Sequence analysis and structural implications of rotavirus capsid proteins.

Rotavirus is the major cause of severe virus-associated gastroenteritis worldwide in children aged 5 and younger. Many children lose their lives annually due to this infection and the impact is particularly pronounced in developing countries. The mature rotavirus is a non-enveloped triple-layered nucleocapsid containing 11 double stranded RNA segments. Here a global view on the sequence and structure of the three main capsid proteins, VP2, VP6 and VP7 is shown by generating a consensus sequence for each of these rotavirus proteins, for each species obtained from published data of representative rotavirus genotypes from across the world and across species. Degree of conservation between species was represented on homology models for each of the proteins. VP7 shows the highest level of variation with 14-45 amino acids showing conservation of less than 60%. These changes are localised to the outer surface alluding to a possible mechanism in evading the immune system. The middle layer, VP6 shows lower variability with only 14-32 sites having lower than 70% conservation. The inner structural layer made up of VP2 showed the lowest variability with only 1-16 sites having less than 70% conservation across species. The results correlate with each protein's multiple structural roles in the infection cycle. Thus, although the nucleotide sequences vary due to the error-prone nature of replication and lack of proof reading, the corresponding amino acid sequence of VP2, 6 and 7 remain relatively conserved. Benefits of this knowledge about the conservation include the ability to target proteins at sites that cannot undergo mutational changes without influencing viral fitness; as well as possibility to study systems that are highly evolved for structure and function in order to determine how to generate and manipulate such systems for use in various biotechnological applications.

Characterization and molecular epidemiology of rotavirus strains recovered in Northern Pretoria, South Africa during 2003-2006.

Rotavirus infection is the most common cause of severe dehydrating gastroenteritis in infants and young children and remains a significant clinical problem worldwide. The severity and the burden of rotavirus disease could be reduced through the implementation of an effective vaccine. The aim of this study was to characterize rotavirus strains circulating in the local community as part of an ongoing hospital burden of disease study when a G1P[8] rotavirus vaccine candidate was being evaluated in the same community. From 2003 through 2006, 729 rotavirus-positive stool specimens were collected from children <5 years of age who were treated for diarrhea at Dr George Mukhari Hospital, Ga-Rankuwa, South Africa. Molecular characterization of the strains was performed by polyacrylamide gel electrophoresis and genotyping of the VP4 and VP7 alleles using well-established seminested multiplex reverse-transcription polymerase chain reaction methods. In 2003, 62% of strains exhibited the short rotavirus electropherotype, and the most common rotavirus strain was G2P[4]. In subsequent years, predominant rotavirus strains included G1P[8] and G1P[6] in 2004, G3P[8] and G3P[6] in 2005, and G1P[8] in 2006. For the 4 years of the study, rotavirus strains with P[6] genotype were detected in 25% of all rotavirus-positive specimens. In addition, unusual G12P[6] and G8 strains were detected at a low frequency. These results reflect the diversity of rotavirus strains circulating in South African communities.

The detection and molecular characterization of human G12 genotypes in South Africa.

The last decade has seen an increase in the detection of rotavirus strains other than G1-G4 emerging or even predominating in some settings. The performance of the current rotavirus vaccines against unusual or rare circulating rotavirus serotypes cannot be predicted and continuous monitoring of wild type rotaviruses will remain a priority. Routine molecular rotavirus surveillance conducted in the Gauteng Province, South Africa during 2004, resulted in the detection of strains that could not typed using standard G specific genotyping primers. Sequencing of the first round amplicons revealed 19 serotype G12P[6] strains and one G12P[8] strain. Phylogenetic analyses of the G12 strains indicated that these strains are probably a recent introduction into South Africa and emerged from a strain related to the Indian isolate ISO-5. The association of the South African G12s with the P[6] genotype may suggest a mechanism for unusual strains to become more ecologically suited to local population transmission dynamics. This is the first report of serotype G12 strains on the African continent and continued surveillance will be required to track the emergence of G12 strains in Africa.

Prevalence of rotavirus, adenovirus and astrovirus infection in young children with gastroenteritis in Gaborone, Botswana.

