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Background Enteroviruses are amongst the most common causes of aseptic meningitis. Between November 2018 and May 2019, an outbreak of enterovirus-associated aseptic meningitis cases was noted in the Western and Eastern Cape Provinces, South Africa. Objectives To describe the epidemiology and phylogeography of enterovirus infections during an aseptic meningitis outbreak in the Western and Eastern Cape Provinces of South Africa. Methods Cerebrospinal fluid samples from suspected cases were screened using a polymerase chain reaction targeting the 5’UTR. Confirmed enterovirus-associated meningitis samples underwent molecular typing through species–specific VP1/VP2 primers and pan-species VP1 primers. Results Between November 2018 and May 2019, 3497 suspected cases of aseptic meningitis were documented in the Western and Eastern Cape Provinces. Median age was 8 years (range 0–61), interquartile range (IQR=4–13 years), 405/735 (55%) male. 742/3497 (21%) cases were laboratory – confirmed enterovirus positive by routine diagnostic PCR targeting the 5’UTR. 128/742 (17%) underwent molecular typing by VP1 gene sequencing. Echovirus 4 (E4) was detected in 102/128 (80%) cases. Echovirus 9 was found in 7%, Coxsackievirus A13 in 3%. 10 genotypes contributed to the remaining 10% of cases. Synonymous mutations were found in most cases, with sporadic amino acid changes in 13 (12.7%) cases. Conclusion The aseptic meningitis outbreak was associated with echovirus 4. Stool samples are valuable for molecular typing in CSF confirmed EV-associated aseptic meningitis.

Sub-Saharan Africa has approximately 10.15 million people viraemic with chronic hepatitis C virus infection, extensive genotype and sub-genotype diversity is present, in addition to novel hepatitis C genotypes. Many of the unusual genotypes have extensive baseline resistance associated substitutions with direct acting antiviral therapy treatment outcome data, limited. We report a patient found to have a novel genotype 7b variant with extensive baseline resistance associated substitutions. There is a clear need for a better understanding of the virological characteristics of hepatitis C populations in sub-Saharan Africa to guide best optimal treatment decisions in national hepatitis C elimination programmes.

The SARS-CoV-2 pandemic has resulted in shortages of both critical reagents for nucleic acid purification and highly trained staff as supply chains are strained by high demand, public health measures and frequent quarantining and isolation of staff. This created the need for alternate workflows with limited reliance on specialised reagents, equipment and staff. We present here the validation and implementation of such a workflow for preparing samples for downstream SARS-CoV-2 RT-PCR using liquid handling robots. The rapid sample preparation technique evaluated, which included sample centrifugation and heating prior to RT-PCR, showed a 97.37% (95% CI: 92.55-99.28%) positive percent agreement and 97.30% (95% CI: 90.67-99.52%) negative percent agreement compared to nucleic acid purification-based testing. This method was subsequently adopted as the primary sample preparation method in the Groote Schuur Hospital Virology Diagnostic Laboratory in Cape Town, South Africa.

