African Isolates Research Articles

Structural effects of HIV-1 subtype C integrase mutations on the activity of integrase strand transfer inhibitors in South African patients

HIV-1 integrase enzyme is responsible for the integration of viral DNA into the host genomic DNA. Integrase strand transfer inhibitors (INSTIs) are highly potent antiretroviral agents that inhibit this process, and are internationally approved for the treatment of both naïve and treated HIV-1 patients. However, their long-term efficacy is threatened by development of drug resistance strains resulting in resistance mutations. This work aimed to examine the effect of INSTI resistance-associated mutations (RAMs) and polymorphisms on the structure of HIV-1 subtype C (HIV-1C) integrase. Genetic analysis was performed on seven HIV-1C infected individuals with virologic failure after at least 6 months of INSTI-based antiretroviral therapy, presenting at the King Edward VIII hospital in Durban, South Africa. These were compared with sequences from 41 INSTI-naïve isolates. Integrase structures of selected isolates were modeled on the SWISS model online server. Molecular docking and dynamics simulations were also conducted using AutoDock-Vina and AMBER 18 force fields, respectively. Only one INSTI-treated isolate (14.28%) harboured major mutations (G140A + Q148R) as well as the E157Q minor mutation. Interestingly, S119T and V151I were only found in patients failing raltegravir (an INSTI drug). Molecular modeling and docking showed that RAMs and polymorphisms associated with INSTI-based therapy affect protein stability and this is supported by their weakened hydrogen-bond interactions compared to the wild-type. To the best of our knowledge, this is the first study to identify a double mutant in the 140’s loop region from South African HIV-1C isolates and study its effects on Raltegravir, Elvitegravir, and Dolutegravir binding. Communicated by Ramaswamy H. Sarma

Genetic response to nitrogen starvation in the aggressive Eucalyptus foliar pathogen Teratosphaeria destructans.

Teratosphaeria destructans is one of the most aggressive foliar pathogens of Eucalyptus. The biological factors underpinning T. destructans infections, which include shoot and leaf blight on young trees, have never been interrogated. Thus, the means by which the pathogen modifies its host environment to overcome host defences remain unknown. By applying transcriptome sequencing, the aim of this study was to compare gene expression in a South African isolate of T. destructans grown on nitrogen-deficient and complete media. This made it possible to identify upregulated genes in a nitrogen-starved environment, often linked to the pathogenicity of the fungus. The results support the hypothesis that nitrogen starvation in T. destructans likely mirrors an in planta genetic response. This is because 45% of genes that were highly upregulated under nitrogen starvation have previously been reported to be associated with infection in other pathogen systems. These included several CAZymes, fungal effector proteins, peptidases, kinases, toxins, lipases and proteins associated with detoxification of toxic compounds. Twenty-five secondary metabolites were identified and expressed in both nitrogen-deficient and complete conditions. Additionally, the most highly expressed genes in both growth conditions had pathogenicity-related functions. This study highlights the large number of expressed genes associated with pathogenicity and overcoming plant defences. As such, the generated baseline knowledge regarding pathogenicity and aggressiveness in T. destructans is a valuable reference for future in planta work.

Application of quantitative immunofluorescence assays to analyze the expression of cell contact proteins during Zika virus infections

Zika Virus (ZIKV) is an RNA virus that belongs to the Flavivirus (FV) genus. In the last years, several unique characteristics of ZIKV among FV have been revealed, as the multiple routes of transmission and its ability to reach different human tissues, including the central nervous system. Thus, one of the most intriguing features of ZIKV biology is its ability to cross diverse complex biological barriers. The main aim of this study is to contribute to the understanding of the still unclear mechanisms behind this viral activity. We investigated an African strain and two South American ZIKV isolates belonging to the Asian lineage, in order to characterize possible differences regarding their ability to disturb intercellular junctions. The Asian isolates correspond to an imported (Venezuelan) and an autochthonous (Argentinian) ZIKV strain for which there is still no data available. We focused on occludin and DLG1 expression as markers of tight and adherent junctions, respectively. For this, we applied a quantitative immunofluorescence assay that can ascertain alterations in the cell junction proteins expression in the infected cells. Our findings indicated that the different ZIKV strains were able to reduce the levels of both polarity proteins without altering their overall cell distribution. Moreover, the grade of this effect was strain-dependent, being the DLG1 reduction higher for the African and Asian Venezuelan isolates and, on the contrary, occludin down-regulation was more noticeable for the Argentinian strain. Interestingly, among both junction proteins the viral infection caused a relative larger reduction in DLG1 expression for all viruses, suggesting DLG1 may be of particular relevance for ZIKV infections. Taken together, this study contributes to the knowledge of the biological mechanisms involved in ZIKV cytopathogenesis, with a special focus on regional isolates.

