collected from commercial breedersbroilers and free-range chickens from different regions ofBrazil Whether these chickens are infected with CAV eithernaturally or through vaccination programs is not known

Phylogenetic analysis of the VP1 region provides furtherevidence that CAV detected in this study may originate fromBrazil All the South African isolates of either genotype Dor A clustered together with Brazilian isolates Sourcing ofimported chicken products from the earlier time of humansample collection to confirm this association was not possiblein 2013

5 Conclusion

The role and clinical relevance of gyroviruses in human dis-ease require further investigation Larger studies on patientswith a variety of medical conditions are needed to elucidatethe pathogenesis of gyrovirus infection

Conflict of Interests

The author declares that there is no conflict of interestsregarding the publication of this paper

References

[1] P Biagini M Bendinelli S Hino et al Eds Virus TaxonomyNinth Report of the International Committee on Taxonomy ofViruses Elsevier Academic press New York NY USA 2011

[2] P Biagini ldquoRestructuring and expansion of the family Anell-oviradae Anellviradae-Circoviradae study grouprdquo Internatio-nal Committee on Taxonomy of Viruses ICTV files and discus-sion httptalkictvonlineorgfilesproposalstaxonomy-pro-posalsvertebrat1mvert013911aspx

[3] F A M Rijsewijk H F dos Santos T F Teixeira et al ldquoDiscov-ery of a genome of a distant relative of chicken anemia virusreveals a newmember of the genusGyrovirusrdquoArchives of Virol-ogy vol 156 no 6 pp 1097ndash1100 2011

[4] V Sauvage J Cheval V Foulongne et al ldquoIdentification of thefirst human Gyrovirus a virus related to chicken anemia virusrdquoJournal of Virology vol 85 no 15 pp 7948ndash7950 2011

[5] T G Phan L Li M G OrsquoRyan et al ldquoA third gyrovirus speciesin human faecesrdquo Journal of General Virology vol 93 no 6 pp1356ndash1361 2012

[6] DKWChu L LM Poon S S S Chiu et al ldquoCharacterizationof a novel gyrovirus in human stool and chicken meatrdquo Journalof Clinical Virology vol 55 no 3 pp 209ndash213 2012

[7] T G Phan N P Vo K Sdiri-Loulizi et al ldquoDivergent gyro-viruses in the feces of Tunisian childrenrdquo Journal of Virologyvol 446 no 1-2 pp 346ndash348 2013

[8] K A Schat ldquoChicken anemia virusrdquo Current Topics in Microbi-ology and Immunology vol 331 pp 151ndash183 2009

[9] C J Snoeck G F Komoyo B P Mbee et al ldquoEpidemiology ofchicken anaemia virus in Central Africa Republic and Cam-eroonrdquo Journal of Virology vol 9 pp 189ndash197 2012

[10] A M Hegazy F M Abdallah L K Abd-El Samie and A ANazim ldquoChicken infectious anemia virus (CIAV) in broilers andlaying hens in Sharkia Province Egyptrdquo Journal of AmericanScience vol 6 pp 752ndash758 2010

[11] D O Oluwayelu D Todd and O D Olaleye ldquoSequence andphylogenetic analysis of chicken anaemia virus obtained frombackyard and commercial chickens in Nigeriardquo OnderstepoortJournal of Veterinary Research vol 75 no 4 pp 353ndash357 2008

[12] M F Ducatez A A Owoade J O Abiola and C P MullerldquoMolecular epidemiology of chicken anemia virus in NigeriardquoArchives of Virology vol 151 no 1 pp 97ndash111 2006

[13] B O Emikpe D O Oluwayelu O G Ohore O A Oladeleand A T Oladokun ldquoSerological evidence of chicken anaemiavirus infection in Nigerian indigenous chickensrdquoOnderstepoortJournal of Veterinary Research vol 72 no 1 pp 101ndash103 2005

[14] D O Oluwayelu D Todd N W Ball et al ldquoIsolation and pre-liminary characterization of chicken anemia virus from chick-ens inNigeriardquoAvianDiseases vol 49 no 3 pp 446ndash450 2005