Background. The epidemiology of hepatitis C virus (HCV) in the general population of South Africa (SA) is incompletely understood. A high HCV prevalence in key populations is known, but data are limited in terms of a broader understanding of transmission risks in our general population. Objectives. To investigate a patient cohort with HCV infection clustering in a rural SA town, in order to identify possible HCV transmission risks, virological characteristics, phylogenetic data and treatment outcomes. Methods. A cluster of patients with positive HCV serology, previously identified from laboratory records, were contacted by a local district hospital and offered confirmatory testing for HCV viraemia where needed. Those with confirmed HCV RNA were invited to a local hospital visit, where relevant demographic information was recorded, clinical assessment performed and a confidential questionnaire administered. HCV population-based sequencing was performed on HCV NS3/4A, NS5A and NS5B using polymerase chain reaction-specific or M13 universal primers, and sequences were aligned using BioEdit 7.2.5. Phylogenetic trees were constructed. Clinical assessments included liver fibrosis determination with FibroScan (cut-off ≥12.5 kPa = F4). Patients were offered treatment, and sustained virological response (SVR) was confirmed by undetectable HCV RNA at least 12 weeks after the end of treatment. Results. Twenty-one patients, all from the same town, median (interquartile range (IQR)) age 64 (59 - 70) years, 57% female, were evaluated. Of these, 24% ( n =5) were HIV co-infected, stable on antiretrovirals. The median (IQR) alanine aminotransferase level was 51 (31 - 89) U/L, with fibrosis distribution including 29% F1, 29% F2, 9% F3 and 33% F4 METAVIR fibrosis. Virologically, two genotypes were observed: 62% ( n =13) genotype (GT) 1b and 38% ( n =8) GT5a. No patient had ever used injecting drugs, 14% ( n =3) had received blood products before 1992, and 9.5% ( n =2) had undergone traditional healer-administered scarification. All ( n =21) reported attendance at a single primary care clinic in the past, with most ( n =20) recalling having received parenteral therapies at the clinic. Phylogenetic analysis of the HCV NS5A and NS5B regions confirmed GT1b and GT5a genotypes and formed two separate clusters within their respective genotypes, suggesting a common source for each genotype infection. Most patients received treatment with sofosbuvir/daclatasvir, 1 was treated with sofosbuvir/velpatasvir, and 1 was re-treated with sofosbuvir/velpatasvir/voxilaprevir. Per protocol SVR was 95%, with the non-SVR patient successfully re-treated. Conclusions. Data from a rural town cluster of patients suggest parenteral medical exposure as the probable common source of hepatitis C transmission risk. The cohort was of older age with a significant number having advanced fibrosis or cirrhosis, suggesting HCV acquisition in the distant past. Using a simplified care approach, treatment outcomes were very good.

Background Human pegivirus-1 (HPgV-1) infection in the brain has not been extensively examined and its association with disease remains unconfirmed. In a high throughput sequencing study to look for infectious agents that could play a role in HIV-associated neurocognitive disorder (HAND), this virus was detected in 3 of 8 CSF samples. Objectives To determine the significance of this finding, additional patients were screened and the viral load and viral diversity in blood and CSF were examined. Study design Nested PCR of the viral 5′NCR region was performed on blood and CSF pairs from 16 HAND patients. PCR products were cloned, sequenced and analysed to determine viral diversity in blood and CSF. HPgV-1 viral loads were determined in paired blood and CSF of 2 patients by digital droplet PCR. Nested PCR was also performed on CSF samples from patients with other brain disorders. Results Virus was detected in both blood and CSF in 3 of 16 HAND patients. Viral loads were very high in blood (8.81 and 10.56 log copies/ml) and 4–5 logs lower in CSF (4.68 and 5.84 log copies/ml). Sequence analysis of 5′NCR clones in blood and CSF showed limited variation. The dominant viral variant (based on clonal sequence identity) in blood and CSF was usually identical. HPgV-1 was detected in CSF from patients with other brain disorders at a similar frequency (15% versus 18.75% in HAND patients). Conclusion While several studies have reported HPgV-1 detection in CSF of patients with brain disease, this is the only study that has examined both blood and CSF compartments simultaneously. Our findings show that virus in CSF always coincided with viraemia and levels were 4–5 logs higher in blood. While a rare, but specific brain tropism cannot be excluded, blood is the more probable source of virus in HAND patients.

