A comparative genome analysis of Rift Valley Fever virus isolates from foci of the disease outbreak in South Africa in 2008-2010.

Rift Valley fever (RVF) is a re-emerging zoonotic disease responsible for major losses in livestock production, with negative impact on the livelihoods of both commercial and resource-poor farmers in sub-Sahara African countries. The disease remains a threat in countries where its mosquito vector thrives. Outbreaks of RVF usually follow weather conditions which favour increase in mosquito populations. Such outbreaks are usually cyclical, occurring every 10-15 years. Recent outbreaks of the disease in South Africa have occurred unpredictably and with increased frequency. In 2008, outbreaks were reported in Mpumalanga, Limpopo and Gauteng provinces, followed by 2009 outbreaks in KwaZulu-Natal, Mpumalanga and Northern Cape provinces and in 2010 in the Eastern Cape, Northern Cape, Western Cape, North West, Free State and Mpumalanga provinces. By August 2010, 232 confirmed infections had been reported in humans, with 26 confirmed deaths.To investigate the evolutionary dynamics of RVF viruses (RVFVs) circulating in South Africa, we undertook complete genome sequence analysis of isolates from animals at discrete foci of the 2008-2010 outbreaks. The genome sequences of these viruses were compared with those of the viruses from earlier outbreaks in South Africa and in other countries. The data indicate that one 2009 and all the 2008 isolates from South Africa and Madagascar (M49/08) cluster in Lineage C or Kenya-1. The remaining of the 2009 and 2010 isolates cluster within Lineage H, except isolate M259_RSA_09, which is a probable segment M reassortant. This information will be useful to agencies involved in the control and management of Rift Valley fever in South Africa and the neighbouring countries.

Expression of Rift Valley fever virus N-protein in Nicotiana benthamiana for use as a diagnostic antigen.

BACKGROUND: Rift Valley fever virus (RVFV), the causative agent of Rift Valley fever, is an enveloped single-stranded negative-sense RNA virus in the genus Phlebovirus, family Bunyaviridae. The virus is spread by infected mosquitoes and affects ruminants and humans, causing abortion storms in pregnant ruminants, high neonatal mortality in animals, and morbidity and occasional fatalities in humans. The disease is endemic in parts of Africa and the Arabian Peninsula, but is described as emerging due to the wide range of mosquitoes that could spread the disease into non-endemic regions. There are different tests for determining whether animals are infected with or have been exposed to RVFV. The most common serological test is antibody ELISA, which detects host immunoglobulins M or G produced specifically in response to infection with RVFV. The presence of antibodies to RVFV nucleocapsid protein (N-protein) is among the best indicators of RVFV exposure in animals. This work describes an investigation of the feasibility of producing a recombinant N-protein in Nicotiana benthamiana and using it in an ELISA. RESULTS: The human-codon optimised RVFV N-protein was successfully expressed in N. benthamiana via Agrobacterium-mediated infiltration of leaves. The recombinant protein was detected as monomers and dimers with maximum protein yields calculated to be 500-558 mg/kg of fresh plant leaves. The identity of the protein was confirmed by liquid chromatography-mass spectrometry (LC-MS) resulting in 87.35% coverage, with 264 unique peptides. Transmission electron microscopy revealed that the protein forms ring structures of ~ 10 nm in diameter. Preliminary data revealed that the protein could successfully differentiate between sera of RVFV-infected sheep and from sera of those not infected with the virus. CONCLUSIONS: To the best of our knowledge this is the first study demonstrating the successful production of RVFV N-protein as a diagnostic reagent by Agrobacterium-mediated transient heterologous expression in N. benthamiana. Preliminary testing of the antigen showed its ability to distinguish RVFV-positive animal sera from RVFV negative animal sera when used in an enzyme linked immunosorbent assay (ELISA). The cost-effective, scalable and simple production method has great potential for use in developing countries where rapid diagnosis of RVFV is necessary.

Anomalous High Rainfall and Soil Saturation as Combined Risk Indicator of Rift Valley Fever Outbreaks, South Africa, 2008-2011.

