African Isolates Research Articles

Low nucleotide diversity of the Plasmodium falciparum AP2-EXP2 gene among clinical samples from Ghana.

PfAP2-EXP2 is located within chromosome 6 of Plasmodium falciparum recently identified to be undergoing an extensive selective sweep in West African isolates. The gene encoding this transcription factor, PfAP2-EXP2, is essential and thus likely subject to purifying selection that limits variants in the parasite population despite its genomic location. 72 Plasmodium falciparum field samples and 801 clinical sequences from the Pf6 MalariaGEN dataset of Ghanaian origin, were integrated and analysed. A total of 14 single nucleotide variants of which 5 were missense variants, were identified after quality checks and filtering. Except for one, all identified variants were rare among the clinical samples obtained in this study (Minor allelic frequency < 0.01). Further results revealed a considerably low dN/dS value (0.208) suggesting the presence of purifying selection. Further, all the mutant amino acids were wildtype residues in AP2-EXP2 orthologous proteins-tentatively suggesting a genus-level conservation of amino acid residues. Computational analysis and predictions corroborated these findings. Despite the recent extensive selective sweep within chromosome 6 of West African isolates, PfAP2-EXP2 of Ghanaian origin exhibits low nucleotide diversity and very low dN/dS consistent with purifying selection acting to maintain the function of an essential gene. The conservation of AP2-EXP2 is an important factor that makes it a potential drug target.

Assembled genome of an Ethiopian Plasmodium vivax isolate generated using GridION long-read technology.

Plasmodium vivax causes ~20% of malaria cases in Ethiopia. Here, we report a long-read genome assembly generated from an individual collected in 2022. P. vivax is genetically diverse across endemic regions; thus, the genome assembly of an African isolate is an important resource for the malaria research community.

Wide variation in aggressiveness and growth in South African Fusarium circinatum isolates with geographical origin as the primary determinant

Fusarium circinatum is a globally important fungal pathogen that causes pitch canker on a wide range of Pinus species. In this study, we considered mycelial growth in culture and aggressiveness of a South African population of F. circinatum isolates. The specific aim was to determine how these phenotypes are correlated with one another and other isolate characteristics, including mating type, host species and geographic origin of the isolates. The study involved the selection of 102 isolates of F. circinatum from five provinces of South Africa based on genotype, mating type and original host from which they were isolated, after which pathogenicity tests were carried out on Pinus patula seedlings. Mycelial growth in culture was also determined for all the isolates at three different temperatures. In the pathogenicity tests, most of the isolates produced significant lesions. While seven had low levels of aggressiveness, other isolates tested were as or more aggressive than a standard set of isolates routinely used for resistance/ tolerance screening. Most F. circinatum isolates grew best at 25 °C, followed by growth at 20 °C and 30 °C. A notable correlation between aggressiveness and the geographical origin was identified. Moreover, the study highlighted a moderately positive relationship between the growth rate of F. circinatum isolates and their reported aggressiveness. The extensive data gathered on the aggressiveness of F. circinatum will offer a broad range of isolates for future comparative studies using other genotypes of F. circinatum and will also aid in the consideration of F. circinatum isolates used for tolerance screening studies.

Four new Geosmithia species from bark beetles infesting indigenous South African trees

Over the past two decades, numerous Geosmithia fungi have been isolated from the bodies and galleries of wood–boring beetles. However, this genus of asexual Sordariomycetes remains taxonomically and ecologically understudied, especially in the Southern Hemisphere. In South Africa, two prior surveys reported Geosmithia species from bark beetles, but neither thoroughly investigated species identities. In this study, we collected bark beetles from native trees in the Western Cape Province of South Africa and isolated, identified and described their associated Geosmithia species. Geosmithia spp. previously collected in South Africa were also re-examined. The ITS sequences of Geosmithia isolates from 13 beetle taxa infesting 10 host species were considered. Additional gene regions, BT, EF1a and RPB2, were sequenced for a subset of isolates. Four previously described species, G. flava, G. langdonii, G. omnicola and G. pumila were identified by phylogenetic analyses. Additionally, four novel taxa were identified and are here described as G. capensis, G. multisociorum, G. oroboidis and G. stellenboschiana. Geosmithia multisociorum appears to be a generalist associated with multiple beetle–host combinations in the Northern and Southern Hemisphere, whereas G. oroboidis is currently known only from a single beetle and tree host in South Africa. South African isolates of G. capensis and G. stellenboschiana appeared to be restricted to Lanurgus spp. and Hypothenemus sp. beetles, respectively, but both species are also known from beetles and hosts in other countries.