Genetic Diversity of the Fusarium oxysporum Complex Isolated from the Grassland Biome of South Africa.

The genetic diversity of pathogenic members of the Fusarium oxysporum species complex (FOSC) has been intensively studied worldwide, yet strains occurring in native soils with low anthropogenic disturbance remain poorly understood. This study focused on 355 F. oxysporum isolates from soils with low anthropogenic activity obtained from the grassland biome of South Africa. Analysis of the translation elongation factor 1-alpha (tef-1α) gene revealed high levels of sequence type diversity within the soil population in comparison with the global dataset. Phylogenetic relationships of the South African isolates revealed that four nested within FOSC clade 1. This is the first report of members of the basal clade recovered from ecosystems with low anthropogenic disturbance from Sub-Saharan Africa. The remaining strains nested within clades 2 to 5. This study contributes significantly to our understanding of the distribution of the FOSC in natural systems as we show that FOSC populations in the South African grassland biome are genetically diverse. This fills in our knowledge gap because previous studies reported only on the occurrence and diversity of the FOSC isolated from plant debris in South Africa. This is the first comprehensive survey of fusaria from grassland soils with low anthropogenic disturbance in South Africa.

Neuroinvasiveness of the MR766 strain of Zika virus in IFNAR-/-mice maps to prM residues conserved amongst African genotype viruses.

Zika virus (ZIKV) strains are classified into the African and Asian genotypes. The higher virulence of the African MR766 strain, which has been used extensively in ZIKV research, in adult IFNα/β receptor knockout (IFNAR-/-) mice is widely viewed as an artifact associated with mouse adaptation due to at least 146 passages in wild-type suckling mouse brains. To gain insights into the molecular determinants of MR766’s virulence, a series of genes from MR766 were swapped with those from the Asian genotype PRVABC59 isolate, which is less virulent in IFNAR-/- mice. MR766 causes 100% lethal infection in IFNAR-/- mice, but when the prM gene of MR766 was replaced with that of PRVABC59, the chimera MR/PR(prM) showed 0% lethal infection. The reduced virulence was associated with reduced neuroinvasiveness, with MR766 brain titers ≈3 logs higher than those of MR/PR(prM) after subcutaneous infection, but was not significantly different in brain titers of MR766 and MR/PR(prM) after intracranial inoculation. MR/PR(prM) also showed reduced transcytosis when compared with MR766 in vitro. The high neuroinvasiveness of MR766 in IFNAR-/- mice could be linked to the 10 amino acids that differ between the prM proteins of MR766 and PRVABC59, with 5 of these changes affecting positive charge and hydrophobicity on the exposed surface of the prM protein. These 10 amino acids are highly conserved amongst African ZIKV isolates, irrespective of suckling mouse passage, arguing that the high virulence of MR766 in adult IFNAR-/- mice is not the result of mouse adaptation.

Identification and phylogenetic analysis of a collection of Beauveria spp. Isolates from Central America and Puerto Rico

The genus Beauveria comprises economically important entomopathogenic fungi, widely used for biological control in agriculture. Interest in these organisms in Costa Rica prompted surveys and establishment of collections in the past two decades. However, there was neither a formal identification nor a characterization of the isolates. With that purpose, the morphology and genetic variation by microsatellites and partial sequencing of Bloc, TEF-1α and RPB2 regions were studied for 32 isolates of Beauveria, which included 26 from Costa Rica, five from Puerto Rico and one from Honduras. The isolates were identified as B. bassiana (29) and B. caledonica (3). Ninety-three percent of B. bassiana isolates belonged to a monophyletic group of African and Neotropical isolates. A total of 105 alleles were recorded with 11 SSR markers, and the results suggested high diversity within the collection. Mantel tests showed low association between geographic origin and the variation among isolates.