Rift Valley fever (RVF), a zoonotic vectorborne viral disease, causes loss of life among humans and livestock and an adverse effect on the economy of affected countries. Vaccination is the most effective way to protect livestock; however, during protracted interepidemic periods, farmers discontinue vaccination, which leads to loss of herd immunity and heavy losses of livestock when subsequent outbreaks occur. Retrospective analysis of the 2008-2011 RVF epidemics in South Africa revealed a pattern of continuous and widespread seasonal rainfall causing substantial soil saturation followed by explicit rainfall events that flooded dambos (seasonally flooded depressions), triggering outbreaks of disease. Incorporation of rainfall and soil saturation data into a prediction model for major outbreaks of RVF resulted in the correctly identified risk in nearly 90% of instances at least 1 month before outbreaks occurred; all indications are that irrigation is of major importance in the remaining 10% of outbreaks.

Development of three triplex real-time reverse transcription PCR assays for the qualitative molecular typing of the nine serotypes of African horse sickness virus.

Blood samples collected as part of routine diagnostic investigations from South African horses with clinical signs suggestive of African horse sickness (AHS) were subjected to analysis with an AHS virus (AHSV) group specific reverse transcription quantitative polymerase chain reaction (AHSV RT-qPCR) assay and virus isolation (VI) with subsequent serotyping by plaque inhibition (PI) assays using AHSV serotype-specific antisera. Blood samples that tested positive by AHSV RT-qPCR were then selected for analysis using AHSV type specific RT-qPCR (AHSV TS RT-qPCR) assays. The TS RT-qPCR assays were evaluated using both historic stocks of the South African reference strains of each of the 9 AHSV serotypes, as well as recently derived stocks of these same viruses. Of the 503 horse blood samples tested, 156 were positive by both AHSV RT-qPCR and VI assays, whereas 135 samples that were VI negative were positive by AHSV RT-qPCR assay. The virus isolates made from the various blood samples included all 9 AHSV serotypes, and there was 100% agreement between the results of conventional serotyping of individual virus isolates by PI assay and AHSV TS RT-qPCR typing results. Results of the current study confirm that the AHSV TS RT-qPCR assays for the identification of individual AHSV serotypes are applicable and practicable and therefore are potentially highly useful and appropriate for virus typing in AHS outbreak situations in endemic or sporadic incursion areas, which can be crucial in determining appropriate and timely vaccination and control strategies.

Validation of an ELISA for the concurrent detection of total antibodies (IgM and IgG) to Rift Valley fever virus.

Rift Valley fever virus (RVFV) infects humans and livestock, causing haemorrhaging and abortions in animals. Three major RVF epizootics have occurred in South Africa since the 1950s and the outbreak in 2010 had a mortality rate of 10.7% in humans. Accurate and early detection is therefore essential for management of this zoonotic disease. Enzyme-linked immunosorbent assays (ELISAs) have been developed for the detection of either IgM or IgG antibodies to RVFV in animal sera. In this study, data are presented on the validation of a double-antigen ELISA for the simultaneous detection of both classes of antibodies to RVFV ina single test. ELISA plates were coated with a recombinant nucleoprotein. The nucleoprotein,conjugated to horseradish peroxidase, was used as the detecting reagent. A total of 534 sera from sheep and cattle were used in the validation. The sheep sera were collected during a RVF pathogenesis study at the Agricultural Research Council (ARC) - Onderstepoort Veterinary Institute and the cattle sera were collected during an outbreak of RVF in 2008 at the ARC -Animal Production Institute in Irene, Pretoria. The ELISA had a diagnostic sensitivity of 98.4% and a specificity of 100% when compared to a commercial cELISA. This convenient and fast assay is suitable for use in serological surveys or monitoring immune responses in vaccinated animals.

Validation of an IgM antibody capture ELISA based on a recombinant nucleoprotein for identification of domestic ruminants infected with Rift Valley fever virus.

The presence of competent vectors in some countries currently free of Rift Valley fever (RVF) and global changes in climate, travel and trade have increased the risk of RVF spreading to new regions and have emphasised the need for accurate and reliable diagnostic tools for early diagnosis during RVF outbreaks. Highly sensitive viral detection systems like PCR have a limited use during outbreaks because of the short duration of viraemia, whereas antibodies like specific IgM which are serological indicators of acute infection, can be detected for up to 50 days after infection. Using the highly conserved and immunogenic recombinant nucleoprotein of RVF virus in an IgM capture ELISA, the risk of laboratory infection associated with traditional serological methods is avoided. The use of pre-coated/pre-blocked ELISA plates and the conjugation of the recombinant nucleoprotein with horseradish peroxidase simplified and shortened the assay procedure. Results showed the assay to be highly reproducible with a lower detection limit equal to that of a commercial competition ELISA. By receiver operating characteristic (ROC) curve analysis the area under curve (AUC) index was determined as 1.0 and the diagnostic sensitivity and specificity at a PP cut-off value of 4.1 as 100% and 99.78% respectively. The results of this study demonstrated that the IgM capture ELISA is a safe, reliable and highly accurate diagnostic tool which can be used on its own or in parallel with other methods for the early diagnosis of RVF virus infection and also for monitoring of immune responses in vaccinated domestic ruminants.