[15] A A Owoade D O Oluwayelu O A Fagbohun W Ammer-laan M N Mulders and C P Muller ldquoSerologic evidence ofchicken infectious anemia in commercial chicken flocks insouthwest Nigeriardquo Avian Diseases vol 48 no 1 pp 202ndash2052004

[16] H A Hussein M Z Sabry E A El-Ibiary M El-Safty and AI Abd El-Hady ldquoChicken infectious anaemia virus in Egyptmolecular diagnosis by PCR and isolation of the virus frominfected flocksrdquo Arab Journal of Biotechnology vol 5 no 2 pp263ndash274 2002

[17] J DWicht and S B Maharaj ldquoChicken anaemia agent in SouthAfricardquo Veterinary Record vol 133 no 6 pp 147ndash148 1993

[18] F Maggi L Macera D Focosi et al ldquoHuman gyrovirus DNA inhuman blood Italyrdquo Emerging Infectious Diseases vol 18 no 6pp 956ndash959 2012

[19] J D Thompson D G Higgins and T J Gibson ldquoCLUSTALW improving the sensitivity of progressive multiple sequencealignment through sequence weighting position-specific gappenalties and weight matrix choicerdquoNucleic Acids Research vol22 no 22 pp 4673ndash4680 1994

[20] K Tamura D Peterson N Peterson G Stecher M Nei andS Kumar ldquoMEGA5 molecular evolutionary genetics analysisusing maximum likelihood evolutionary distance and max-imum parsimony methodsrdquo Molecular Biology and Evolutionvol 28 no 10 pp 2731ndash2739 2011

[21] S Natesan JMKataria KDhama S Rahul andN BaradhwajldquoBiological and molecular characterization of chicken anaemiavirus isolates of Indian originrdquo Virus Research vol 118 no 1-2pp 78ndash86 2006


[22] Y M Eltahir K Qian W Jin P Wang and A Qin ldquoMolecularepidemiology of chicken anemia virus in commercial farms inChinardquo Journal of Virology vol 8 pp 145ndash152 2011

[23] S Yamaguchi T Imada N Kaji et al ldquoIdentification of a geneticdeterminant of pathogenicity in chicken anaemia virusrdquo Journalof General Virology vol 82 no 5 pp 1233ndash1238 2001

[24] P Biagini S BedaridaM Touinssi V Galicher and P deMiccoldquoHuman gyrovirus in healthy blood donors Francerdquo EmergingInfectious Diseases vol 19 no 6 pp 1014ndash1015 2013

[25] F A Seeliger M L Brugmann L Kruger et al ldquoPorcinecircovirus type 2-associated cerebellar vasculitis in postweaningmultisystemic wasting syndrome (PMWS)-affected pigsrdquo Vet-erinary Pathology vol 44 no 5 pp 621ndash634 2007

[26] S Honma S Nakata Y Sakai M Tatsumi K Numata-Kin-oshita and S Chiba ldquoLow prevalence of TT virus in the cere-brospinal fluid of viremic patients with central nervous systemdisordersrdquo Journal of Medical Virology vol 65 no 2 pp 418ndash422 2001

[27] S L Smits E E Zijlstra J J van Hellemond et al ldquoNovelcyclovirus in human cerebrospinal fluid Malawi 2010-2011rdquoEmerging Infectious Diseases vol 19 no 9 pp 1511ndash1513 2013

[28] L V Tan H R van Doorn H D Nghia et al ldquoIdentification ofa new cyclovirus in cerebrospinal fluid of patients with acutecentral nervous system infectionsrdquo MBio vol 4 no 3 ppe00231ndashe00213 2013

[29] D Esterhuizen ldquoSouth African poultry update the supply anddemand for broiler meat in South Africardquo httpwwwthepoul-trysitecomreportsid=1795

[30] S Simionatto C A da Veiga Lima-Rosa E Binneck A P Rav-azzolo and C W Canal ldquoCharacterization and phylogeneticanalysis of Brazilian chicken anaemia virusrdquo Virus Genes vol33 no 1 pp 5ndash10 2006