South African Buffalo-Derived Theileria parva Is Distinct From Other Buffalo and Cattle-Derived T. parva.

Theileria parva is a protozoan parasite transmitted by the brown-eared ticks, Rhipicephalus appendiculatus and Rhipicephalus zambeziensis. Buffaloes are the parasite’s ancestral host, with cattle being the most recent host. The parasite has two transmission modes namely, cattle–cattle and buffalo–cattle transmission. Cattle–cattle T. parva transmission causes East Coast fever (ECF) and January disease syndromes. Buffalo to cattle transmission causes Corridor disease. Knowledge on the genetic diversity of South African T. parva populations will assist in determining its origin, evolution and identify any cattle–cattle transmitted strains. To achieve this, genomic DNA of blood and in vitro culture material infected with South African isolates (8160, 8301, 8200, 9620, 9656, 9679, Johnston, KNP2, HL3, KNP102, 9574, and 9581) were extracted and paired-end whole genome sequencing using Illumina HiSeq 2500 was performed. East and southern African sample data (Chitongo Z2, Katete B2, Kiambu Z464/C12, Mandali Z22H10, Entebbe, Nyakizu, Katumba, Buffalo LAWR, and Buffalo Z5E5) was also added for comparative purposes. Data was analyzed using BWA and SAMtools variant calling with the T. parva Muguga genome sequence used as a reference. Buffalo-derived strains had higher genetic diversity, with twice the number of variants compared to cattle-derived strains, confirming that buffaloes are ancestral reservoir hosts of T. parva. Host specific SNPs, however, could not be identified among the selected 74 gene sequences. Phylogenetically, strains tended to cluster by host with South African buffalo-derived strains clustering with buffalo-derived strains. Among the buffalo-derived strains, South African strains were genetically divergent from other buffalo-derived strains indicating possible geographic sub-structuring. Geographic sub- structuring was also observed within South Africa strains. The knowledge generated from this study indicates that to date, ECF is not circulating in buffalo from South Africa. It also shows that T. parva has historically been present in buffalo from South Africa before the introduction of ECF and was not introduced into buffalo during the ECF epidemic.

Cassava mosaic virus in Africa: Functional analysis of virus coat proteins based on evolutionary processes and protein structure

African cassava mosaic virus (ACMV) has been identified as a typical example of a pathogenic microorganism that constitutes a global menace to cassava's profitable production, especially in Africa, where the highest economic yield loss of 34 million tons usually is recorded annually. Functional analysis of virus coat proteins based on evolutionary processes and structure of ACMV isolates from fifteen African countries was conducted with Indian cassava mosaic virus and East African cassava mosaic Malawi virus as checks. The study revealed that ACMV coat proteins (CPs) belong to the nuclear export factor BR1 family in the Gemini-coat protein superfamily. Moreover, the phylogenetic analysis performed shows a very strong affinity amongst the African isolates within their geographical regions without evidence of evolutionary divergence amongst all the isolates. Multiple sequence alignment of the genes revealed a highly conserved domain and three motifs. Thirteen of the isolates from Africa share similar physicochemical properties and possess DNA-binding property. A three-dimensional homology model structure was built for ACMV CPs with Ramachandran plot values between 99 and 100%. The gene annotation analysis predicted most of the coat proteins associated with the virion and virus capsid, undertaking mainly organic compound and nucleic acids binding molecular functions and shuttling as the dominant biological process. Therefore, this study established that ACMV isolates have common ancestral descent. These findings form a strong basis that could be harnessed by applying molecular techniques for effective detection and management of ACMV isolates, causing substantial cassava losses recorded annually across several continents of the globe.