During a measles virus (MeV) epidemic in 2009 in South Africa, measles inclusion body encephalitis (MIBE) was identified in several HIV-infected patients. Years later, children are presenting with subacute sclerosing panencephalitis (SSPE). To investigate the features of established MeV neuronal infections, viral sequences were analyzed from brain tissue samples of a single SSPE case and compared with MIBE sequences previously obtained from patients infected during the same epidemic. Both the SSPE and the MIBE viruses had amino acid substitutions in the ectodomain of the F protein that confer enhanced fusion properties. Functional analysis of the fusion complexes confirmed that both MIBE and SSPE F protein mutations promoted fusion with less dependence on interaction by the viral receptor-binding protein with known MeV receptors. While the SSPE F required the presence of a homotypic attachment protein, MeV H, in order to fuse, MIBE F did not. Both F proteins had decreased thermal stability compared to that of the corresponding wild-type F protein. Finally, recombinant viruses expressing MIBE or SSPE fusion complexes spread in the absence of known MeV receptors, with MIBE F-bearing viruses causing large syncytia in these cells. Our results suggest that alterations to the MeV fusion complex that promote fusion and cell-to-cell spread in the absence of known MeV receptors is a key property for infection of the brain. IMPORTANCE Measles virus can invade the central nervous system (CNS) and cause severe neurological complications, such as MIBE and SSPE. However, mechanisms by which MeV enters the CNS and triggers the disease remain unclear. We analyzed viruses from brain tissue of individuals with MIBE or SSPE, infected during the same epidemic, after the onset of neurological disease. Our findings indicate that the emergence of hyperfusogenic MeV F proteins is associated with infection of the brain. We also demonstrate that hyperfusogenic F proteins permit MeV to enter cells and spread without the need to engage nectin-4 or CD150, known receptors for MeV that are not present on neural cells.

Background Human enteroviruses (HEVs) are common causal agents of aseptic meningitis in young children. Laboratory and syndromic surveillance during December 2015 and January 2016 noted an unusually high number of paediatric aseptic meningitis cases at a hospital in Mossel Bay, Western Cape Province, South Africa. HEV was detected in clinical samples, prompting an outbreak investigation. Methods Epidemiological investigations were conducted to ascertain possible linkage between cases. Amplification, sequencing and phylogenetic analysis of the 5’UTR and VP1 regions was undertaken to determine the HEV serotype associated with the outbreak as well as other cases of aseptic meningitis in the area in the preceding 6 weeks. Results Over the 2-month period, 63 CSF samples were available for testing. A total of 43 outbreak cases (68.3%) were observed, and the 26 (60.5%) that could be typed were coxsackie virus A9 (CVA9). Children attending three crèche facilities were epidemiologically linked, accounting for 60.5% (26/43) of the CVA9 cases. The majority of patients were under 10 years of age (55/63, 87.3%) and there was a male predominance (66%). Nucleotide sequence analysis of the 5’UTR and VP1 regions identified 2 lineages of CVA9 co-circulating during the outbreak, although the VP1 capsid protein sequence was identical as all nucleotide differences were synonymous. There was a unique isoleucine at position 64 and all outbreak viruses had a valine to threonine change in the hypervariable BC loop of VP1. Other HEV types circulating in the preceding period were echovirus 30 (n = 4), echovirus 5 (n = 3) and 1 each of echovirus 6, echovirus 9 and echovirus 15. Conclusion CVA9 was identified as the pathogen responsible for the large outbreak of aseptic meningitis, with 2 distinct co-circulating lineages. Electronic supplementary material The online version of this article (10.1186/s12879-018-3641-4) contains supplementary material, which is available to authorized users.