First detection of recombinant variant of African swine fever virus in the Russian Federation (brief communication)

As part of extensive molecular and genetic research into African swine fever virus isolates circulating in Russia, a recombinant variant with a mosaic genome structure has been identified. The one that caused an outbreak on a pig farm in the Primorsky Krai, in 2023. The characterized strain ASFV/Primorsky_2023/DP-4560.Recdemonstrates hemadsorption, active propagation in porcine primary macrophage cell culture, 99.9917% identity with the first recombinant isolates from the People’s Republic of China, recovered in 2021. Recombination sites included 79 open reading frames homologous to genotype II isolates; 49 ones homologous to genotype I and 12 mixed ones. Testing biomaterial from dead pigs in real-time polymerase chain reaction showed no changes in sensitivity or specificity, despite significant genetic distinctions between the recombinant and genotype II isolates that are enzootic to the Russian Federation. However, in 2023, D. Zhao et al. reported on high virulence of the virus related variants as revealed by the challenge tests in domestic pigs. Given the accelerating rates of AFSV molecular evolution in the East Asian countries (China, Vietnam and the Far Eastern regions of Russia), it is required to improve control measures, general and specific prevention, national and international surveillance over the economically significant animal disease.

The Tuberculosis Drug Candidate SQ109 and Its Analogs Have Multistage Activity against Plasmodium falciparum.

Toward repositioning the antitubercular clinical candidate SQ109 as an antimalarial, analogs were investigated for structure-activity relationships for activity against asexual blood stages of the human malaria parasite Plasmodium falciparum pathogenic forms, as well as transmissible, sexual stage gametocytes. We show that equipotent activity (IC50) in the 100-300 nM range could be attained for both asexual and sexual stages, with the activity of most compounds retained against a multidrug-resistant strain. The multistage activity profile relies on high lipophilicity ascribed to the adamantane headgroup, and antiplasmodial activity is critically dependent on the diamine linker. Frontrunner compounds showed conserved activity against genetically diverse southern African clinical isolates. We additionally validated that this series could block transmission to mosquitoes, marking these compounds as novel chemotypes with multistage antiplasmodial activity.

An unusual two-strain cholera outbreak in Lebanon, 2022-2023: a genomic epidemiology study

Cholera is a life-threatening gastrointestinal infection caused by a toxigenic bacterium, Vibrio cholerae. After a lull of almost 30 years, a first case of cholera was detected in Lebanon in October 2022. The outbreak lasted three months, with 8007 suspected cases (671 laboratory-confirmed) and 23 deaths. In this study, we use phenotypic methods and microbial genomics to study 34 clinical and environmental Vibrio cholerae isolates collected throughout this outbreak. All isolates are identified as V. cholerae O1, serotype Ogawa strains from wave 3 of the seventh pandemic El Tor (7PET) lineage. Phylogenomic analysis unexpectedly reveals the presence of two different strains of the seventh pandemic El Tor (7PET) lineage. The dominant strain has a narrow antibiotic resistance profile and is phylogenetically related to South Asian V. cholerae isolates and derived African isolates from the AFR15 sublineage. The second strain is geographically restricted and extensively drug-resistant. It belongs to the AFR13 sublineage and clusters with V. cholerae isolates collected in Yemen. In conclusion, the 2022-2023 Lebanese cholera outbreak is caused by the simultaneous introduction of two different 7PET strains. Genomic surveillance with cross-border collaboration is therefore crucial for the identification of new introductions and routes of circulation of cholera, improving our understanding of cholera epidemiology.