Replication Variance of African and Asian Lineage Zika Virus Strains in Different Cell Lines, Mosquitoes and Mice.

Since the epidemic in 2007, studies on vector competence for Zika virus (ZIKV) have intensified, showing that the transmission efficiency varies depending on the vector population, ZIKV strain, and dose of the infectious blood meal. In this study, we aimed to investigate the replication of African and Asian ZIKV strains in vitro and in vivo in order to reveal their phenotypic differences. In addition, we investigated the vector competence of Cambodian Aedes aegypti (Ae. aegypti) mosquitoes (urban and rural) for these ZIKV strains. We observed a significantly higher pathogenicity of the African ZIKV strain in vitro (in mosquito and mammalian cells), and in vivo in both Ae. aegypti and mice. Both mosquito populations were competent to transmit ZIKV as early as 7 days p.i., depending on the population and the ZIKV strain. Ae. aegypti from rural habitats showed significant higher transmission and survival rates than those from urban. We observed the highest transmission efficiency for the African ZIKV isolate (93.3% 14 days p.i.) and for the Cambodian ZIKV isolate (80% 14 days p.i.). Overall, our results highlight the phenotypic differences of the ZIKV lineages and the potential risk of ZIKV transmission by Ae. aegypti mosquitoes. Further investigations of Cambodian mosquito species and ZIKV specific surveillance in humans is necessary in order to improve the local risk assessment.

Middle East respiratory syndrome coronavirus Spike protein variants exhibit geographic differences in virulence

Human Middle East respiratory syndrome (MERS) cases were detected primarily in the Middle East before a major outbreak occurred in South Korea in 2015. The Korean outbreak was initiated by a single infected individual, allowing studies of virus evolution in the absence of further MERS-CoV introduction into human populations. In contrast, MERS is primarily a camel disease on the Arabian Peninsula and in Africa, with clinical disease in humans only in the former location. Previous work identified two mutations in the South Korean MERS-CoV, D510G and I529T on the Spike (S) protein, that led to impaired binding to the receptor. However, whether these mutations affected virulence is unknown. To address this question, we constructed isogenic viruses expressing mutations found in the S protein from Korean isolates and showed that isogenic viruses carrying the Korean MERS-CoV mutations, D510G or I529T, were attenuated in mice, resulting in greater survival, less induction of inflammatory cytokines, and less severe lung injury. In contrast, isogenic viruses expressing S proteins from African isolates were nearly fully virulent; other studies showed that West African camel isolates carry mutations in MERS-CoV accessory proteins, which may limit human transmission. These data indicate that following a single-point introduction of the virus, MERS-CoV S protein evolved rapidly in South Korea to adapt to human populations, with consequences on virulence. In contrast, the mutations in S proteins of African isolates did not change virulence, indicating that S protein variation likely does not play a major role in the lack of camel-to-human transmission in Africa.

Optimization of cereal Blend (wheat and yellow maize flours (Zea maize)) enriched with African walnut (Tetracarpidium conophorum) protein isolate for cookie making

This study examined the effect of African walnut protein isolate, wheat and yellow maize flours on the general acceptability of cookie production. In this study, blends of African walnut protein isolate, wheat and yellow maize flours were studied in a completely randomized design assessment of the proximate composition. The variables were wheat flour (30-50%), yellow maize flour (20-30%) and African walnut protein isolate (10-20%) to generate 20 composite blends. The proximate composition and functional properties of blends and flours were determined using standard methods. Three blends of samples 50.00 g wheat flour, 30.00 g yellow maize flour, 20.00 g African walnut protein isolate; 45.24 g wheat flour, 28.27 g yellow maize flour, 26.48 g African walnut protein isolate and 45.24 g wheat flour, 37.79 g yellow maize flour and 16.97 g African walnut protein isolate as assessed by the highest protein and fiber and low fat contents, were selected from optimization results of proximate composition for production and sensory evaluation of cookies. The proximate composition of flours revealed that African walnut protein isolate was significantly higher in protein than other flours. Fat in African walnut protein isolate was the least, while crude fiber and carbohydrate contents were not detected in African walnut protein isolate. Functional analysis revealed that yellow maize flour had the highest value in water absorption capacity; yellow maize flour and African walnut protein isolate had the highest oil absorption capacity, while African walnut protein isolate scored the least in bulk density. The sample 45.24 g wheat flour, 28.27 g yellow maize flour and 26.48 g African walnut protein isolate had the highest protein and ash, with the lowest fat content among the blends. However, cookie prepared from sample 45.24 g wheat flour, 37.79 g yellow maize flour and 16.97 g African walnut protein isolate had the best overall acceptability. However, the study recommends the flour proportion of sample 45.24 g wheat flour, 37.79 g yellow maize flour and 16.97 g African walnut protein isolate in cookie production by establishing the inclusion in Nigerian food industry and as a way of reducing or alleviate malnutrition.

