Marianne Wolfaardt

Phylogenetic analysis of the caliciviruses

A phylogenetic portrait of the genus Calicivirus in the family Caliciviridae was developed based upon published sequences and newly characterized calicivirus (CV) strains, including additional Sapporo‐like HuCV strains in pediatric diarrhea stool specimens from South Africa, the United Kingdom, and the United States. Distance and parsimony methods were applied to nucleotide and amino acid sequences of human and animal calicivirus 3D RNA‐dependent RNA polymerase (∼470nt) and capsid hypervariable regions (∼1,200nt) to generate phylogenetic trees. Pairwise amino acid identity in the 3D region among the Sapporo‐like strains ranged from 61%; to 100%. Human and animal caliciviruses (HuCVs and AnCVs) separated into five genogroups: small round‐structured viruses (SRSV), Sapporo‐like, and hepatitis E virus (HEV)‐like HuCVs and rabbit‐, and vesicular exanthema of swine virus (VESV)‐like AnCVs, each with a distinct genome organization. Each genogroup, including the Sapporo‐like HuCVs, subdivided further into subgenogroups. The capsid region trees had higher levels of confidence than the 3D region trees and limited conclusions about genogroups could be drawn from the 3D region analyses. This analysis suggested that CVs include five potential virus subfamilies. J. Med. Virol. 52:419–424, 1997.

Incidence of human calicivirus and rotavirus infection in patients with gastroenteritis in South Africa

Human caliciviruses (HuCVs) are reportedly responsible for 2.5–4% of nonbacterial sporadic gastroenteritis. The incidence of HuCV infection in South Africa is unknown. Stool specimens from 1,296 South African patients with sporadic gastroenteritis were screened for the presence of HuCVs using electron microscopy, recombinant enzyme immunoassays for Norwalk (NV) and Mexican (MX) viruses, and the reverse transcriptase‐polymerase chain reaction (RT‐PCR). RT‐PCR products were sequenced to ascertain which HuCV genogroups were present. HuCVs were detected in 43/1,296 (3.3%) specimens examined, with RT‐PCR proving to be the most sensitive detection method. Genetic analysis of the isolates indicated that 81% were Snow Mountain Agent, or MX‐like; 8% were NV‐like; and 11% were HuCV/Sapporo‐like. This study indicates that a combination of assays is needed for the accurate detection of HuCVs. Comparative data on hospitalised patients showed that the incidence of rotavirus infection was approximately ten times greater than that of HuCV infection. J. Med. Virol. 51:290–296, 1997.

Evidence of a Recombinant Wild-Type Human Astrovirus Strain from a Kenyan Child with Gastroenteritis

ABSTRACT A human astrovirus (HAstV) strain from Kenya was characterized by nucleotide sequence analysis. Sequences from open reading frame 1a (ORF1a) clustered with genotype 6/7, those from ORF1b clustered with genotype 3, and those from ORF2 clustered with genotype 2. A recombination point in the ORF1b-ORF2 junction was identified, with a second possible recombination point within the ORF1a region.

Rotavirus G and P types circulating in the eastern region of Kenya: predominance of G9 and emergence of G12 genotypes.

Background: The World Health Organization has recommended that rotavirus (RV) vaccines be included in all national immunization programs as part of a strategy to control RV-associated diarrheal diseases. Hospital-based surveillance of RV infection is therefore crucial in monitoring the impact pre- and post-vaccine introduction and also to document changes in genotype distribution. This study sought to determine the RV genotypes circulating in the eastern region of Kenya before introduction of the RV vaccine. Methods: During September 2009 to August 2011, 500 stool samples were collected from children <5 years of age admitted for acute diarrhea in hospitals in the eastern region of Kenya and analyzed for the presence of group A RV using an enzyme immunoassay. G and P genotypes were determined using hemi-nested reverse transcriptase polymerase chain reaction. Results: One hundred and eighty nine out of 500 (38%) samples analyzed were positive for rotavirus. The following G types were detected: G9 (50.9%), G1 (26.8%), G8 (12.1%), G12 (3.1%), G2 (0.6%), mixed G (1.3%) and 5.1% were G nontypeable. P types detected included: P[8] (63.7%), P[4] (12.1%), P[6] (4.5%), mixed P (7.6%) and 12.1% were P nontypeable. The most dominant strain was G9P[8] (35%), followed by G1P[8] (26.8%), G8P[4] (9.6%), G12P[6] (2.5%), G9P[6] (1.9%), G9P[4] (1.3%), G8P[8] (1.3%), and G2P[4] (0.6%). Conclusions: The present study demonstrates the recurring changing genotypes of RV circulating in Kenya, with genotypes G9, G1 and G8 being the dominant strains circulating in the eastern region of Kenya between 2009 and 2011. Additionally, G12 genotype was detected for the first time in Kenya.