Background. An estimated 600 000 South Africans are chronically infected with hepatitis C virus (HCV). To date, accurate prevalence data are lacking, but emerging data suggest a significant burden in key populations. Historically, pegylated interferon and ribavirin treatment was challenging, with access limited. The advent of all-oral, short-course direct-acting antiviral (DAA) therapy has revolutionised the management of HCV, being well tolerated and highly effective, although initial cost was a prohibitive factor. Objectives. To report our initial 2-year experience with DAA therapy at the University of Cape Town/Groote Schuur Hospital Liver Clinic, South Africa (SA). Methods. Patients who were viraemic for HCV were offered access to DAA therapy. All relevant demographic, virological, serological and clinical laboratory data were captured in a registry. Liver fibrosis was assessed non-invasively with the FibroScan. DAA regimens were prescribed according to current guidance based on HCV genotype (GT), prior treatment history and degree of fibrosis. On treatment, virological response was recorded and a sustained virological response (SVR) was defined as an undetectable HCV RNA at least 12 weeks after the end of treatment. Results. We report on the first 210 patients treated. Their median (interquartile range (IQR)) age was 52 (42 - 61) years and 65% were male, with men significantly younger than women at 50 (42 - 59) years v. 58 (47 - 67) years, respectively ( p =0.001). All GTs were observed, with 1 and 5 most prevalent at 45% and 20%, respectively, and GTs 2, 3 and 4 frequencies of 7%, 11% and 17%, respectively. Extensive subtype diversity for GTs 2 and 4 was present. The median (IQR) HCV viral load was log10 5.9 IU/mL (5.4 - 6.5). A significant proportion of patients (39%) had advanced fibrosis or cirrhosis, with 11% F3 fibrosis and 28% F4. Of those with cirrhosis, 12% were decompensated with Childs-Pugh B or C disease. Of the patients, 19% were HIV co-infected and 2% HBV co-infected. In total, 13% were treatment experienced. The majority of patients were treated with sofosbuvir and ledipasvir (38%), daclatasvir (36%) or velpatasvir (± voxilaprevir, 9%). Less frequent combinations included partitaprevir, ritonavir, ombitasvir ± dasbuvir (11%) and sofosbuvir/ribavirin (5%). The per-protocol SVR was 96% (98% if sofosbuvir/ribavirin is excluded). The majority of treatment failures occurred with GT-4, notably subtype 4r. Mild side-effects were reported in 10% of patients, with none discontinuing therapy. Conclusions. DAA therapy for HCV in a pan-genotypic group of patients, many with advanced liver disease, was highly effective. Our outcomes correspond with existing trial and real-world data for similar treatment. DAA therapy and access need rapid upscaling in SA, especially targeting key populations at point of care.

The prevalence of hepatitis B virus (HBV) infection in pregnant women is high in South Africa (SA), yet prophylaxis to prevent mother-to-child transmission (MTCT) falls short of international recommendations. We describe a 10-week-old infant who developed fulminant hepatic failure following MTCT. The mother was hepatitis e-antibody positive and had a viral load of only 760 IU/mL. Genetic analysis of virus from mother and infant showed that both had the G1896A mutation in the preC/C gene, which truncates hepatitis e antigen (HBeAg) during translation, causing an HBeAg-negative phenotype. HBeAg attenuates antiviral immune responses, and its absence was probably responsible for the infant’s fulminant hepatitis, due to an uncontrolled immune attack on infected liver cells. Pregnant women are not tested for HBV infection in SA and MTCT rates are unknown. Addition of a birth dose of vaccine, HBV screening of pregnant women and antiviral prophylaxis to positive mothers should be prioritised.

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.

Parvovirus B19 comprises three distinct genotypes (1, 2, and 3). The distribution of B19 genotypes has not before been examined in South Africa. Two hundred thirty-nine laboratory samples submitted to a diagnostic virology laboratory for parvovirus DNA detection were analyzed retrospectively. Of the 53 PCR-positive samples investigated, 40 (75.4%) were identified as genotype 1 by genotype-specific PCR or consensus NS1 PCR and sequencing and 3 (5.7%) as genotype 2 and 10 (18.9%) as genotype 3 by analysis of NS1 sequences. Furthermore, phylogenetic analysis identified two genotype 1 sequences which were distinct from the previously described genotypes 1A and 1B. Interestingly, a genotype 2 virus was detected in the serum of an 11-year-old child, providing evidence for its recent circulation. This is the first study to demonstrate the concurrent circulation of all three genotypes of B19 in South Africa and the provisional identification of a novel subtype of genotype 1. The implications of parvovirus B19 variation are discussed.

Background Human coronavirus NL63 (HCoV-NL63) is a novel respiratory virus which is associated with respiratory tract infections in children. Objective To determine the role of HCoV-NL63 in infants and young children hospitalised with acute respiratory tract infections (ARI) in Cape Town, South Africa. Methods Respiratory specimens were collected from 1055 infants and young children hospitalised with ARI in 2003–2004. Samples were screened by RT-PCR to detect HCoV-NL63 and human metapneumovirus (hMPV). Standard shell vial culture and immunofluoresence was used to detect the common respiratory viruses including RSV, influenza A and B viruses, parainfluenza viruses 1, 2, 3, adenovirus and CMV. Results A respiratory virus was found in 401/1055 (38·0%) samples. HCoV-NL63 was detected in 9/1055 (0·85%) with peak activity during autumn (67%). Most patients had a diagnosis of pneumonia or lower respiratory tract infection (6/9; 67%). Conclusions This is the first report of HCoV-NL63 infections in hospitalised children in Africa. During the 2-year period HCoV-NL63 played a minor role in ARI in children.