Molecular Analysis of East African Lumpy Skin Disease Viruses Reveals a Mixed Isolate with Features of Both Vaccine and Field Isolates.

Lumpy skin disease (LSD), an economically significant disease in cattle caused by lumpy skin disease virus (LSDV), is endemic to nearly all of Africa. Since 2012, LSDV has emerged as a significant epizootic pathogen given its rapid spread into new geographical locations outside Africa, including the Middle East, Eastern Europe, and Asia. To assess the genetic diversity of LSDVs in East Africa, we sequenced and analyzed the RPO30 and GPCR genes of LSDV in twenty-two archive samples collected in Ethiopia, Kenya, and Sudan before the appearance of LSD in the Middle East and its incursion into Europe. We compared them to publicly available sequences of LSDVs from the same region and those collected elsewhere. The results showed that the East African field isolates in this study were remarkably similar to each other and to previously sequenced field isolates of LSDV for the RPO30 and GPCR genes. The only exception was LSDV Embu/B338/2011, a field virus collected in Kenya, which displayed mixed features between the LSDV Neethling vaccine and field isolates. LSDV Embu/B338/2011 had the same 12-nucleotide insertion found in LSDV Neethling and KS-1 vaccines. Further analysis of the partial EEV glycoprotein, B22R, RNA helicase, virion core protein, NTPase, and N1R/p28-like protein genes showed that LSDV Embu/B338/2011 differs from previously described LSDV variants carrying the 12-nucleotide insertion in the GPCR gene. These findings highlight the importance of the constant monitoring of genetic variation among LSDV isolates.

Radical genome remodelling accompanied the emergence of a novel host-restricted bacterial pathogen.

The emergence of new pathogens is a major threat to public and veterinary health. Changes in bacterial habitat such as a switch in host or disease tropism are typically accompanied by genetic diversification. Staphylococcus aureus is a multi-host bacterial species associated with human and livestock infections. A microaerophilic subspecies, Staphylococcus aureus subsp. anaerobius, is responsible for Morel’s disease, a lymphadenitis restricted to sheep and goats. However, the evolutionary history of S. aureus subsp. anaerobius and its relatedness to S. aureus are unknown. Population genomic analyses of clinical S. aureus subsp. anaerobius isolates revealed a highly conserved clone that descended from a S. aureus progenitor about 1000 years ago before differentiating into distinct lineages that contain African and European isolates. S. aureus subsp. anaerobius has undergone limited clonal expansion, with a restricted population size, and an evolutionary rate 10-fold slower than S. aureus. The transition to its current restricted ecological niche involved acquisition of a pathogenicity island encoding a ruminant host-specific effector of abscess formation, large chromosomal re-arrangements, and the accumulation of at least 205 pseudogenes, resulting in a highly fastidious metabolism. Importantly, expansion of ~87 insertion sequences (IS) located largely in intergenic regions provided distinct mechanisms for the control of expression of flanking genes, including a novel mechanism associated with IS-mediated anti-anti-sense decoupling of ancestral gene repression. Our findings reveal the remarkable evolutionary trajectory of a host-restricted bacterial pathogen that resulted from extensive remodelling of the S. aureus genome through an array of diverse mechanisms in parallel.