Alternative oxidase (AOX) genes of African trypanosomes: phylogeny and evolution of AOX and plastid terminal oxidase families.

To clarify evolution and phylogenetic relationships of trypanosome alternative oxidase (AOX) molecules, AOX genes (cDNAs) of the African trypanosomes, Trypanosoma congolense and Trypanosoma evansi, were cloned by PCR. Both AOXs possess conserved consensus motifs (-E-, -EXXH-). The putative amino acid sequence of the AOX of T. evansi was exactly the same as that of T. brucei. A protein phylogeny of trypanosome AOXs revealed that three genetically and pathogenically distinct strains of T. congolense are closely related to each other. When all known AOX sequences collected from current databases were analyzed, the common ancestor of these three Trypanosoma species shared a sister-group position to T. brucei/T. evansi. Monophyly of Trypanosoma spp. was clearly supported (100% bootstrap value) with Trypanosoma vivax placed at the most basal position of the Trypanosoma clade. Monophyly of other eukaryotic lineages, terrestrial plants + red algae, Metazoa, diatoms, Alveolata, oomycetes, green algae, and Fungi, was reconstructed in the best AOX tree obtained from maximum likelihood analysis, although some of these clades were not strongly supported. The terrestrial plants + red algae clade showed the closest affinity with an alpha-proteobacterium, Novosphingobium aromaticivorans, and the common ancestor of these lineages, was separated from other eukaryotes. Although the root of the AOX subtree was not clearly determined, subsequent phylogenetic analysis of the composite tree for AOX and plastid terminal oxidase (PTOX) demonstrated that PTOX and related cyanobacterial sequences are of a monophyletic origin and their common ancestor is linked to AOX sequences.

Probabilistic order in antigenic variation of Trypanosoma brucei.

Antigenic variation in African trypanosomes displays a degree of order that is usually described as 'semi-predictable' but which has not been analysed in statistical detail. It has been proposed that, during switching, the variable antigen type (VAT) being inactivated can influence which VAT is subsequently activated. Antigenic variation proceeds by the differential activation of members of the large archive of distinct variable surface glycoprotein (VSG) genes. The most popular model for ordered expression of VATs invokes differential activation probabilities for individual VSG genes, dictated in part by which of the four types of genetic locus they occupy. We have shown, in pilot experiments in cattle, correlation between the timing of appearance of VSG-specific mRNA and of lytic antibodies corresponding to seven VSGs encoded by single-copy genes. We have then determined the times of appearance of VAT-specific antibodies, as a measure of appearance of the VATs, in a statistically significant number of mouse infections (n=22). There is a surprisingly high degree of order in temporal appearance of the VATs, indicating that antigenic variation proceeds through order in the probability of activation of each VAT. In addition, for the few examples of each available, the locus type inhabited by the silent 'donor' VSG plays a significant role in determination of order. We have analysed in detail previously published data on VATs appearing in first relapse peaks, and find that the variant being switched off does not influence which one is being switched on. This differs from what has been reported for Plasmodium falciparum var antigenic variation. All these features of trypanosome antigenic variation can be explained by a one-step model in which, following an initial deactivation event, the switch process and the imposition of order early in infection arise from the inherent activation probabilities of the specific VSG being switched on.

Identification of a genetic marker for isometamidium chloride resistance in Trypanosoma congolense.