Molecular characterization and identification of African Swine Fever virus isolates from emerging cases of infection in Central Luzon, Philippines

A study that evaluated the molecular characteristics to confirm the identity of the African Swine Fever (ASF) pathogen from emerging cases of infection in Central Luzon, Philippines was initiated. The study involved the collection of 125 blood samples from pigs during the four-month duration of the study when clinical signs of ASF was prevalent in the four out of seven provinces of Central Luzon, Philippines. This work included the amplification of the p72 gene in DNA samples through PCR and confirmation of the identity of the ASF pathogen through DNA sequencing. The DNA sequences of the p72 gene of the ASF pathogen are made up of 412 nucleotides and used as a basis in identifying the Philippine ASF pathogens as ASF virus PH/CL 4/2022, ASF virus PH/CL 3/2022, ASF virus PH/CL 2/2022 and ASF virus PH/CL 1/2022. Philippines ASF virus isolates showed 100% sequence similarity with sequences reported from Malaysia ASF isolate MVZT218/2021, Vietnam ASF isolate D/ASFV/VN/DN-VC/2019, and India ASF isolate ASF/MZ/IND/3 based on BLASTN homology search in the NCBI database. The results of the study revealed close phylogenetic relationships among ASF isolates from Vietnam, Malaysia, India and the Philippines.

Extensive genetic diversity in Plasmodium vivax from Sudan and its genetic relationships with other geographical isolates

Plasmodium vivax, traditionally overlooked has experienced a notable increase in cases in East Africa. This study investigated the geographical origin and genetic diversity of P. vivax in Sudan using 14 microsatellite markers. A total of 113 clinical P. vivax samples were collected from two different ecogeographical zones, New Halfa and Khartoum, in Sudan. Additionally, 841 geographical samples from the database were incorporated for a global genetic analysis to discern genetic relationships among P. vivax isolates on regional and worldwide scales. On the regional scale, our findings revealed 91 unique and 8 shared haplotypes among the Sudan samples, showcasing a remarkable genetic diversity compared to other geographical isolates and supporting the hypothesis that P. vivax originated from Africa. On a global scale, distinct genetic clustering of P. vivax isolates from Africa, South America, and Asia (including Papua New Guinea and Solomon Island) was observed, with limited admixture among the three clusters. Principal component analysis emphasized the substantial contribution of African isolates to the observed global genetic variation. The Sudanese populations displayed extensive genetic diversity, marked by significant multi-locus linkage disequilibrium, suggesting an ancestral source of P. vivax variation globally and frequent recombination among the isolates. Notably, the East African P. vivax exhibited similarity with some Asian isolates, indicating potential recent introductions. Overall, our results underscore the effectiveness of utilizing microsatellite markers for implementing robust control measures, given their ability to capture extensive genetic diversity and linkage disequilibrium patterns.

Differentiation in detoxification gene complements, including neofunctionalization of duplicated cytochrome P450 genes, between lineages of cotton bollworm, Helicoverpa armigera.

Here we investigate the evolutionary dynamics of five enzyme superfamilies (CYPs, GSTs, UGTs, CCEs and ABCs) involved in detoxification in Helicoverpa armigera. The reference assembly for an African isolate of the major lineages, H. a. armigera, has 373 genes in the five superfamilies. Most of its CYPs, GSTs, UGTs and CCEs and a few of its ABCs occur in blocks and most of the clustered genes are in subfamilies specifically implicated in detoxification. Most of the genes have orthologues in the reference genome for the Oceania lineage, H. a. conferta. However, clustered orthologues and subfamilies specifically implicated in detoxification show greater sequence divergence and less constraint on non-synonymous differences between the two assemblies than do other members of the five superfamilies. Two duplicated CYPs, which were found in the H. a. armigera but not H. a. conferta reference genome, were also missing in 16 Chinese populations spanning two different lineages of H. a. armigera. The enzyme produced by one of these duplicates has higher activity against esfenvalerate than a previously described chimeric CYP mutant conferring pyrethroid resistance. Various transposable elements were found in the introns of most detoxification genes, generating diverse gene structures. Extensive resequencing data for the Chinese H. a. armigera and H. a. conferta lineages also revealed complex copy number polymorphisms in 17 CCE001s in a cluster also implicated in pyrethroid metabolism, with substantial haplotype differences between all three lineages. Our results suggest that cotton bollworm has a versatile complement of detoxification genes which are evolving in diverse ways across its range.