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International Journal of

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Advances in

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Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


The aim of this study was to determine if some of thenovel gyroviruses and CAV are present in the South Africanpopulation and in chickens from this region

2 Materials and Methods

21 Clinical Samples Anonymized stool samples from 49healthy children aged 6ndash36 months were obtained withparentalguardian consent from a creche over 2 periodssummer (FebruaryMarch 2006) and winter (JulyAugust2006) Stool samples from patients with diarrhoea submittedto the virology and microbiology diagnostic laboratory overthe same time periods in 2006 were also examined (119899 = 149)

246 nasopharyngeal respiratory samples from childrenaged 1ndash60 months previously screened for the 7 commonrespiratory viruses (adenovirus human respiratory syncytialvirus human metapneumovirus influenza A and B parain-fluenza virus 1ndash3 and human rhinovirus A) were tested ofwhich 152 were negative and the remainder (119899 = 94) werepositive for one of the above viruses

Nasal swabs (119899 = 48) collected in May 2004 from chil-dren aged 6ndash25 months with acute wheezing were alsoscreened Informed consent from a parent or guardian hadbeen obtained

The presence of gyrovirus in plasma from HIV-infectedpatients (119899 = 48) was evaluated as Maggi et al [18] hadidentified HGyV1 in one Italian patient The CD4120583L countranged from 13 to 1065 with a mean of 397

94 cerebrospinal fluid (CSF) samples from patients withsuspected meningitis were also screened

22 Chicken Meat Fresh chicken meat for human consump-tionwas purchased inAugust 2012 from4 retail stores inCapeTown South Africa These included 4 thighs and 4 drumsticks from3of 4 stores and 4 thighs from the remaining store

A sterile scalpel was used to remove a small piece of theskin and underlying meat from each sample This was placedin a sterile 15mL Eppendorf tube and stored at minus20∘C untilextracted

23 DNA Extraction Total nucleic acid was extracted fromnasopharyngeal respiratory samples using the MagNA PureLC automated extraction method as per manufacturerrsquosinstructions (Roche Diagnostics GmbH Penzberg Ger-many) Nucleic acid was extracted from stool nasal swabCSF plasma and chicken meat samples using the QIAampDNA stool mini kit and QIAamp DNA mini kit with proto-cols appropriate for body fluid or tissue as per manufacturerrsquosinstructions (Qiagen Hilden Germany)

24 PCR for Detection of Gyrovirus or CAV Consensus prim-ers targeting the conserved VP2 region of the gyrovirusesCAV AGV2HGyV1 GyV3 and GyV6 were used as outerprimers as described by Phan et al [5] Nested primersspecific for either CAV (CAV F1n 51015840 GGCAGTGAATCG-GCGCTTAGCCG and CAV R1n 51015840 AGTCGCTTGAGGTG-GTGCCACCG) or AGV2HGyV1 and GyV3 (ConGy F1n51015840 GGCAGTGAATTGCCGCTTAGGC and ConGy R1n 51015840

CGCAGTCTGTGTCTCCAGTGC) were used to improvesensitivity The inner PCR product was sim550 bp in size ThePCR conditions for the first round were as follows denatura-tion at 95∘C for 3minutes 40 cycles of 95∘C for 15 sec 45∘C for25 sec and 72∘C for 35 sec followed by a final 72∘C elongationstep for 7min The second round of amplification was thesame as above with an increase in the annealing temperatureto 55∘C PCR products were visualised by electrophoresisthrough a 2agarose gel ethidiumbromide staining andUVillumination GyV4 GyV5 and GyV6 are not amplified withthe nested primers and therefore this study did not measuretheir prevalence

25 Sanger Sequencing The VP2 amplicons were sequenceddirectly in both directions using either CAV or GyV nestedPCR primers The BigDye terminator cycle sequencing kitwas used (Applied Biosystems Foster City CA USA) TheVP2 genes were aligned with reference sequences from Gen-Bank using ClustalW [19] and phylogenetic trees constructedusing MEGA 505 [20] with 1000 bootstrap resamplingsBLAST analysis was also undertaken