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. © 2008 Wiley‐Liss, Inc.

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.

Introduction. Chicken anaemia virus, CAV, was until recently the only member of theGyrovirusgenus. 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.

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.

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.

Forty strains ofH. fennelliaecollected from paediatric blood and stool samples over an 18 year period at a children's hospital in Cape Town, South Africa, were amplified by PCR of the 16S rRNA. Two distinct genotypes ofH. fennelliaewere 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 novelHelicobacterstrain (accession number AF237612) isolated in Australia, while threeH. fennelliaetype 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 ofH. fennelliae. The South AfricanH. fennelliaeisolates 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 ofH. fennelliae.

Aim: To describe the clinical presentation and outcomes of hospitalised patients infected with human metapneumovirus (hMPV) and human respiratory syncytial virus (hRSV) in a tertiary hospital in Cape Town, South Africa. Methods: hMPV was identified in 17 respiratory specimens submitted for viral studies during the period 2001–2003. These patients’ medical folders were retrospectively reviewed for clinical, radiological and laboratory data, together with a convenience sample of 20 hRSV-infected patients. Results: hMPV-infected patients were older than those infected with hRSV (P = 0.04) and required a longer hospital stay (P = 0.02). Presenting clinical signs and symptoms were similar between groups. Fourteen (87.5%) hMPV- and 16 (80%) hRSV-infected patients presented with co-morbid and/or immunosuppressive conditions (P ≥ 0.5). The most common abnormalities on chest radiographs in both groups were bronchial wall thickening, focal consolidation and atelectasis. Six (37.5%) hMPV- and 11 (55%) hRSV-infected patients required admission to the paediatric intensive care unit (P > 0.1) with five (31.3%) hMPV- and eight (40%) hRSV-infected patients requiring intubation and ventilation (P > 0.5). Three (18.7%) hMPV-patients and three (15%) hRSV-infected patients died during this admission (P > 0.5). All hMPV-infected patients who died had significant co-morbid conditions. Conclusions: These data confirm that hMPV is a significant respiratory pathogen in this setting, with similar presentation and outcome to hRSV infection. This is the largest report of hMPV infection causing significant morbidity, prolonged hospital stay and death, associated with underlying risk factors.