Molecular genotyping of Babesia caballi

Babesia caballi is an intra-erythrocytic parasite causing equine piroplasmosis. Three B. caballi genotypes (A, B, and C) have been identified based on the 18 S rRNA and rhoptry-associated protein (rap-1) gene sequences. These variant parasite genotypes compromise the diagnostic utility of the WOAH-recommended serological assays in declaring horses free of equine piroplasmosis. Although a gene encoding a spherical body protein 4 (sbp4) has recently been identified as a potential antigen for the serological detection of B. caballi, the ability of this antigen to detect the different geographical strains has not been determined. The molecular distinction between variant B. caballi genotypes is limited and therefore we developed molecular typing assays for the rapid detection and quantification of distinct parasite genotypes. Field samples were screened for the presence of B. caballi using an established multiplex equine piroplasmosis qPCR assay. In this study, B. caballi genotype A was not detected in any field samples screened. However, phylogenetic analysis of the amplified sbp4 and 18 S rRNA genes confirmed the phylogenetic groupings of the South African isolates into either B. caballi genotypes B or C. A multiple sequence alignment of the sbp4 gene sequences obtained in this study together with the published sbp4 sequences representing B. caballi genotype A, were used to identify conserved regions within the gene to design three primer pairs and three genotype-specific TaqMan minor-groove binder (MGB™) probes. The qPCR assays were shown to be specific and efficient in the detection and differentiation between B. caballi genotypes A, B, and C and could be used as a diagnostic assay to prevent the unintentional spread of variant B. caballi genotypes globally.

First Report of Rust Caused by Phakopsora nishidana on Creeping Fig, Ficus pumila, in South Africa.

Phakopsora nishidana has recently been reported as the causal organism of rust on edible fig, Ficus carica in South Africa (SA) (Boshoff et al. 2022). This contradicted reports by Doidge (1927, 1950) and Verwoerd (1929) who listed Cerotelium fici as the causal organism of the disease in SA. Similarities in urediniospore morphology and differing taxonomic interpretations most likely contributed to the use of both pathogen names as the causal agents for fig rust (Boshoff et al. 2022; Padamsee and McKenzie 2024). In January 2023 rust was commonly observed on nursery specimens of creeping fig, Ficus pumila, a popular evergreen and fast-growing garden plant in SA. Ficus pumila is native to southern China, Indochina, and eastern Asia but has been introduced to many countries worldwide (https://powo.science.kew.org/ accessed 20 March 2024). The F. pumila rust isolate collected in Somerset West (34°07'00.70"S, 18°52'41.75"E; Western Cape (WC) province) was identified using the sequenced 5.8S rRNA-ITS2-28S rRNA locus. When used in a BLAST analysis, this F. pumila isolate (GenBank accession number OR835538) shared the best homology with the Vermont P. nishidana isolate (34°24'33.37"S, 19°08'59.24"E) (MZ047090; e-value 0.0, 99.7% identity, 1400/1404 bp) reported on edible fig by Boshoff et al. (2022). Other P. nishidana isolates with excellent homology were KY764080 (e-value 0.0, 99.8% identity, 1012/1014 bp), KY764081 (e-value 0.0, 99% identity, 882/885 bp) and MF580676 (e-value 0.0, 99.3% identity, 981/988 bp). Accessions were used in a phylogenetic study that included four other rust samples collected from F. carica trees in Somerset West, WC (OR835534; 34°03'37.26"S, 18°51'02.18"E), Onrus River, WC (OR835535; 34°24'43.10"S, 19°09'57.99"E), Elgin, WC (OR835536; 34°14'31.37"S, 19°03'05.38"E), and Bloemfontein, Free State province (OR835537; 29°05'05.1"S 26°09'09.5"E). Reference sequences were as described in Boshoff et al. (2022). Despite limited variation, all six South African isolates, including the F. pumila isolate, grouped with the three P. nishidana reference isolates, but separate from the three P. myrtacearum isolates collected from eucalyptus trees (Maier et al. 2016). Uredinia on F. pumila leaves were mostly hypophyllous and surrounded by a halo of brown, necrotic tissue. On the upper leaf surface, the lesions appeared as small, dark leaf spots with infrequent sporulation. Urediniospores were echinulate, mostly obovoid or elliptical and their cell walls pale-yellow to yellowish-brown. The mean width and length of urediniospores were 16.7 x 21.3 µm. No telia were observed. Applying standard rust inoculation and incubation procedures (Boshoff et al. 2022), F. pumila plants inoculated with P. nishidana isolate PREM63073 produced sporulating uredinia on the abaxial surface of leaves. Likewise, inoculation of F. carica cv. Kadota leaves with the F. pumila isolate OR835538 yielded sporulating lesions containing urediniospores typical of P. nishidana, on the lower surface. Based on both the DNA sequence data and controlled infection studies, our study confirmed the host status of F. pumila for P. nishidana. The occurrence of rust on creeping fig plants in nurseries, the evergreen status of the creeper, and thus the potential to harbor the pathogen during winter, are likely to contribute to the spread of fig rust in SA.