OBJECTIVE: To determine the prevalence of three enteric viruses, namely rotavirus, adenovirus and astrovirus, as agents of diarrhoea in and around Gaborone, Botswana. DESIGN: The sample were categorised into four groups according to the age of the patient: 0-3 months, 4-6 months, 7-12 months and 25-60 months. Total monthly samples across age groups formed basis for calcultating seasonal prevalence of rotavirus infection. SETTING: Stool samples were collected from three medical laboratories in Gaborone and one in the town of Mochudi. These were collected from children under the age of five years with gastroenteritis. SUBJECTS: Stool samples were collected between March 2001 and February 2002 from 346 children less than five years of age suffering from gastroenteritis. These samples had been sent to medical laboratories for microbiological examination. METHODS: The samples were screened for rotavirus (RV), adenovirus (Ad) and astrovirus (AsV) antigens using commercially available ELISA kits. The Ad positive samples were further analysed by commercially available group specific Ad type 40/41 Enzyme Immuno Assays (EIA). RESULTS: Shedding of RV was detected in 9.2%, Ad in 7.8% and AsV in 2.7% of the samples analysed. The enteric Ad (types 40 and 41) were detected in 2% of the samples and the remaining 5.8% of Ad positive samples were non-enteric Ad. An increase of RV was noted in the autumn-winter season but no seasonal pattern was observed in Ad shedding. Seasonal prevalence of AsV could not be determined. The average age of children infected with these agents was less than one year. CONCLUSION: The incidence of rotavirus infection amongst children in Botswana appears to be relatively low. The prevalence rate of adenovirus and astrovirus is similar to other studies in parts of Southern Africa. However, continued enteric virus surveillance and epidemiology amongst this group is required.

A cost-effective particle agglutination assay to detect viral antibodies in dried blood spots--a simple solution to HIV and HCV screening.

OBJECTIVES: To conduct a serological survey of human immunodeficiency virus (HIV) and hepatitis C virus (HCV) in Gabon and Ga-Rankuwa, South Africa. A secondary objective was to test a novel, simple, inexpensive agglutination assay for anti-HIV IgG and anti-HCV IgG from blood samples stored as spots dried onto filter paper. DESIGN: Blood from heel pricks was dried onto filter paper and stored. Blood was eluted from the spots and serum antibody was then assayed using a modified agglutination assay--blood was added to gelatin agglutination beads that had been sensitised with viral antigen. A positive result showed as an agglutination pattern while a negative result appeared as a tight bead. SUBJECT: This was a hospital-based study involving 271 neonates at Ga-Rankuwa Hospital, South Africa, and 856 patients ranging in age from three months to over 50 years who attended clinics in Gabon. RESULTS: Seroprevalence to HIV was determined in Ga-Rankuwa to be just under 14% (13.8%). Antibodies to HCV were not detected. In Gabon, the prevalence to HIV was just under 1% (0.82%) with a relatively high incidence of HCV, nearing 4% (3.79%). CONCLUSION: The sensitivity of the agglutination assay compared favourably to enzyme immune assay (EIA) with respect to sensitivity, simplicity and cost. This assay may be useful in sero-epidemilogical assays in developing countries.

Polymerized serum albumin beads possessing slow release properties for use in vaccines.

Experimental vaccines have been made by co-polymerizing virus particles and virus subunits into rabbit serum albumin beads. When injected into rabbits these model vaccines induced specific humoral antibody production and mimicked vaccines prepared using Freund's incomplete adjuvant. No adverse reactions occurred in experimental rabbits and no antibodies reacting with polymerized rabbit serum albumin beads were found. Immunostimulation is attributed to a slow release of antigen coinciding with the gradual breakdown of bead structure.

Biodegradable serum albumin polymers for the sustained release of virus antigens in vaccines. A preliminary report.

In an experimental vaccine, virus particles were chemically cross-linked into rabbit serum albumin beads. This vaccine was injected into rabbits without adverse reactions and the humoral antibody levels achieved were comparable to those obtained using virus emulsified in Freund's adjuvant. The antibodies produced were capable of neutralizing virus infectivity.

Differences in the genotypes and plasma concentrations of the INTERLEUKIN-1 receptor antagonist in black and white South African asthmatics and control subjects.

The allelic frequency of a variable tandem repeat (VNTR) polymorphism in intron 2 of the IL-1 Ra gene was studied in black and white patients with asthma as well as control individuals. The plasma IL-1 Ra concentration was also determined in asthmatics and compared to control individuals. The 410-bp allele of the IL-1 Ra was significantly increased in all black subjects (90%) as compared to all white subjects (74%, P<0.0001), while the 240-bp allele was significantly reduced in all black subjects (11%) as compared to all white subjects (27%, P<0.0001). There was no difference in the frequency of the VNTR of the IL-1 Ra between black asthmatics and black controls and between white asthmatics and white controls. The IL-1 Ra levels were significantly increased in black and white patients with severe or moderate asthma as compared to patients with mild asthma. Increased plasma concentrations of the IL-1 Ra was found to be associated with disease severity in all asthmatic patients.