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

To the Editor: In recent years, several novel respiratory viruses have been identified. These include human metapneumovirus (HMPV) (1), severe acute respiratory syndrome–associated coronavirus (2), human coronavirus (HCoV) NL63 (3,4), HCoV HKU1 (5), and, most recently, human bocavirus (HBoV) (6). The latter belongs to the Parvoviridae family and is most closely related to bovine parvovirus and canine minute virus (CnMV), which are members of the genus Bocavirus (6). Parvovirus B19 and HBoV are the only 2 parvoviruses known to be pathogenic to humans, but the relevance of HBoV infection in the clinical setting is not known. In this retrospective study, 341 nasopharyngeal and bronchoalveolar lavage samples were taken from children (age 2 days–12 years) hospitalized with respiratory tract infections in 2004 in the Red Cross War Memorial Children's Hospital, Cape Town, South Africa. Samples were originally screened by using an indirect immunofluorescence assay (Light Diagnostics, Chemicon International, Temecula, CA, USA) for common respiratory viruses, including respiratory syncytial virus; influenza virus A and B; parainfluenza viruses 1, 2, and 3; adenovirus; and cytomegalovirus. Subsequently, HMPV and HCoV NL63 were detected by using reverse transcription–PCR (1,3). Samples were also screened for HBoV DNA. DNA was extracted by using the QIAamp DNA blood mini kit according to the manufacturer's instructions (Qiagen Inc., Valencia, CA, USA). PCR amplification of a region of the NP-1 gene and the 3´ portion of the VP1/2 capsid gene of HBoV was performed. Briefly, 10 μL DNA was added to a 50-μL PCR mix containing 2 IU Supertherm polymerase (JMR Holdings, Kent, UK), 1.5 mmol/L MgCl2, 200 μmol/L each dNTP, and 0.2 μmol/L primers NP-1 s1 (5´-TAACTGCTCCAGCAAGTCCTCCA) and NP-1 as1 (5´-GGAAGCTCTGTGTTGACTGAAT). To improve sensitivity, a second seminested reaction with 2.5 μL outer product and NP-1 as1 primer and NP-1 s2 (5´-CTCACCTGCGAGCTCTGTAAGTA) primer was performed at an annealing temperature of 55°C. Negative controls were used, and appropriate measures were taken to prevent contamination (7). Samples with an NP-1–specific PCR product of 368 bp were confirmed by amplifying a 980-bp product of the VP1/2 capsid gene in a similar seminested PCR amplification protocol (primers VP s1 5´-GCACTTCTGTATCAGATGCCTT, VP as1 5´-CGTGGTATGTAGGCGTGTAG, and VP s2 5´-CTTAGAACTGGTGAGAGCACTG). A selection of the inner VP1/2 amplicons obtained from samples taken over the year were sequenced directly and aligned in ClustalX, and a phylogenetic tree was constructed with the Kimura 2-parameter neighbor-joining method with 1,000 bootstrap resamplings. Comparative sequences were obtained from GenBank and included HBoV isolate st1 ({"type":"entrez-nucleotide","attrs":{"text":"DQ000495","term_id":"66356128","term_text":"DQ000495"}}DQ000495), HBoV isolate st2 ({"type":"entrez-nucleotide","attrs":{"text":"DQ000496","term_id":"66356133","term_text":"DQ000496"}}DQ000496), and a CnMV isolate ({"type":"entrez-nucleotide","attrs":{"text":"NC_004442","term_id":"27151463","term_text":"NC_004442"}}NC_004442). Nucleotide sequences from this study were deposited into GenBank ({"type":"entrez-nucleotide-range","attrs":{"text":"DQ317539-DQ317561","start_term":"DQ317539","end_term":"DQ317561","start_term_id":"83835448","end_term_id":"83835492"}}DQ317539-DQ317561). HBoV DNA was detected in 38 (11%) samples from 35 children, all

BACKGROUND: During 2009/10 a major measles epidemic caused by genotype B3 occurred in South Africa. Measles inclusion body encephalitis (MIBE) was diagnosed in a number of highly immuno-compromised HIV patients. The diagnosis was based on typical clinical and MRI findings and positive measles virus PCR in brain or CSF.To characterize the brain virus, nucleoprotein, matrix, fusion and haemagglutinin genes from 4 cases was compared with virus from acutely infected patients. METHODS: cDNA was synthesized using random primers and viral genes were amplified by nested RT-PCR. PCR products were sequenced in the forward and reverse direction and a contig of each gene was created. Sequences were aligned with reference sequences from GenBank and other local sequences. RESULTS: Brain virus was very similar to the South African epidemic virus. Features characteristic of persistent measles virus in the brain were absent. Mutation frequency in brain virus was similar to epidemic virus and had the same substitution preference (U to C and C to U). The virus of 2 patients had the same L454W mutation in the fusion protein. CONCLUSION: The brain virus was very similar to the epidemic strain. The relatively few mutations probably reflect the short time from infection to brain disease in these highly immuno-compromised patients.

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.

