Heidi Smuts

Evidence that the GBV‐C/hepatitis G virus is primarily a lymphotropic virus

GB virus‐C and the hepatitis G virus (GBV‐C/HGV) are variants of the same positive sense RNA flavivirus, initially thought to be associated with hepatitis. The tissue tropism of GBV‐C/HGV in normal subjects has not been evaluated to date using an extended tissue spectrum. Therefore, the sites of GBV‐C/HGV replication were investigated in serum and twenty‐three tissues collected during post‐mortem examination of four apparently healthy individuals who died accidental deaths, who were infected with GBV‐C/HGV. All were anti‐HIV and anti‐HCV negative and three out of four were HBsAg negative. Tissues were collected carefully to prevent cross contamination. A highly strand‐specific RT‐PCR assay was employed for the detection of either GBV‐C/HGV positive strand RNA (virion) or negative strand RNA (replicative intermediary). Strand specificity of the RT‐PCR assay was assessed with synthetic positive‐and negative strand GBV‐C/HGV RNA generated from a plasmid, using T7 and T3 RNA polymerases. The spleen and bone marrow biopsies were found to be uniformly positive for both negative‐and positive strand GBV‐C/HGV RNA. In addition, one cadaver was positive for both RNA strands in the kidney, and another positive for both in the liver. No negative strand RNA was detected in the following: brain, muscle, heart, thyroid, salivary gland, tonsil, lung, lymph nodes, gall bladder, pancreas, oesophagus, stomach, small bowel, large bowel, adrenal gland, gonad, aorta, skin and cartilage. This preliminary study concludes that GBV‐C/HGV is a lymphotropic virus that replicates primarily in the spleen and bone marrow. J. Med. Virol. 61:52–58, 2000.

Role of human metapneumovirus, human coronavirus NL63 and human bocavirus in infants and young children with acute wheezing

The role of the novel respiratory viruses, human metapneumovirus (hMPV), human coronavirus NL63 (HCoV NL63) and human bocavirus (HBoV), in wheezing illness in children has not been well studied, especially in Africa. The aim of this study was to investigate the prevalence of hMPV, HCoV NL63 and HBoV in South African children with acute wheezing. A prospective study of consecutive children presenting with acute wheezing to a pediatric hospital from May 2004 to November 2005 was undertaken. A nasal swab was taken for reverse transcription‐polymerase chain reaction (RT‐PCR) and PCR for hMPV, HCoV NL63 and HBoV; when positive, the genes were sequenced. Shell vial culture for RSV, influenza A and B viruses, adenovirus and parainfluenza viruses 1, 2, 3 was performed on every 5th sample. Two hundred and forty two nasal swabs were collected from 238 children (median age 12.4 months). A novel respiratory virus was found in 44/242 (18.2%). hMPV, HBoV, and HCoV NL63 was found in 20 (8.3%), 18 (7.4%), and 6 (2.4%) of samples, respectively. Fifteen of 59 (25%) samples were positive for other respiratory viruses. Viral co‐infections, occurred in 6/242 (2.5%). Phylogenetic analysis showed co‐circulation of hMPV and HCoV NL63 A and B lineages, although only HBoV genotype st2 was found. Viruses are an important cause of wheezing in preschool children; hMPV, HCoV NL63, and HBoV are less common than the usual respiratory pathogens. J. Med. Virol. 80:906–912, 2008.

GBV-C/HGV genotypes: Proposed nomenclature for genotypes 1–5

The GB virus‐C and hepatitis G virus (GBV‐C/HGV) are variants of the same flavivirus. This proposal attempts to clarify the conflicting nomenclature for GBV‐C/HGV genotypes. The first three genotypes described were genotype 1 (West Africa); genotype 2 (US/Europe) and genotype 3 (Asia). Subsequently, two groups published data from South Africa and Southeast Asia both stating the presence of a novel “4th genotype.” These isolates are distinct phylogenetically. It is proposed that the nomenclature for genotypes 1–3 remains as per previous publications, and that the Southeast Asian isolates be known as genotype 4, and the South African isolates as genotype 5. J. Med. Virol. 62:82–83, 2000.