Improved phylogenetic resolution of CAV positive sam-ples was investigated by amplification of the 1349 bp of theVP1 and overlapping VP2 and VP3 genes using primers pre-viously described by Natesan et al [21] and Eltahir et al [22]Increased sensitivity was achieved by the design of a nestedVP1 forward primer (51015840CCGCAAGAAGTATAAGAC)

3 Results

31 Gyrovirus in Clinical Samples AGV2HGyV1 or GyV3was detected in stools from both healthy children (1749347) and patients with diarrhoea (22149 148) (Table 1)The detection rate in respiratory samples was considerablylower at 13 (2152) in samples negative for the com-mon respiratory viruses and 11 (194) in positive samples(Table 1) In the latter sample parainfluenza 1ndash3 was previ-ously detected using the Seeplex RV7 assay (Seegene SeoulSouth Korea) NoAGV2HGyV1 or GyV3was detected in thenasal swabs from children with acute wheezing in plasmafrom HIV-infected individuals or in the CSF from patientswith suspected meningitis

32 CAV in Stool Samples CAV was detected in 51 (2549)stool samples from healthy children and 161 (24149)in diarrhoea samples (Table 1) Nasopharyngeal respiratorysamples nasal swabs HIV-positive plasma andCSFwere notscreened for the presence of CAV

Dual infection with both CAV and AGV2HGyV1GyV3was found in 1049 (204) and 8149 (54) stools fromhealthy children and patients with diarrhoea respectivelyCodetection of CAV and AGV2HGyV1GyV3 was not sta-tistically linked (119875 = 085)

33 CAVGyrovirus in Chicken Meat Screening of 28chicken samples for the presence of CAV DNA or AGV2HGyV1GyV3DNA showed a considerably higher prevalenceof gyrovirus (2028 714) compared to CAV (128 36)




CAVNumber ()









4 Discussion







JQ308212 HGyV1

STH-46-healthy

Chicken-P6L

STH-4-healthy

Chicken-C5L

Chicken-P1

STH-39-healthy

FR823283 HGyV1

Chicken-S1

NC015630 HGyV1

Chicken-P2

Chicken-W1

STH-53-healthy

STH-31-healthy

STH-42-healthy

STH-35-healthy

V06415-diarrhoea

JQ690763 AGV2

STH-47-healthy


RV4078-respiratory



RV5842-respiratory

STH-6-healthy

V083133-diarrhoea

STH-44-healthy

RV8347-respiratory

NC015396 AGV2

HM590588 AGV2

STH-33-healthy

Chicken-W5L

Chicken-W2

Chicken-W6L

STH-54-healthy

STH-41-healthy


V062119-diarrhoea

NC017091 GyV3

JQ308210 GyV399

73

81

0001

(a)

Figure 1 Continued


AY855081 Brazil


AY855080 Brazil

L14767USA A1

AY855084 Brazil

AF313470 USA A2

AY846844 China A3

AB046590 Japan A3

AY855086 Brazil

STH23-healthy

AF475908 China A2

AY855079 Brazil



AB031296 Japan A2

AY855085 Brazil

AY843527 China A2


M81223 Germany A2

M55918 Germany A2

D31965 Japan B



AF311900 USA D1

AY855083 Brazil

AB119448 Japan D2


U65414 Australia D2

AB027470 Japan D2

STH44-healthy

STH33-healthy

STH19-healthy

AY855087 Brazil

STH28-healthy

AY855082 Brazil

STH47-healthy

AY855088 Brazil

STH27-healthy

100

99

99

98

95

88

86

78

87

84

93

0005

75

(b)







5 Conclusion




References







































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




CAVNumber ()