High prevalence of species D human adenoviruses in fecal specimens from Urban Kenyan children with diarrhea

Human adenoviruses (HAdVs) cause a wide range of clinical syndromes and are classified in seven species, A–G, comprising 52 serotypes. HAdV‐A31, ‐F40, and ‐F41 have been associated with diarrhea in infants and young children. In developing countries gastroenteritis is a major cause of morbidity and mortality in children and, in comparison to rotaviruses, there are no data on the HAdVs associated with diarrhea in pediatric patients in Kenya. This study investigates the prevalence and genotypes of HAdVs in 278 stool specimens (211 diarrheal; 67 non‐diarrheal) from children ≤14 years of age in urban and rural areas in Kenya. Stool specimens were screened for HAdVs using a nested polymerase chain reaction and the HAdVs genotyped by sequence analysis of a conserved hexon gene fragment. HAdVs were detected in 104/278 (37.4%) of the stool specimens: 35/43 (81.4%) of diarrheal and 10/61 (16.4%) of non‐diarrheal stool specimens from children in an urban hospice; 25/94 (26.6%) of diarrheal specimens from urban children and 34/80 (42.5%) of diarrheal specimens from children in a rural area. Species D HAdVs were identified as the most prevalent HAdV species in diarrheal stool specimens from urban children comprising 18/37 (48.6%) of the strains identified. In contrast HAdV species F predominated in pediatric diarrheal specimens from the rural area, being identified in 7/16 (43.8%) of the characterized strains. This study provides valuable new data on the prevalence and distribution of HAdV genotypes in diarrheal stool specimens in Kenya and Africa, and highlights the necessity for further investigations. J. Med. Virol. 82:77–84, 2010.

An investigation of the role of oral epithelial cells and Langerhans cells as possible HIV viral reservoirs

BACKGROUND The role of the oral mucosa as a target of human immunodeficiency virus (HIV-1) infection and persistence is unclear. HIV-1 has been reported in oral epithelial cells, but this has not been confirmed. Cellular reservoirs may impede antiretroviral therapies and should be identified. This study was performed to determine the presence of HIV-1 in oral epithelial and Langerhans cells (LCs) of HIV-1-positive antiretroviral naïve patients. Non-invasive brush biopsy technique for future in vivo HIV research was also evaluated. METHODS Oral mucosal cells were harvested from the buccal mucosae, dorsal tongue and the gingiva of the mandibular teeth of 35 HIV-1-positive patients using a Cytobrush Plus cell collector. Epithelial cells were purified from the samples by flow cytometric cell sorting using cytokeratin stains after which the epithelial cell samples were further purified and divided into superficial and deep epithelial cells by laser microdissection on Pap stained cytospin smears. LCs were picked up individually by laser microdissection from CD1a stained cytospin smears. Purified epithelial and LC samples were tested for the presence of HIV-1 DNA by polymerase chain reaction analysis. RESULTS Ten of the patients had HIV-1 DNA in one or more of the sampled anatomical locations. No HIV-1 DNA could be demonstrated in any of the purified superficial or deep epithelial or LC samples. CONCLUSIONS HIV-DNA can be found using non-invasive oral brush biopsies and should be investigated further as an experimental model for in vivo oral HIV research. Better ways to purify the different cell types should be investigated.