Molecular characterization of the 5' non-coding region of South African GBV-C/HGV isolates: major deletion and evidence for a fourth genotype.

GB virus C/hepatitis G virus (GBV‐C/HGV) has been characterised as a novel flavivirus, and to date three known genotypes have been cloned. Greater genetic variation of GBV‐C/HGV has been demonstrated in West African isolates, but no major deletions have been shown in the 5′ non‐coding region (NCR). The 5′NCR regulates protein translation via an internal ribosomal entry site (IRES). We cloned, sequenced, and analysed a 344‐bp polymerase chain reaction (PCR) product, representing >60% of the 5′NCR, from 32 GBV‐C/HGV PCR‐positive volunteers. Wild‐type virus amplicons were detected in all samples. However, 5/32 (15.6%) also amplified another fragment of between 205 and 231 bp. Sequence analysis showed all cloned PCR fragments to be GBV‐C/HGV‐specific. A typical deletion of 113–131 bp with minor variation was detected in isolates generating the smaller bands. RNA secondary structure analysis showed the deletions to be over domains II and III. This finding suggests that nucleotides 303–444 may be non‐essential for 5′NCR functioning. Phylogenetic analysis demonstrated a novel fourth South African genotype, distinct from genotypes 1–3 with DNA distances of >0.1000. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) values for the wild‐type and mutant samples were normal. This study documents the first major deletion in the 5′NCR of GBV‐C/HGV, and suggests that bases 303–444 may not be essential for viral replication and ribosomal entry. A fourth GBV‐C/HGV genotype appears to predominate in South Africa. J. Med. Virol. 59:52–59, 1999.

Human rhinovirus infection in young African children with acute wheezing.

BackgroundInfections caused by human rhinoviruses (HRVs) are important triggers of wheezing in young children. Wheezy illness has increasingly been recognised as an important cause of morbidity in African children, but there is little information on the contribution of HRV to this. The aim of this study was to determine the role of HRV as a cause of acute wheezing in South African children.MethodsTwo hundred and twenty children presenting consecutively at a tertiary childrens hospital with a wheezing illness from May 2004 to November 2005 were prospectively enrolled. A nasal swab was taken and reverse transcription PCR used to screen the samples for HRV. The presence of human metapneumovirus, human bocavirus and human coronavirus-NL63 was assessed in all samples using PCR-based assays. A general shell vial culture using a pool of monoclonal antibodies was used to detect other common respiratory viruses on 26% of samples. Phylogenetic analysis to determine circulating HRV species was performed on a portion of HRV-positive samples. Categorical characteristics were analysed using Fishers Exact test.ResultsHRV was detected in 128 (58.2%) of children, most (72%) of whom were under 2 years of age. Presenting symptoms between the HRV-positive and negative groups were similar. Most illness was managed with ambulatory therapy, but 45 (35%) were hospitalized for treatment and 3 (2%) were admitted to intensive care. There were no in-hospital deaths. All 3 species of HRV were detected with HRV-C being the most common (52%) followed by HRV-A (37%) and HRV-B (11%). Infection with other respiratory viruses occurred in 20/128 (16%) of HRV-positive children and in 26/92 (28%) of HRV-negative samples.ConclusionHRV may be the commonest viral infection in young South African children with acute wheezing. Infection is associated with mild or moderate clinical disease.

Novel Hybrid Parvovirus-Like Virus, NIH-CQV/PHV, Contaminants in Silica Column-Based Nucleic Acid Extraction Kits

The discovery of a novel hybrid parvovirus-like virus, NIH-CQV, in non-A to -E (non-A-E) hepatitis virus samples by Xu et al. ([1][1]) as a possible causative agent of seronegative hepatitis prompted our investigation of samples collected from patients with a range of liver diseases, including acute

Prevalence of myocarditis and cardiotropic virus infection in Africans with HIV-associated cardiomyopathy, idiopathic dilated cardiomyopathy and heart transplant recipients : a pilot study : cardiovascular topic