Early Embryonic Loss Following Intravaginal Zika Virus Challenge in Rhesus Macaques.

Zika virus (ZIKV) is an arthropod-borne virus (arbovirus) and is primarily transmitted by Aedes species mosquitoes; however, ZIKV can also be sexually transmitted. During the initial epidemic and in places where ZIKV is now considered endemic, it is difficult to disentangle the risks and contributions of sexual versus vector-borne transmission to adverse pregnancy outcomes. To examine the potential impact of sexual transmission of ZIKV on pregnancy outcome, we challenged three rhesus macaques (Macaca mulatta) three times intravaginally with 1 x 107 PFU of a low passage, African lineage ZIKV isolate (ZIKV-DAK) in the first trimester (~30 days gestational age). Samples were collected from all animals initially on days 3 through 10 post challenge, followed by twice, and then once weekly sample collection; ultrasound examinations were performed every 3-4 days then weekly as pregnancies progressed. All three dams had ZIKV RNA detectable in plasma on day 3 post-ZIKV challenge. At approximately 45 days gestation (17-18 days post-challenge), two of the three dams were found with nonviable embryos by ultrasound. Viral RNA was detected in recovered tissues and at the maternal-fetal interface (MFI) in both cases. The remaining viable pregnancy proceeded to near term (~155 days gestational age) and ZIKV RNA was detected at the MFI but not in fetal tissues. These results suggest that sexual transmission of ZIKV may represent an underappreciated risk of pregnancy loss during early gestation.

Nutritional, antioxidant, angiotensin-converting-enzyme and carbohydrate-hydrolyzing-enzyme inhibitory activities of underutilized leafy vegetable: African wild lettuce (Lactuca taraxacifolia Willd)

BackgroundAfrican wild lettuce (Lactuca taraxacifolia Willd.) is an underutilised indigenous leafy vegetable containing essential nutrients and medicinal properties. Hence, this study aimed to determine the chemical composition, antioxidant activities, α-amylase, α-glucosidase and angiotensin converting enzyme inhibitory potentials of wild lettuce leaves powder samples.MethodsFreshly harvested Wild Lettuce leaves were processed into whole leafy powder (WLF), extracted powder (WLE), residue (WLR) and leaf protein isolate (WPI). Chemical composition, antioxidant activities, α-amylase, α-glucosidase and angiotensin-converting enzyme inhibitory potentials of the powder samples were determined.ResultsCrude protein of Wild Lettuce leaves ranged from 23.27 to 46.57 and crude fiber from 4.17–37.37 g/100 g. Phosphorous was the most abundant element, while zinc had the lowest concentration. The samples essential amino acids, protein efficiency ratio, essential amino acid index and biological values were 39.83–50.65 mg/100 g protein. 2.79–3.51, 77.03–92.36% and 72.26–88.97%, respectively. Saponin, tannin, oxalate, phytate, terpennoids, flavonoid and phenol in the leafy vegetable samples were within tolerable levels. The African wild lettuce leaf protein isolate (WPI) had higher DPPH antioxidant activity (91.88%), percentage inhibitory properties on α-amylase (26.11%), α-glucosidase (64.24%) and angiotensin-1-converting enzyme (97.53%) than WLF (18.28, 25.44, 55.41 and 67.56), WLE (70.85, 24.97, 62.53 and 93.27) and WLR (53.07, 24.68, 50.03 and 85.28) respectively.ConclusionAfrican wild lettuce leaf samples, particularly protein isolate, contain essential nutrients, antioxidant activities and ability to inhibit angiotensin-1-converting, α-amylase and α-glucosidase enzymes Therefore, the leafy vegetable samples, particularly WPI, may be suitable as antioxidant, antidiabetic and antihypertensive agent.