Molecular characterisation of hepatitis A virus strains from water sources in South Africa.

Hepatitis A virus (HAV) strains found in selected South African (SA) surface waters were characterised to establish what HAV types are circulating in the environment, thus reflecting circulation in the surrounding communities. Surface water samples used for irrigation or domestic purposes, and water samples from the outflow of wastewater plants were collected from six provinces. Viruses were recovered from the samples using a glass wool adsorption-elution method and then further concentrated using polyethylene glycol/sodium chloride precipitation. After automated nucleic acid extraction, samples were analysed for HAV by real-time reverse-transcriptase polymerase chain reaction. HAV strains were genotyped by nucleotide sequence analysis of the capsid gene VP1 and the VP1/P2B junction. HAVs were detected in 76% (16/21) of the surface water samples and in 37% (19/51) of the samples from the wastewater plants. Strains were characterised from 32 of the 35 samples and classified within genotype IB. The presence of genotype IB in the water sources confirms human faecal contamination. Hence, these faecally-contaminated water sources may be a potential transmission route of HAV infection and a potential source of contamination of irrigated fresh produce in SA.

Molecular characterisation of enteroviruses and clinical findings from a cluster of paediatric viral meningitis cases in Tshwane, South Africa 2010–2011

BACKGROUND Human enteroviruses (HEVs) are the most common viral pathogen associated with paediatric aseptic meningitis. From October 2010 to February 2011 a cluster of HEV-associated meningitis cases was identified in paediatric patients who had presented at two large tertiary hospitals in Pretoria in the Tshwane Metropolitan Area, Gauteng, South Africa (SA). OBJECTIVES The aim of this study was to review the clinical features and to characterise the HEV strains associated with this cluster of meningitis cases. STUDY DESIGN In this retrospective study HEVs, detected by real time reverse transcription-polymerase chain reaction in acute phase cerebrospinal fluid specimens from 30 patients with aseptic meningitis, were characterised and the clinical presentations of these patients were described. RESULTS Fever (83%), headache (70%) and vomiting (67%) were the most prominent symptoms with signs of meningeal irritation recorded in 67% of the patients. There was a neutrophil predominance in the cerebrospinal fluid of 57% of the patients with pleocytosis. Based on partial nucleotide sequence analysis of the HEV viral protein 1 gene, echovirus (E) serotype 4 (E-4) was identified in 80% (24/30) of specimens with E-9 (3/30) and coxsackie virus B5 (1/30) detected less frequently. CONCLUSION In this cluster of aseptic meningitis cases E-4 was the predominant strain with E-9, and to a lesser extent other HEVs, identified less frequently.

Genetic characterization of a novel hepatitis a virus strain in irrigation water in South Africa.

Hepatitis A virus (HAV) was detected, by real‐time reverse transcription‐polymerase chain reaction, in irrigation water from a dam on a commercial fresh produce farm in South Africa (SA). The virus was characterized by nucleotide sequence and phylogenetic analysis of a consensus sequence spanning the VP1 and VP1/P2B genomic regions. Amino acid sequence and phylogenetic analysis indicated that the HAV strain was closely related to HAV genotype V and possibly of simian origin. This suggests that a novel HAV may be circulating in SA and its presence in irrigation water highlights the potential for zoonotic or anthroponotic cross‐species transmission via environmental food and water sources. J. Med. Virol. 88:734–737, 2016.

Molecular detection and characterisation of mumps virus in cerebrospinal fluid in a Gauteng laboratory

The study aimed to determine the presence of mumps virus (MuV) in cerebrospinal fluid (CSF) specimens and to genetically characterise detected MuV strains. A real-time reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the MuV F gene, and characterisation was performed by sequencing of the SH gene. Mumps virus was detected in 1.2% (3/260) of specimens. Phylogenetic analysis of one MuV strain revealed that it clustered with the Jeryl-Lynn and RIT4385 vaccine strains. As far as the authors could ascertain this is the first study to provide viral proof that these vaccine-like strains may be associated with aseptic meningitis.