Global Spread, Genetic Differentiation, and Selection of Barley Spot Form Net Blotch Isolates.

Spot form net blotch, caused by Pyrenophora teres f. maculata, is a significant necrotrophic disease of barley that spread worldwide in the twentieth century. Genetic relationships were analyzed to determine the diversity, survival, and dispersal of a diverse collection of 346 isolates from Australia, Southern Africa, North America, Asia Minor, and Europe. The results, based on genome-wide DArTseq data, indicated that isolates from Turkey were the most differentiated with regional sub-structuring, together with individuals closely related to geographically distant genotypes. Elsewhere, population subdivision related to country of origin was evident, although low levels of admixturing was found that may represent rare genotypes or migration from unsampled populations. Canadian isolates were the next most diverged, and Australian and South African the most closely related. With the exception of Turkish isolates, multiple independent Cyp51A mutation events (which confer insensitivity to demethylation inhibitor fungicides) between countries and within regions was evident, with strong selection for a transposable element insertion at the 3' end of the promoter and counterselection elsewhere. Individuals from Western Australia shared genomic regions and Cyp51A haplotypes with South African isolates, suggesting a recent common origin. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Full genome sequence analysis of African swine fever virus isolates from Cameroon.

African swine fever (ASF) is a devastating disease of domestic pigs that has spread across the globe since its introduction into Georgia in 2007. The etiological agent is a large double-stranded DNA virus with a genome of 170 to 180 kb in length depending on the isolate. Much of the differences in genome length between isolates are due to variations in the copy number of five different multigene families that are encoded in repetitive regions that are towards the termini of the covalently closed ends of the genome. Molecular epidemiology of African swine fever virus (ASFV) is primarily based on Sanger sequencing of a few conserved and variable regions, but due to the stability of the dsDNA genome changes in the variable regions occur relatively slowly. Observations in Europe and Asia have shown that changes in other genetic loci can occur and that this could be useful in molecular tracking. ASFV has been circulating in Western Africa for at least forty years. It is therefore reasonable to assume that changes may have accumulated in regions of the genome other than the standard targets over the years. At present only one full genome sequence is available for an isolate from Western Africa, that of a highly virulent isolate collected from Benin during an outbreak in 1997. In Cameroon, ASFV was first reported in 1981 and outbreaks have been reported to the present day and is considered endemic. Here we report three full genome sequences from Cameroon isolates of 1982, 1994 and 2018 outbreaks and identify novel single nucleotide polymorphisms and insertion-deletions that may prove useful for molecular epidemiology studies in Western Africa and beyond.

Near-complete genome sequences of multiple genotype 1 African swine fever virus isolates from 2016 to 2018 in Cameroon.

African swine fever virus has been endemic in Cameroon since 1982. Here, we announce the sequences of Cameroon/2016/C1, Cameroon/2016/C5, Cameroon/2017/C-A2, Cameroon/2018/C02, and Cameroon/2018/CF3, five genotype 1 African swine fever virus genomes collected from domestic pigs between 2016 and 2018.