English

The molecular diversity of Tomato spotted wilt virus (TSWV) isolates from Pakistan was investigated based on nucleocapsid protein (N) gene. It revealed that the Pakistani isolates shared 98.2–99.3% (nt) and 95.7–97.4% (aa) identities with each other and 97.2–99.3% (nt) and 92.7–97.8% (aa) with other TSWV isolates retrieved from Genbank. In a neighbour-joining phylogenetic reconstruction, four Pakistani isolates clustered with South Korean and Turkish isolates and one with Australian and South African isolates. All the Pakistani isolates shared maximum genetic diversity (0.024–0.039 and 0.026–0.029) with South African (AJ296600) and Australian (AY879109) isolates and the lowest values of genetic diversity (0.009–0.013) was recorded with Czech Republican isolate (AJ296599). The gene differentiation co-efficient value (Fst) was 0.33 less than standard value noticed as 0.05145, between globally reported and Pakistani TSWV isolates considering it a frequent gene flow. The significant values of statistical tests based on three genetic differentiation analysis; Ks⃰; 4.86231, Z; 34.27778 and Snn; 0.55556 were observed. The negative values were recorded according to statistical test as Fu, & Li’s D ⃰ (-2.55465), Fu, & Li’s F ⃰ (-2.78103) and one positive value from Tajima’s D (2.10697). These results indicate low polymorphism frequency in TSWV population of Pakistan. © 2021 Friends Science Publishers

Molecular surveillance of anti-malarial resistance pfcrt, pfmdr1, and pfk13 polymorphisms in African Plasmodium falciparum imported parasites to Wuhan, China

BackgroundImported malaria parasites with anti-malarial drug resistance (ADR) from Africa is a serious public health challenge in non-malarial regions, including Wuhan, China. It is crucial to assess the ADR status in African Plasmodium falciparum isolates from imported malaria cases, as this will provide valuable information for rational medication and malaria control.MethodsDuring 2017–2019, a cross-sectional study was carried out in Wuhan, China. Peripheral blood 3 ml of returned migrant workers from Africa was collected. The target fragments from pfcrt, pfmdr1, and k13 propeller (pfk13) genes were amplified, sequenced, and analysed.ResultsIn total, 106 samples were collected. Subsequently, 98.11% (104/106), 100% (106/106), and 86.79% (92/106) of these samples were successfully amplified and sequenced for the pfcrt (72–76), pfmdr1, and pfk13 genes, respectively. The prevalence of the pfcrt 76 T, pfmdr1 86Y, and pfmdr1 184F mutations was 9.62, 4.72, and 47.17%, respectively. At codons 72–76, the pfcrt locus displayed three haplotypes, CVMNK (wild-type), CVIET (mutation type), CV M/I N/E K/T (mixed type), with 87.50%, 9.62%, and 2.88% prevalence, respectively. For the pfmdr1 gene, NY (wild type), NF and YF (mutant type), N Y/F, Y Y/F, and N/Y Y/F (mixed type) accounted for 34.91, 43.40, 3.77, 15.09, 0.94, and 1.89% of the haplotypes, respectively. A total of 83 isolates with six unique haplotypes were found in pfcrt and pfmdr1 combined haplotypes, of which NY-CVMNK and NF-CVMNK accounted for 40.96% (34/83) and 43.37% (36/83), respectively. Furthermore, 90 cases were successfully sequenced (84.91%, 90/106) at loci 93, 97, 101, and 145, and 78 cases were successfully sequenced (73.58%, 78/106) at loci 343, 353, and 356 for pfcrt. However, the mutation was observed only in locus 356 with 6.41%. For pfk13, mutations reported in Southeast Asia (at loci 474, 476, 493, 508, 527, 533, 537, 539, 543, 553, 568, 574, 578, and 580) and Africa (at loci 550, 561, 575, 579, and 589) were not observed.ConclusionsThe present data from pfcrt and pfmdr1 demonstrate that anti-malarial drugs including chloroquine, amodiaquine, and mefloquine, remain effective against malaria treatment in Africa. The new mutations in pfcrt related to piperaquine resistance remain at relatively low levels. Another source of concern is the artemether-lumefantrine resistance-related profiles of N86 and 184F of pfmdr1. Although no mutation in pfk13 is detected, molecular surveillance must continue.