Polymorphisms of the beta chain of the high-affinity immunoglobulin E receptor (Fcepsilon RI-beta) in South African black and white asthmatic and nonasthmatic individuals.

We used amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) to document the prevalence of three mutations in the beta chain of the high-affinity IgE receptor (Fcepsilon RI-beta): I181L, V183L, and E237G in a sample of black and white asthmatic and control subjects in South Africa to determine whether these variants contribute to the enhanced IgE responses in these groups and also to determine whether the discrepancy in the prevalence of atopy in these groups could be attributed to these variants, as whites tend to be more atopic than blacks. There was a significant difference in the frequency of I181L between white asthmatics (28%) and white control subjects (3%) (p = 0.00001), and between black control subjects (16%) and white control subjects (p = 0.002); no difference in the frequency of I181L was observed between black asthmatics (22%) and black control subjects (16%). V183L was found in one black asthmatic who was also positive for I181L and E237G. There was a significant difference in the frequency of E237G between black asthmatics (20%) and white asthmatics (12%) (p = 0.05), and between control subjects (20%) and white control subjects (5%) (p = 0.003). E237G was more prevalent in blacks (20%) than in whites (8.5%) (p = 0.001). I181L might predispose to atopy in the white population, but not in the black population. The significantly higher prevalence of E237G in blacks than in whites might explain why blacks tend to have more severe asthma than whites and might offer more insight into the higher asthma mortality rate in the black population as compared with the white population.

Ethnic differences in allelic associations of the interleukin-1 gene cluster in South African patients with inflammatory bowel disease (IBD) and in control individuals.

The allelic frequencies of TaqI, PstI, and variable number of tandem repeat (VNTR) polymorphisms of the IL-1beta, IL-1 receptor (IL-1Re), and IL-1 receptor antagonist (IL-1Ra) respectively, were investigated in black and white patients with inflammatory bowel diseases (IBD) and compared with control individuals. Plasma concentrations of IL-1beta and IL-1Ra were also determined in these individuals. The IL-1beta TaqI(-) allele was significantly more frequent in 50 white IBD patients (60%) compared with 47 white controls (17%), and 20 black patients (20%) (P=0.00001 and P=0.0001, respectively). The IL-1Re PstI(-) allele was significantly more frequent in 20 black patients (75%) compared with 50 white patients (44%) (P=0.0001). The frequency of the IL-1Ra 240-bp allele was lower in black (12%) compared with white controls (25%), (P=0.0151), and the 410-bp allele was more frequent in black (87%) compared with white (73%) controls (P=0.0096). Linkage disequilibrium was found in black individuals homozygous for the 410-bp allele of IL-1Ra, and the PstI(-) allele of IL-1Re (84%) (P=0.0032). There was a significantly increased level of IL-1Ra in black patients compared with white patients and black controls (P=0.0006 and P=0.0008, respectively). The population differences in allelic frequencies of the IL-1 gene cluster and IL-1Ra concentrations suggest that genetic and environmental factors play an important role in susceptibility to IBD.

Differences in the prevalence of a TaqI RFLP in the 3' flanking region of the alpha 1-proteinase inhibitor gene between asthmatic and non-asthmatic black and white South Africans.

The prevalence of the Taq I(-) allele in variants of alpha 1-proteinase inhibitor (alpha 1PI) was investigated in a group of 28 black asthmatic patients and 32 black control individuals, and was compared to 43 white asthmatic patients and 32 white control individuals. The plasma concentration of alpha 1PI was determined in eight black and 14 white asthmatics without the Taq I(-) allele, and compared to seven black and three white asthmatics with the Taq I(-) allele. Alpha-1-PI concentration was also determined in 10 black and 29 white control individuals without the Taq I(-) allele and compared to seven black and three white controls with the Taq I(-) allele. There was a highly significant difference in the frequency of the Taq I(-) allele between black South Africans (24.1%) and white South Africans (6%) (p < 0.00001) and a significant difference in the frequency of the Taq I(-) allele between black asthmatics and white asthmatics (p = 0.0004) and between black controls and white controls (p = 0.011). The Taq I(-) allele was significantly associated with the M1 (Val213) variant as compared to the M1 (Ala213) of alpha 1PI (p = 0.0042). There was no difference in the concentration of alpha 1PI between the asthmatics (black and white) lacking the Taq I(-) allele and the asthmatics (black and white) with the allele. However, a significant increase in plasma alpha 1PI concentration was found in the asthmatics compared to the controls (p = 0.011). The Taq I(-) allele did not seem to interfere with the basal expression of alpha 1PI in the groups of asthmatic patients in this study.