Human T-lymphotropic virus proteins and post-translational modification pathways

Cell life from the cell cycle to the signaling transduction and response to stimuli is finely tuned by protein post-translational modifications(PTMs).PTMs alter the conformation,the stability,the localization,and hence the pattern of interactions of the targeted protein.Cell pathways involve the activation of enzymes,like kinases,ligases and transferases,that,once activated,act on many proteins simultaneously,altering the state of the cell and triggering the processes they are involved in.Viruses enter a balanced system and hijack the cell,exploiting the potential of PTMs either to activate viral encoded proteins or to alter cellular pathways,with the ultimate consequence to perpetuate through their replication.Human T-lymphotropic virus type 1(HTLV-1)is known to be highly oncogenic and associates with adult T-cell leukemia/lymphoma,HTLV-1-associated myelopathy/tropical spastic paraparesis and other inflammatory pathological conditions.HTLV-1 protein activity is controlled by PTMs and,in turn,viral activity is associated with the modulation of cellular pathways based on PTMs.More knowledge is acquired about the PTMs involved in the activation of its proteins,like Tax,Rex,p12,p13,p30,HTLV-I basic leucine zipper factorand Gag.However,more has to be understood at the biochemical level in order to counteract the associated fatal outcomes.This review will focus on known PTMs that directly modify HTLV-1 components and on enzymes whose activity is modulated by viral proteins.

An Optimized Protocol for Myxosporidia (Cnidaria: Myxosporea) DNA Extraction for Molecular Studies

Myxosporidia constitute a major group of fish parasites which have a significant negative impact on wild and cultured fish. The used of DNA in Myxosporidia studies has progressed rapidly over the last twenty years, especially in their identification and characterization as well as determination of species diversity and investigation of their evolutionary relationships. Extraction and isolation of pure and high quality DNA are essential for any molecular study, but constitute a challenge for many laboratories especially in low and middle income countries. Myxosporidia plasmodia filled with mature myxospores were isolated from different tissues of Labeo batesii Boulenger, 1911. DNA from myxosporidia myxospores were extracted using a Livak optimized DNAs extraction protocol. Four particular phases of the original protocol were optimized. Yield and absorbance ratios of extracted DNA were determined using spectrophotometer. DNA samples were used as template for the amplification of the 18S rDNA region and amplicons resolved on 1.5% agarose gel for determination of fragment sizes and purity evaluation. The concentration of extracted DNA from all Myxosporidia species ranged from 4.6 to 26 ng/μl with purity indices ranging from 1.88 to 2.12. We successfully amplified the 1050 bp DNA fragment as targeted. The intensity, thickness and clarity of the bands were evidences of non-degradation of DNA. The optimized Livak protocol is simple, low-cost and manageable. Regarding the quantity, purity and quality of extracted DNA, the optimized Livak protocol is highly recommended for Myxosporidia studies.

Efficacy of the Microbial Larvicide VectoMax^®G against <i>Anopheles gambiae</i>s.l. and <i>Culex</i>spp. Larvae under Laboratory and Open Field Trial Experiments in the City of Yaoundé, Cameroon

Background: With the rapid expansion of insecticide resistance limiting the effectiveness of insecticide-based vector control interventions, integrated control strategies associating larviciding could be appropriate to improve current control efforts. The present experimental study assesses laboratory and field efficacy of the larvicide VectoMax®G on Anopheline and Culicine larval stages in Yaoundé. Methods: The effect of the larvicide VectoMax®G, a combination of Bacillus thuringiensis var. israelensis (Bti) and Bacillus sphaericus (Bs), on larval development was assessed during both laboratory and open field trial experiments. Laboratory experiments permitted the evaluation of five different concentrations with four replicates/experiments. Laboratory experiments were conducted with Anopheles coluzzii “Ngousso” and Culex quinquefasciatus laboratory strains. Open field trials were conducted using sixteen plastic containers with a diameter of 0.31 m buried in an array of four rows with 4 containers each. Distance between rows and between containers in a row was 1 meter. This experiment permitted to test the effect of the microbial larvicide VectoMax®G under operational application conditions on field mosquito populations. Results: The time to induce 100% mortality after exposure to serial concentrations of the larvicide varied according to the dose from 4 - 12 hours for An. coluzzii and 6 - 9 hours for Cx. quinquefasciatus in laboratory experiments. Measurements of the residual activity indicated that all VectoMax®G concentrations were still active after 35 days and killed 86% - 100% of larvae. Lethal dose of VectoMax®G killing 50% of larvae was estimated at 5.24 × 10-8 mg/m2 for An. coluzzii and 1.25 × 10-8 mg/m2 for Cx. quinquefasciatus. The lethal concentration inducing 95% mortality was estimated at 3.13 × 10-7 mg/m2 for An. coluzzii and 2.5 × 10-8 mg/m2 for Cx. quinquefasciatus. Open field trials tests indicated that sub-lethal concentrations of VectoMax®G successfully killed 100% An. gambiae s.l. larvae within 24 hours, while with Culex spp. larvae, 100% mortality was recorded after 48 hours post-treatment. Natural recolonization of water containers by larvae was recorded between 3 and 6 days respectively after the treatment with sublethal doses. Late instar larvae were recorded 5 and 6 days after treatment. When the jars were treated with reference dosage or supra doses of VectoMax®G, recolonization of water containers was observed six days after treatments. No pupae of both species were found 6 and 7 days post-treatment. Conclusions: The study indicated high efficacy of the microbial larvicide VectoMax®G against Anopheline and Culex larvae. Microbial larvicides such as VectoMax®G could be appropriate for controlling mosquito population particularly in areas experiencing high insecticide resistance or outdoor biting mosquitoes.