Genetic Diversity of Rice stripe necrosis virus and New Insights into Evolution of the Genus Benyvirus.

The rice stripe necrosis virus (RSNV) has been reported to infect rice in several countries in Africa and South America, but limited genomic data are currently publicly available. Here, eleven RSNV genomes were entirely sequenced, including the first corpus of RSNV genomes of African isolates. The genetic variability was differently distributed along the two genomic segments. The segment RNA1, within which clusters of polymorphisms were identified, showed a higher nucleotidic variability than did the beet necrotic yellow vein virus (BNYVV) RNA1 segment. The diversity patterns of both viruses were similar in the RNA2 segment, except for an in-frame insertion of 243 nucleotides located in the RSNV tgbp1 gene. Recombination events were detected into RNA1 and RNA2 segments, in particular in the two most divergent RSNV isolates from Colombia and Sierra Leone. In contrast to BNYVV, the RSNV molecular diversity had a geographical structure with two main RSNV lineages distributed in America and in Africa. Our data on the genetic diversity of RSNV revealed unexpected differences with BNYVV suggesting a complex evolutionary history of the genus Benyvirus.

First report of entomopathogenic nematode Heterorhabditis bacteriophora (Rhabditida: Heterorhabditidae) from South Africa

We present the first report of Heterorhabditis bacteriophora entomopathogenic nematodes isolated from sandy loam soil obtained from Walkerville, South of Johannesburg, Gauteng province, South Africa. A survey was conducted to collect soil samples and isolate entomopathogenic nematodes between 2012 and 2016. Entomopathogenic nematodes are insect-killing microscopic worms found in soil and have a symbiotic relationship with insect pathogenic bacteria. Molecular and morphological techniques including genomic DNA extractions, polymerase chain reaction and sequencing of the 18S rDNA, phylogenetic analysis, light microscopy and scanning electron microscopy were used to identify entomopathogenic nematodes from the collected soil samples. H. bacteriophora was recovered in one out of 80 soil samples from an uncultivated grassland and the frequency of its isolation from soil samples in our study was 2%. Phylogenetic tree analysis revealed that the South African isolate was grouped with the strain H. bacteriophora strain 56-C. H. bacteriophora. The body length, body width and tail of the isolate are slightly shorter than the previously described H. bacteriophora. This is the first H. bacteriophora species to be isolated and reported from South African soil and future studies will explore its potential as a biological control agent of problematic insect pests in agricultural industries.

New species and records of Trichoderma isolated as mycoparasites and endophytes from cultivated and wild coffee in Africa

A survey for species of the genus Trichoderma occurring as endophytes of Coffea, and as mycoparasites of coffee rusts (Hemileia), was undertaken in Africa; concentrating on Cameroon and Ethiopia. Ninety-four isolates of Trichoderma were obtained during this study: 76 as endophytes of healthy leaves, stems and berries and, 18 directly from colonized rust pustules. A phylogenetic analysis of all isolates used a combination of three genes: translation elongation factor-1α (tef1), rpb2 and cal for selected isolates. GCPSR criteria were used for the recognition of species; supported by morphological and cultural characters. The results reveal a previously unrecorded diversity of Trichoderma species endophytic in both wild and cultivated Coffea, and mycoparasitic on Hemileia rusts. Sixteen species were delimited, including four novel taxa which are described herein: T. botryosum, T. caeruloviride, T. lentissimum and T. pseudopyramidale. Two of these new species, T. botryosum and T. pseudopyramidale, constituted over 60% of the total isolations, predominantly from wild C. arabica in Ethiopian cloud forest. In sharp contrast, not a single isolate of Trichoderma was obtained using the same isolation protocol during a survey of coffee in four Brazilian states, suggesting the existence of a ‘Trichoderma void’ in the endophyte mycobiota of coffee outside of Africa. The potential use of these African Trichoderma isolates in classical biological control, either as endophytic bodyguards—to protect coffee plants from Hemileia vastatrix, the fungus causing coffee leaf rust (CLR)—or to reduce its impact through mycoparasitism, is discussed, with reference to the on-going CLR crisis in Central America.