Background: The prevalence of myocarditis and cardiotropic viral infection in human immunodeficiency virus (HIV)-associated cardiomyopathy is unknown in Africa. Methods Between April 2002 and December 2007, we compared the prevalence of myocarditis and cardiotropic viral genomes in HIV-associated cardiomyopathy cases with HIV-negative idiopathic dilated cardiomyopathy patients (i.e. negative controls for immunodeficiency) and heart transplant recipients (i.e. positive controls for immunodeficiency) who were seen at Groote Schuur Hospital, Cape Town, South Africa. Myocarditis was sought on endomyocardial biopsy using the imunohistological criteria of the World Heart Federation in 33 patients, 14 of whom had HIV-associated cardiomyopathy, eight with idiopathic dilated cardiomyopathy and 11 heart transplant recipients. Results Myocarditis was present in 44% of HIV-associated cardiomyopathy cases, 36% of heart transplant recipients, and 25% of participants with idiopathic dilated cardiomyopathy. While myocarditis was acute in 50% of HIV- and heart transplant-associated myocarditis, it was chronic in all those with idiopathic dilated cardiomyopathy. Cardiotropic viral infection was present in all HIV-associated cardiomyopathy and idiopathic dilated cardiomyopathy cases, and in 90% of heart transplant recipients. Multiple viruses were identified in the majority of cases, with HIV-associated cardiomyopathy, heart transplant recipients and idiopathic dilated cardiomyopathy patients having an average of 2.5, 2.2 and 1.1 viruses per individual, respectively. Conclusions Acute myocarditis was present in 21% of cases of HIV-associated cardiomyopathy, compared to none of those with idiopathic dilated cardiomyopathy. Infection with multiple cardiotropic viruses may be ubiquitous in Africans, with a greater burden of infection in acquired immunodeficiency states.

Molecular Characterization of Duck Hepatitis B Virus Isolates from South African Ducks

The objective of the study was to characterize the genome of duck hepatitis B virus (DHBV) isolates from South African Pekin ducks. Duck serum and liver samples were collected from two commercial duck farms from geographically distinct regions of South Africa. In total, 498 duck serum samples were tested for the presence of DHBV DNA using either sub-genomic or full-length polymerase chain reaction (PCR) assays. The overall prevalence of DHBV infection in South African ducks was 47%. In addition, 30% of 59 liver tissues tested were DHBV DNA-positive. Six randomly selected serum or liver samples were used to clone and sequence the genomes of the South African DHBV strains. All six isolates had DHBV genomes of 3,021 nucleotides with three characteristic overlapping reading frames encoding the polymerase, surface and core gene products. No X-like gene with a traditional start codon was found. Following phylogenetic analysis, the South African DHBV isolates clustered with DHBV isolates from other “Western” countries, including United States of America, Canada, Germany and India. On translation of the open reading frames, the South African isolates were found to share signature amino acids in the polymerase and surface genes with the “Western” country isolates as opposed to those of Chinese DHBV isolates.

Molecular Characterization of the 16S rRNA Gene of Helicobacter fennelliae Isolated from Stools and Blood Cultures from Paediatric Patients in South Africa.

Forty strains of H. fennelliae collected from paediatric blood and stool samples over an 18 year period at a childrens hospital in Cape Town, South Africa, were amplified by PCR of the 16S rRNA. Two distinct genotypes of H. fennelliae were identified based on the phylogenetic analysis. This was confirmed by sequencing a portion of the beta subunit of the RNA polymerase (rpoB) gene. All isolates from South Africa clustered with a proposed novel Helicobacter strain (accession number AF237612) isolated in Australia, while three H. fennelliae type strains from the northern hemisphere, NCTC 11612, LMG 7546 and CCUG 18820, formed a separate branch. A large (355bp) highly conserved intervening sequence (IVS) in the 16S rRNA was found in all isolates. Predicted secondary structures of the IVS from the 16S rRNA and 23S rRNA were characterised by a primary stem structure formed by base pairing of the 3′ and 5′ ends and internal loops and stems. This phylogenetic analysis is the largest undertaken of H. fennelliae. The South African H. fennelliae isolates are closely related to an Australian isolate previously reported to be a possible novel species of Helicobacter. This study suggests that the latter is strain of H. fennelliae.

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

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.