Isometamidium chloride has remained a very important prophylactic and therapeutic drug against trypanosomosis in cattle since its introduction into the market in the 1950s with, unfortunately, a concomitant development of resistance in trypanosomosis endemic areas. Amplified Fragment Length Polymorphism (AFLP) was used to compare two isogenic clones of Trypanosoma congolense. The parent clone, sensitive to isometamidium, has a CD50 (the curative dose that gives complete cure in 50% of the animals) in the mouse of 0.018 mg/kg and its derivative exposed to increasing doses of isometamidium, has a CD50 that is 94-fold higher. Sixty-four combinations of eight Eco RI and eight Mse I primers were used in comparative AFLP analysis to detect subtle genetic differences between the two clones. Thirty-five polymorphic fragments of DNA that were observed only in the resistant clone were purified and then sequenced. The nucleotide sequences were used in searching the GeneDB T. congolense database to find surrounding sequences upstream of an open reading frame and downstream to a stop codon. The sequences of the open reading frames were subsequently compared to the sequences in the genomic databases. A predicted gene coding for an 854 amino acids protein was thus identified. The protein contains a putative ATP binding site, Walker B and LSGG motifs and eight predicted trans-membrane domains. The gene in the resistant strain of T. congolense has a triplet insertion coding for an extra lysine. Using polymerase chain reaction-restriction fragment length polymorphism, the insertion was sought in the genomes of 35 T. congolense strains isolated from different geographic origins and whose response to isometamidium chloride had been determined through single dose mouse tests. The presence of the insertion, specifying an extra codon was found to always be present in the genomes of T. congolense clones that were resistant to isometamidium chloride.

Variable Surface Glycoprotein RoTat 1.2 PCR as a specific diagnostic tool for the detection of Trypanosoma evansi infections.

BACKGROUND: Based on the recently sequenced gene coding for the Trypanosoma evansi (T. evansi) RoTat 1.2 Variable Surface Glycoprotein (VSG), a primer pair was designed targeting the DNA region lacking homology to other known VSG genes. A total of 39 different trypanosome stocks were tested using the RoTat 1.2 based Polymerase Chain Reaction (PCR). RESULTS: This PCR yielded a 205 bp product in all T. evansi and in seven out of nine T. equiperdum strains tested. This product was not detected in the DNA from T. b. brucei, T. b. gambiense, T. b. rhodesiense, T. congolense, T. vivax and T. theileri parasites. The Rotat 1.2 PCR detects as few as 10 trypanosomes per reaction with purified DNA from blood samples, i.e. 50 trypanosomes/ml. CONCLUSION: PCR amplification of the RoTat 1.2 VSG gene is a specific marker for T. evansi strains, except T. evansi type B, and is especially useful in dyskinetoplastic strains where kDNA based markers may fail to amplify. Furthermore, our data support previous suggestions that some T. evansi stocks have been previously misclassified as T. equiperdum.

Population genetic structure and cladistic analysis of Trypanosoma brucei isolates.

Using a novel multilocus DNA marker analysis method, we studied the population genetic structure of Trypansoma brucei stocks and derived clones isolated from animal and rhodesiense sleeping sickness patients during a national sleeping sickness control program in Mukono district, Uganda. We then performed a cladistic analysis to trace relationships and evolution, using stocks and clones recovered from geographically and temporally matched hosts, including inter-strain comparisons with T. b. gambiense stocks and clones. Our results show that while there was close genetic relatedness among parasite populations from the same geographical region, micro-heterogeneities exist between different stocks. Data are presented that indicate that not every human sleeping sickness focus may be associated with a particular human-infective trypanosome strain responsible for long-term stability of the reference focus. We provide evidence of genetic sub-structuring among type 1 T. b. gambiense stocks, which has potentially important implications for molecular epidemiology of T. brucei.

Multiplex-endonuclease genotyping approach (MEGA): a tool for the fine-scale detection of unlinked polymorphic DNA markers.

Restriction enzyme-detectable polymorphisms have been used for assessing genetic differences and generating informative genetic markers. The most detailed fingerprinting analyses have been obtained using the AFLP (amplified fragment length polymorphism) technique, which accesses subsets of polymorphisms at one or two restriction sites. To combine increased discriminatory power with the stringency of polymerase chain reaction amplification, it would be beneficial to access additional independent restriction sites per analysis, and to amplify subsets of DNA restriction fragments with only one pair of oligonucleotide primers. We have now developed a unique approach that permits the simultaneous use of four or more endonucleases in combination with one pair of adapters/primers, and applied it to genotype 21 trypanosome populations to subspecific level. The approach takes advantage of the fact that some endonucleases create cohesive ends that are compatible with the overhang sites created by other endonucleases. We demonstrate the greater resolution of identifiable polymorphic fragments over the conventional ligation-mediated restriction analysis method, and discuss the value of the approach as a tool for fine genetic mapping of Trypanosoma brucei. Finally, we propose use of the method for fine characterisation and for identifying co-dominant genetic markers in a variety of other taxa.