<i>Anopheles leesoni</i>Evans 1931, a Member of the <i>Anopheles funestus</i>Group, Is a Potential Malaria Vector in Cameroon

Background: Understanding the biology of Anopheles malaria vector species is essential to planning effective and sustainable malaria control strategies in endemic countries. This study reported the implication of Anopheles leesoni in malaria transmission in Cameroon, Central Africa. Methods: Mosquitoes were collected in three localities from May 2015 to March 2018 using electric aspirators and Centers for Disease Control light traps (CDC-LT). Anopheles funestus sensu lato (s.l.) mosquitoes were identified as species using polymerase chain reaction assay (PCR). Furthermore, Plasmodium falciparum infection status was determined using the enzyme-linked immunosorbent assay (ELISA) method. Results: A total of 12,744 Anopheles mosquitoes were collected by electric aspirator (N = 4844) and CDC-LT (N = 7900). Anopheles funestus s.l. (86.95%) was the major species and the main malaria vector in rural savannah and rural forest sites followed by A. gambiae s.l. (13.05%) whereas in urban areas, A. gambiae s.l. was by far the most abundant representing 91.45% of Anopheles mosquitoes collected. Two members of the A. funestus group were identified among 1389 analysed by PCR: 1307 A. funestus sensu stricto (s.s.) (94.10%) and 82 A. leesoni (5.9%). Plasmodium falciparum infection rate was 21.04% in A. funestus s.s. For the first time, A. leesoni was found positive for P. falciparum (infection rate: 10.98%) in Cameroon. Conclusion: A very high P. falciparum infection rate was observed in this study in A. funestus s.s., highlighting its high implication in malaria transmission in Cameroon. Furthermore, the detection of P. falciparum infection in A. leesoni calls for more attention towards this neglected vector species.

High efficacy of chlorfenapyr-based net Interceptor■G2 against pyrethroid-resistant malaria vectors from Cameroon

Background The increasing reports of resistance to pyrethroid insecticides associated with reduced efficacy of pyrethroid-only interventions highlight the urgency of introducing new non-pyrethroid-only control tools.Here,we investigated the performance of piperonyl-butoxide(PBO)-pyrethroid[Permanet 3.0(P3.0)]and dual active ingredients(AI)nets[Interceptor G2(IG2):containing pyrethroids and chlorfenapyr and Royal Guard(RG):containing pyrethroids and pyriproxyfen]compared to pyrethroid-only net Royal Sentry(RS)against pyrethroid-resistant malaria vectors in Cameroon.Methods The efficacy of these tools was firstly evaluated onAnopheles gambiae s.l.andAnopheles funestus s.l.from Gounougou,Mibellon,Mangoum,Nkolondom,and Elende using cone/tunnel assays.In addition,experimental hut trials(EHT)were performed to evaluate the performance of unwashed and 20 times washed nets in semi-field conditions.Furthermore,pyrethroid-resistant markers were genotyped in dead vs alive,blood-fed vs unfed mosquitoes after exposure to the nets to evaluate the impact of these markers on net performance.The XLSTAT software was used to calculate the various entomological outcomes and the Chi-square test was used to compare the efficacy of various nets.The odds ratio and Fisher exact test were then used to establish the statistical significance of any association between insecticide resistance markers and bed net efficacy.Results Interceptor G2 was the most effective net against wild pyrethroid-resistantAn.funestus followed by Permanet 3.0.In EHT,this net induced up to 87.8%mortality[95%confidence interval(CI):83.5-92.1%)and 55.6%(95%CI:48.5-62.7%)after 20 washes whilst unwashed pyrethroid-only net(Royal Sentry)killed just 18.2%(95%CI:13.4-22.9%)of host-seekingAn.funestus.The unwashed Permanet 3.0 killed up to 53.8%(95%CI:44.3-63.4%)of field-resistant mosquitoes and 47.2%(95%CI:37.7-56.7%)when washed 20 times,and the Royal Guard 13.2%(95%CI:9.0-17.3%)for unwashed net and 8.5%(95%CI:5.7-11.4%)for the 20 washed net.Interceptor G2,Permanet 3.0,and Royal Guard provided better personal protection(blood-feeding inhibition 66.2%,77.8%,and 92.8%,respectively)compared to pyrethroid-only net Royal Sentry(8.4%).Interestingly,a negative association was found betweenkdrw and the chlorfenapyr-based net Interceptor G2(χ^(2)=138;P<0.0001)with homozygote-resistant mosquitoes predominantly found in the dead ones.Conclusions The high mortality recorded with Interceptor G2 against pyrethroid-resistant malaria vectors in this study provides first semi-field evidence of high efficacy against these major malaria vectors in Cameroon encouraging the implementation of this novel net for malaria control in the country.However,the performance of this net should be established in other locations and on other major malaria vectors before implementation at a large scale.