Made available in DSpace on 2013-08-28T14:14:46Z (GMT). No. of bitstreams: 1 WOS000263600000016.pdf: 403547 bytes, checksum: 00bc362fcdd0b972ebb3e4d568306f22 (MD5) Made available in DSpace on 2013-09-30T18:17:16Z (GMT). No. of bitstreams: 2 WOS000263600000016.pdf: 403547 bytes, checksum: 00bc362fcdd0b972ebb3e4d568306f22 (MD5) WOS000263600000016.pdf.txt: 60599 bytes, checksum: b54b4d6932590cfa6f2c627a17471091 (MD5) Previous issue date: 2009-02-01 Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-20T13:33:27Z No. of bitstreams: 2 WOS000263600000016.pdf: 403547 bytes, checksum: 00bc362fcdd0b972ebb3e4d568306f22 (MD5) WOS000263600000016.pdf.txt: 60599 bytes, checksum: b54b4d6932590cfa6f2c627a17471091 (MD5) Made available in DSpace on 2014-05-20T13:33:27Z (GMT). No. of bitstreams: 2 WOS000263600000016.pdf: 403547 bytes, checksum: 00bc362fcdd0b972ebb3e4d568306f22 (MD5) WOS000263600000016.pdf.txt: 60599 bytes, checksum: b54b4d6932590cfa6f2c627a17471091 (MD5) Previous issue date: 2009-02-01 Qiagen, Germany German Ministry of Health National Reference Centre for Tropical Infections at the Bernhard Nocht Institute European Union BackgroundDetection and quantification of hepatitis C virus (HCV) RNA is integral to diagnostic and therapeutic regimens. All molecular assays target the viral 5'-noncoding region (59-NCR), and all show genotype-dependent variation of sensitivities and viral load results. Non-western HCV genotypes have been under-represented in evaluation studies. An alternative diagnostic target region within the HCV genome could facilitate a new generation of assays.Methods and FindingsIn this study we determined by de novo sequencing that the 3'-X-tail element, characterized significantly later than the rest of the genome, is highly conserved across genotypes. To prove its clinical utility as a molecular diagnostic target, a prototype qualitative and quantitative test was developed and evaluated multicentrically on a large and complete panel of 725 clinical plasma samples, covering HCV genotypes 1-6, from four continents (Germany, UK, Brazil, South Africa, Singapore). To our knowledge, this is the most diversified and comprehensive panel of clinical and genotype specimens used in HCV nucleic acid testing (NAT) validation to date. The lower limit of detection (LOD) was 18.4 IU/ml (95% confidence interval, 15.3-24.1 IU/ml), suggesting applicability in donor blood screening. The upper LOD exceeded 10(-9) IU/ml, facilitating viral load monitoring within a wide dynamic range. In 598 genotyped samples, quantified by Bayer VERSANT 3.0 branched DNA (bDNA), X-tail-based viral loads were highly concordant with bDNA for all genotypes. Correlation coefficients between bDNA and X-tail NAT, for genotypes 1-6, were: 0.92, 0.85, 0.95, 0.91, 0.95, and 0.96, respectively; X-tail-based viral loads deviated by more than 0.5 log10 from 5'-NCR-based viral loads in only 12% of samples (maximum deviation, 0.85 log10). The successful introduction of X-tail NAT in a Brazilian laboratory confirmed the practical stability and robustness of the X-tail-based protocol. The assay was implemented at low reaction costs (US$8.70 per sample), short turnover times (2.5 h for up to 96 samples), and without technical difficulties.ConclusionThis study indicates a way to fundamentally improve HCV viral load monitoring and infection screening. Our prototype assay can serve as a template for a new generation of viral load assays. Additionally, to our knowledge this study provides the first open protocol to permit industry-grade HCV detection and quantification in resource-limited settings. Univ Bonn, Inst Virol, D-5300 Bonn, Germany Bernhard Nocht Inst Trop Med, Clin Virol Grp, Hamburg, Germany Universidade Federal da Bahia (UFBA), Univ Hosp Prof Edgard Santos, Infect Dis Res Lab, Salvador, BA, Brazil UNESP, Botucatu Med Sch, Ctr Blood Transfus, Mol Biol Lab, Botucatu, SP, Brazil UNESP, Dept Internal Med, Botucatu, SP, Brazil Univ Edinburgh, Ctr Infect Dis, Virus Evolut Grp, Edinburgh, Midlothian, Scotland Natl Univ Singapore, Yong Loo Lin Sch Med, Dept Pathol, Singapore, Singapore Natl Univ Singapore Hosp, Mol Diag Ctr, Singapore 119074, Singapore Univ Cape Town, Fac Hlth Sci, Dept Clin Lab Sci, Div Med Virol,Natl Hlth Lab Serv, ZA-7925 Cape Town, South Africa GFE Blut MbH, Frankfurt, Germany UNESP, Botucatu Med Sch, Ctr Blood Transfus, Mol Biol Lab, Botucatu, SP, Brazil UNESP, Dept Internal Med, Botucatu, SP, Brazil EU: SSPE-CT-2005-022639

