Novel Gyroviruses, including Chicken Anaemia Virus, in Clinical and Chicken Samples from South Africa

Hindawi Publishing Corporation Advances in Virology Volume 2014, Article ID 321284, 7 pages http://dx.doi.org/10.1155/2014/321284 Research Article Novel Gyroviruses, including Chicken Anaemia Virus, in Clinical and Chicken Samples from South Africa Heidi E. M. Smuts Division Medical Virology/National Health Laboratory Service, Department of Clinical Sciences, Faculty of Health Sciences, University of Cape Town, Observatory 7925, South Africa Correspondence should be addressed to Heidi E. M. Smuts; Received 17 December 2013; Accepted 17 March 2014; Published 29 April 2014 Academic Editor: Finn S. Pedersen Copyright © 2014 Heidi E. M. Smuts. 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. Introduction. Chicken anaemia virus, CAV, was until recently the only member of the Gyrovirus genus. 6 novel gyroviruses, AGV2, HGyV1, and GyV3-6, have since been discovered in human and chicken samples. Methods. PCR amplification of the VP2 gene was used to detect AGV2/HGyV1, GyV3, and CAV in a range of clinical samples including stool, respiratory, CSF, and HIV-positive plasma. Screening of fresh local chicken meat was also performed. Results. AGV2/HGyV1 or GyV3 was detected in stools from healthy children (17/49, 34.7%) and patients with diarrhoea (22/149, 14.8%). 1.2% (3/246) nasopharyngeal respiratory samples were positive. No AGV2/HGyV1 or GyV3 was detected in nasal swabs from wheezing patients, in CSF from patients with meningitis, and in HIVpositive plasma. CAV was found in 51% (25/49) of stools from healthy children and 16% (24/149) in diarrhoea samples. Screening of 28 chicken samples showed a higher prevalence of gyrovirus (20/28, 71%) compared to CAV (1/28, 3.6%). Phylogenetic analysis of the CAV VP1 gene showed South African sequences clustering with Brazilian isolates from genotypes D2 and A2. Conclusion. Novel gyroviruses, including CAV, are present in the South African population with diarrhoea and respiratory illness as well as in healthy children. Their presence suggests an origin from chicken meat consumption. 1. Introduction Until recently chicken anaemia virus (CAV) was the only member of the genus Gyrovirus in the Circoviridae family. This genus is characterized by small nonenveloped DNA viruses with a negative sense single-stranded circular DNA of about 2.3 kb [1]. Circoviruses, in contrast, have an ambisense genome. The similarity of the gyrovirus genome organization to annelloviruses, with 3 overlapping open reading frames (ORFs), has led to the recommendation that gyroviruses become a subfamily, Gyrovirinae, within the Anelloviridaefamily [2]. In early 2011 Rijsewijk et al. [3] reported the discovery of a distant relative to CAV, avian gyrovirus 2 (AGV2), in diseased chicken from Brazil, with only 40% homology to CAV. Later that year, Sauvage et al. [4] identified a very closely related gyrovirus on human skin (HGyV1). Subsequently 4 other novel gyroviruses have been described. Gyrovirus 3 (GyV3) was identified by viral metagenomics in faeces from Chilean children with acute gastroenteritis and also in chicken meat [5]. A phylogenetically distinct gyrovirus (GyV4) was also discovered in both human stool samples and chicken meat by 454 pyrosequencing [6]. Further 2 divergent gyroviruses, GyV5 and GyV6, were found in the stools of Tunisian children with diarrhoea [7]. CAV is an economically important pathogen in the poultry industry causing severe anaemia and immunosuppression in young 2-3-week-old chicken that lack protective maternal antibodies [8]. In adult chickens CAV infection is subclinical, but financial losses may be incurred by poultry farmers due to lack of weight gain and susceptibility to secondary infections. The role of CAV in the African poultry context has been addressed by a number of researchers [9–16]. Seroprevalence in Nigeria, Egypt, Central African Republic, and Cameroon ranged from 37% to 89% depending on age of chicken. Further, CAV DNA could be detected in the majority (77%) of seropositive chickens, indicating persistent virus shedding in the presence of antibodies. To date there has only been one brief report on CAV in South African chickens [17], where 3 broiler chickens were seropositive. 2 The aim of this study was to determine if some of the novel gyroviruses and CAV are present in the South African population and in chickens from this region. 2. Materials and Methods 2.1. Clinical Samples. Anonymized stool samples from 49 healthy children, aged 6–36 months, were obtained with parental/guardian consent from a crèche over 2 periods: summer (February/March 2006) and winter (July/August 2006). Stool samples from patients with diarrhoea submitted to the virology and microbiology diagnostic laboratory over the same time periods in 2006 were also examined (𝑛 = 149). 246 nasopharyngeal respiratory samples from children aged 1–60 months previously screened for the 7 common respiratory viruses (adenovirus, human respiratory syncytial virus, human metapneumovirus, influenza A and B, parainfluenza virus 1–3, and human rhinovirus A) were tested, of which 152 were negative and the remainder (𝑛 = 94) were positive for one of the above viruses. Nasal swabs (𝑛 = 48) collected in May 2004 from children aged 6–25 months with acute wheezing were also screened. Informed consent from a parent or guardian had been obtained. The presence of gyrovirus in plasma from HIV-infected patients (𝑛 = 48) was evaluated as Maggi et al. [18] had identified HGyV1 in one Italian patient. The CD4/𝜇L count ranged from 13 to 1065 with a mean of 397. 94 cerebrospinal fluid (CSF) samples from patients with suspected meningitis were also screened. 2.2. Chicken Meat. Fresh chicken meat for human consumption was purchased in August 2012 from 4 retail stores in Cape Town, South Africa. These included 4 thighs and 4 drum sticks from 3 of 4 stores and 4 thighs from the remaining store. A sterile scalpel was used to remove a small piece of the skin and underlying meat from each sample. This was placed in a sterile 1.5 mL Eppendorf tube and stored at −20∘ C until extracted. 2.3. DNA Extraction. Total nucleic acid was extracted from nasopharyngeal respiratory samples using the MagNA Pure LC automated extraction method as per manufacturer’s instructions (Roche Diagnostics GmbH, Penzberg, Germany). Nucleic acid was extracted from stool, nasal swab, CSF, plasma, and chicken meat samples using the QIAamp DNA stool mini kit and QIAamp DNA mini kit, with protocols appropriate for body fluid or tissue as per manufacturer’s instructions (Qiagen, Hilden, Germany). 2.4. PCR for Detection of Gyrovirus or CAV. Consensus primers targeting the conserved VP2 region of the gyroviruses, CAV, AGV2/HGyV1, GyV3, and GyV6, were used as outer primers as described by Phan et al. [5]. Nested primers specific for either CAV (CAV F1n (...truncated)