Gastropod invasions in anthropogenically impacted impoundments in South Africa: Tracing their origins and exploring field evidence of parasite spillback and amplification

Invasive snails are associated with ecological problems in freshwater bodies worldwide. However, their impact on the transmission of digenean infections remain underreported. In the present study, 1708 specimens representing four snail species were sampled from four impoundments in the Limpopo River system in South Africa. Gyraulus chinensis (Planorbidae), Physella acuta (Physidae) and Pseudosuccinea columella (Lymnaeidae), which are invasive, were found in all the sampling sites. In contrast, the native lymnaeid Radix natalensis occurred at only one study site. Digeneans were observed only from R. natalensis (prevalence = 49%) and Ps. columella (prevalence = 23%). Morphological and genetic analyses revealed four digeneans: Fasciola nyanzae, Orientocreadium sp., Petasiger sp. and Patagifer vioscai. Pseudosuccinea columella was infected by the four digeneans while R. natalensis harboured only Orientocreadium sp. and Petasiger sp. Partial sequences of Orientocreadium sp. from the current study differed from congeners whose DNA data are available on GenBank, by p-distances of at least 1.84 and 2.2% for 28S and the internal transcribed spacer (ITS) rDNA, respectively. Phylogenetic analyses demonstrated that the present species is sister to Orientocreadium batrachoides. Genetic and phylogenetic data based on 28S and ITS rDNA suggested that Petasiger sp. from the present study and isolates of three unidentified Petasiger spp. from Kenya, Hungary and Australia, were representatives of the same species. This is the first known report of Orientocreadium, Petasiger and Patagifer from Ps. columella. The occurrence of F. nyanzae in Ps. columella indicates spillback from R. natalensis. These findings echo the concerns raised in previous studies about the potential role of Ps. columella in the amplification of digenean diseases in its introduced range. Phylogenetic analyses of partial sequences of the cytochrome c oxidase subunit 1 mitochondrial gene (cox1) showed multiple lineages of Ps. columella in North and South America. Pseudosuccinea columella specimens from the present study belong to an invasive genotype that has spread globally and has been reported from Zimbabwe, Egypt, Portugal, Australia, Argentina, Colombia and New Mexico (USA). Physella acuta from the current study had a stronger genetic relationship with isolates from Canada and Iceland, than with isolates from other parts of Africa, suggesting several invasion routes into Africa. This is the first known DNA characterisation of G. chinensis from Africa. Phylogenetic reconstruction indicated multiple exit events of G. chinensis from Asia into Europe and Africa. South African isolates clustered in a recent branch containing isolates from the Czech Republic and Hong Kong, China. Considering the presence of invasive snails in all the sampling sites in the present study, it is necessary to investigate the factors that enhance their establishment and to monitor their effects on the native snail populations.

Genomic insights into Plasmodium vivax population structure and diversity in central Africa

BackgroundThough Plasmodium vivax is the second most common malaria species to infect humans, it has not traditionally been considered a major human health concern in central Africa given the high prevalence of the human Duffy-negative phenotype that is believed to prevent infection. Increasing reports of asymptomatic and symptomatic infections in Duffy-negative individuals throughout Africa raise the possibility that P. vivax is evolving to evade host resistance, but there are few parasite samples with genomic data available from this part of the world.MethodsWhole genome sequencing of one new P. vivax isolate from the Democratic Republic of the Congo (DRC) was performed and used in population genomics analyses to assess how this central African isolate fits into the global context of this species.ResultsPlasmodium vivax from DRC is similar to other African populations and is not closely related to the non-human primate parasite P. vivax-like. Evidence is found for a duplication of the gene PvDBP and a single copy of PvDBP2.ConclusionThese results suggest an endemic P. vivax population is present in central Africa. Intentional sampling of P. vivax across Africa would further contextualize this sample within African P. vivax diversity and shed light on the mechanisms of infection in Duffy negative individuals. These results are limited by the uncertainty of how representative this single sample is of the larger population of P. vivax in central Africa.