4 Discussion







JQ308212 HGyV1

STH-46-healthy

Chicken-P6L

STH-4-healthy

Chicken-C5L

Chicken-P1

STH-39-healthy

FR823283 HGyV1

Chicken-S1

NC015630 HGyV1

Chicken-P2

Chicken-W1

STH-53-healthy

STH-31-healthy

STH-42-healthy

STH-35-healthy

V06415-diarrhoea

JQ690763 AGV2

STH-47-healthy


RV4078-respiratory



RV5842-respiratory

STH-6-healthy

V083133-diarrhoea

STH-44-healthy

RV8347-respiratory

NC015396 AGV2

HM590588 AGV2

STH-33-healthy

Chicken-W5L

Chicken-W2

Chicken-W6L

STH-54-healthy

STH-41-healthy


V062119-diarrhoea

NC017091 GyV3

JQ308210 GyV399

73

81

0001

(a)

Figure 1 Continued


AY855081 Brazil


AY855080 Brazil

L14767USA A1

AY855084 Brazil

AF313470 USA A2

AY846844 China A3

AB046590 Japan A3

AY855086 Brazil

STH23-healthy

AF475908 China A2

AY855079 Brazil



AB031296 Japan A2

AY855085 Brazil

AY843527 China A2


M81223 Germany A2

M55918 Germany A2

D31965 Japan B



AF311900 USA D1

AY855083 Brazil

AB119448 Japan D2


U65414 Australia D2

AB027470 Japan D2

STH44-healthy

STH33-healthy

STH19-healthy

AY855087 Brazil

STH28-healthy

AY855082 Brazil

STH47-healthy

AY855088 Brazil

STH27-healthy

100

99

99

98

95

88

86

78

87

84

93

0005

75

(b)







5 Conclusion




References







































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


JQ308212 HGyV1

STH-46-healthy

Chicken-P6L

STH-4-healthy

Chicken-C5L

Chicken-P1

STH-39-healthy

FR823283 HGyV1

Chicken-S1

NC015630 HGyV1

Chicken-P2

Chicken-W1

STH-53-healthy

STH-31-healthy

STH-42-healthy

STH-35-healthy

V06415-diarrhoea

JQ690763 AGV2

STH-47-healthy


RV4078-respiratory



RV5842-respiratory

STH-6-healthy

V083133-diarrhoea

STH-44-healthy

RV8347-respiratory

NC015396 AGV2

HM590588 AGV2

STH-33-healthy

Chicken-W5L

Chicken-W2

Chicken-W6L

STH-54-healthy

STH-41-healthy


V062119-diarrhoea

NC017091 GyV3

JQ308210 GyV399

73

81

0001

(a)

Figure 1 Continued


AY855081 Brazil


AY855080 Brazil

L14767USA A1

AY855084 Brazil

AF313470 USA A2

AY846844 China A3

AB046590 Japan A3

AY855086 Brazil

STH23-healthy

AF475908 China A2

AY855079 Brazil



AB031296 Japan A2

AY855085 Brazil

AY843527 China A2


M81223 Germany A2

M55918 Germany A2

D31965 Japan B



AF311900 USA D1

AY855083 Brazil

AB119448 Japan D2


U65414 Australia D2

AB027470 Japan D2

STH44-healthy

STH33-healthy

STH19-healthy

AY855087 Brazil

STH28-healthy

AY855082 Brazil

STH47-healthy

AY855088 Brazil

STH27-healthy

100

99

99

98

95

88

86

78

87

84

93

0005

75

(b)







5 Conclusion




References







































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


AY855081 Brazil


AY855080 Brazil

L14767USA A1

AY855084 Brazil

AF313470 USA A2

AY846844 China A3

AB046590 Japan A3

AY855086 Brazil

STH23-healthy

AF475908 China A2

AY855079 Brazil



AB031296 Japan A2

AY855085 Brazil

AY843527 China A2


M81223 Germany A2

M55918 Germany A2

D31965 Japan B



AF311900 USA D1

AY855083 Brazil

AB119448 Japan D2


U65414 Australia D2

AB027470 Japan D2

STH44-healthy

STH33-healthy

STH19-healthy

AY855087 Brazil

STH28-healthy

AY855082 Brazil

STH47-healthy

AY855088 Brazil

STH27-healthy

100

99

99

98

95

88

86

78

87

84

93

0005

75

(b)







5 Conclusion




References







































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






5 Conclusion




References







































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

research article novel gyroviruses, including chicken

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