A summer camp was followed by an outbreak of illness involving around 90 children. Investigations included individual questionnaires, inspection of the camp facilities, and laboratory analysis of water and clinical samples. Contamination of drinking and swimming water was demonstrated. An enterovirus was detected by polymerase chain reaction (PCR) and/or culture in 4/4 cerebrospinal fluid samples, 9/15 (60%) stool samples from symptomatic children and 2/9 (22%) stool samples from asymptomatic children. The virus was identified as an echovirus 3 by sequencing and phylogenetic analysis of a short 5' non-coding region (NCR) PCR product. Viruses from the outbreak clustered closely and an echovirus 3 from a temporally associated non-outbreak case could be readily distinguished. Despite the lack of a standardized approach, direct molecular detection and identification of enteroviruses is an efficient epidemiological tool. Here the 5'-NCR was successfully used for both detection and 'serotyping', and the close genetic relatedness of isolates was proven.

The incidence of persistent hepatitis B surface (HBs) antigenaemia was studied in 114 nephrotic children with glomerulonephritis. Twenty five (24 boys) of 28 cases of membranous glomerulonephritis were HBs antigen (HBsAg) carriers. Only 9 of the remaining 86 patients with nephropathies other than membranous glomerulonephritis were HBsAg positive. HBsAg immune complexes were seen in the sera by electron microscopy. On radioimmunoassay both HBsAg and antibody (anti-HBs), and HBeAg and antibody (anti-HBe) were often detected concurrently, HBsAg was not shown in the glomerular capillary wall. HBs antigenaemia persisted in 80% of patients after recovery from glomerulonephritis but remission of the proteinuria correlated well, although not fully, with seroconversion to anti-HBe. The natural history of hepatitis B virus (HBV) associated glomerulonephritis in childhood is one of slow recovery. A few patients are left with mild asymptomatic proteinuria but progressive renal failure is rare. The 14% incidence of membranous glomerulonephritis in nephrotic children in this area is much higher than that found by the international study of kidney disease in children in well developed countries and is probably related to a high HBV carrier rate. A search for HBV markers should be included in the investigation of persistent glomerulonephritis, particularly in countries with a high prevalence of HBV carriers.

Unconcentrated cerebrospinal fluid (CSF) was mixed in a constant ratio with carrier ampholytes and spacer amino acids and analysed by isotachophoresis. Examination of the cerebrospinal fluids from 113 patients showed characteristic patterns for normal, acute viral and acute bacterial meningitis. CSF from cases of subacute sclerosing panencephalitis (SSPE) and multiple sclerosis (MS) differed from these and from each other in the gamma-globulin region. This technique provides useful information on the integrity of the blood-brain barrier and is able to determine whether immunoglobulins present arise within the central nervous system or enter through a damaged barrier.

Immune complexes of the hepatitis B e-antigen (HBeAg) could be labeled and thus visually identified in the electron microscope by using antibody to HBeAg (anti-HBe) tagged with colloidal gold particles. Circulating immune complexes of HBeAg were detected in sera from patients with acute hepatitis B infections as well as from asymptomatic carriers of hepatitis B surface antigens (HBsAg). Sera positive for rheumatoid factor frequently contained mixed aggregates in which immune complexes of HBsAg were closely bound to immune complexes of HBeAg.

Background Infections 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. Methods Two hundred and twenty children presenting consecutively at a tertiary children's 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 Fisher's Exact test. Results HRV 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. Conclusion HRV may be the commonest viral infection in young South African children with acute wheezing. Infection is associated with mild or moderate clinical disease.