Wheat stripe mosaic virus from Brazil and South Africa evolved as distinct subpopulations with low genetic variability

AbstractWheat stripe mosaic virus (WhSMV) is the causal agent of soilborne wheat mosaic disease (SBWMD) in Brazil, which is a serious threat to wheat production in the southern part of the country. WhSMV also occurs in Paraguay and South Africa. The virus is soilborne, transmitted by Polymyxa graminis, and management strategies are mainly based on genetic resistance. Variation in the reaction (type and severity of symptoms) of cultivars has been reported depending on the region and/or year of cultivation, leading wheat breeding programmes to test their materials in several locations, which increases costs and is time‐consuming. One hypothesis is that this variation in symptoms is a result of the genetic variability of WhSMV. We assessed the genetic variability and population structure of WhSMV infecting wheat in Brazil and South Africa. In field experiments conducted in different locations in southern Brazil, a consistent reproducibility of symptoms was observed in a set of cultivars, and molecular analysis showed a low degree of genetic variability of the Brazilian viral population. The hypothesis that the variation in SBWMD symptoms in Brazil is due to genetic variability of the virus was thus rejected. Comparison of the Brazilian and South African WhSMV isolates indicated that they comprise distinct subpopulations and that the Brazilian subpopulation is more variable than the South African subpopulation. The lower genetic variability of the South African subpopulation suggests genetic stability or a recent emergence of WhSMV in that region.

Loop-mediated isothermal amplification coupled with nanoparticle-based lateral flow biosensor for monkeypox virus detection

Monkeypox virus (MPXV) infection is currently an evolving public health concern, highlighting an urgent need for early and rapid detection of MPXV. Here, we present a diagnostic test called MPXV-LAMP-LFB, which combines loop-mediated isothermal amplification (LAMP) and nanoparticle-based lateral flow biosensor (LFB) for the simple, sensitive and specific detection of MPXV and differentiation of its two clades. The MPXV-LAMP-LFB can be conducted at a heating block and the detection results can be visually indicated with the biosensor without any specialized apparatus. Two sets of LAMP primers targeting the D14L and ATI genes were designed for the Central and West African MPXV isolates, respectively. The optimal amplification condition was 64 °C for 40 min. Thus, the MPXV-LAMP-LFB test can be completed within 1 h, incorporating rapid DNA extraction (∼15 min), LAMP reaction (∼40 min) and result indicating (∼5 min). The MPXV-LAMP-LFB assay could detect down to 5 copies of plasmid template and 12.5 copies of pseudotyped virus in simulated blood samples. Furthermore, the MPXV-LAMP-LFB assay correctly identified all the positive controls and successfully avoided cross-reactivity with the non-MPXV pathogens or clinical samples, demonstrating its high specificity. Overall, the MPXV-LAMP-LFB test developed in this study showed great promise as a rapid, sensitive and accurate molecular tool for diagnosing MPXV infection.

Characterisation of South African field Ehrlichia ruminantium using multilocus sequence typing.

Heartwater, one of the major tick-borne diseases of some domestic and wild ruminants in Africa, is caused byEhrlichia ruminantium. The genetic diversity ofE. ruminantiumisolates renders the available vaccine ineffective against certain virulent isolates. To better understand theE. ruminantiumgenotypes in South Africa, a total of 1004Amblyomma hebraeumtick deoxyribonucleic acid (DNA) samples from cattle in three South African provinces were tested by pCS20 Sol1 real-time polymerase chain reaction (qPCR) and characterised by multilocus sequence typing (MLST) using five housekeeping genes. Out of 1004 samples tested, 222 (22%) were positive forE. ruminantium. The occurrence ofE. ruminantiumin Mpumalanga, KwaZulu-Natal and Limpopo provinces was 19%, 22% and 27%, respectively. TheE. ruminantiumpositive samples were screened for housekeeping genes and sequenced. Phylogenetic analysis revealed three main lineages: clade 1 made up of worldwide isolates (eastern, southern Africa, and Caribbean isolates), clade 2 comprised only West African isolates and clade 3 consisted of Omatjenne, Kümm2 and Riverside. Some study sample sequences were not identical to any of the reference isolates. However, they could all be grouped into the worldwide clade. Genetic variation in the sequenced regions was observed in the form of single nucleotide polymorphisms (SNPs). Using MLST to characteriseE. ruminantiumfield isolates allowed the South African genotypes to be clearly distinguished from the distinct West African isolates.Contribution:Characterisation ofE. ruminantiumfield isolates is important for the control of heartwater and contributes to preliminary knowledge required for the development of a more practical vaccine against heartwater.