Spatial distribution of insecticide resistant Check update populations of Aedes aegypti and Ae. albopictus and first detection of V410L mutation in Ae. aegypti from Cameroon

Background:Dengue(DENV),chikungunya(CHIKV)and Zika virus(ZIKV),are mosquito-borne viruses of medical importance in most tropical and subtropical regions.Vector control,primarily through insecticides,remains the primary method to prevent their transmission.Here,we evaluated insecticide resistance profles and identifed important underlying resistance mechanisms in populations of Aedes aegypti and Ae.albopictus from six different regions in Cameroon to pesticides commonly used during military and civilian public health vector control operations.Methods:Aedes mosquitoes were sampled as larvae or pupae between August 2020 and July 2021 in six locations across Cameroon and reared until the next generation,G1.Ae.aegypti and Ae.albopictus adults from G1 were tested following World Health Organization(WHO)recommendations and Ae.aegypti GO adults screened with real time melting curve qPCR analyses to genotype the F1534C,V1016l and V410L Aedes kdr mutations.Piperonyl butoxide(PBO)assays and real time qPCR were carried out from some cytochrome p450 genes known to be involved in metabolic resistance.Statistical analyses were performed using Chi-square test and generalized linear models.Results:Loss of susceptibility was observed to all insecticides tested.Mortality rates from tests with 0.25%permethrin varied from 24.27%to 85.89%in Ae.aegypti and from 17.35%to 68.08%in Ae.albopictus.Mortality rates for 0.03%deltamethrin were between 23.30%and 88.20%in Ae.aegypti and between 69.47%and 84.11%in Ae.albopictus.We found a moderate level of resistance against bendiocarb,with mortality rates ranging from 69.31%to 90.26%in Ae.aegypti and from 86.75%to 98.95%in Ae.albopictus.With PBO pre-exposure,we found partial or fully restored suscepti bility to pyrethroids and bendiocarb.The genes Cyp9M6F88/87 and Cyp9J10 were overexpressed in Ae.aegypti populations from Douala sites resistant to permethrin and deltamethrin.Cyp6P12 was highly expressed in alphacypermethrin and permethrin resistant Ae.albopictus samples.F1534C and V1016l mutations were detected in A.aegypti mosquitoes and for the first time V410L was reported in Cameroon.Conclusions:This study revealed that Ae.aegypti and Ae albopictus are resistant to multiple insecticide classes with multiple resistance mechanisms implicated.These findings could guide insecticide use to control arbovirus vectors in Cameroon.

High insecticide resistance in the major malaria vector Anopheles coluzzii in Chad Republic

Background:The Sahel region of Chad Republic is a prime candidate for malaria pre-elimination.To facilitate preelimination efforts in this region,two populations o f A nopheles coluzzii from Central Chad Republic were characterized,their insecticide resistance profile and the possible molecular mechanisms driving the resistance in the field investigated.Methods:Bloodfed female Anopheles gom biae s.l.resting indoor,were collected at N'djamena and Massakory,Chad in 2018 and characterized for species composition,and infection rate was determined using the TaqMan assay.Susceptibility to various insecticides was assessed using WHO tube bioassays.Cone bioassays were conducted using various long-lasting insecticidal nets(LLINs).Results were analysed using Chi Square test.Knockdown resistance(kdr)and ace-1 markers were investigated by TaqMan genotyping.Results:Anopheles coluzzi was the major vector found in N’djamena(100%)and Massakory(〜94%).N〇Plasm odium was found in 147 bloodfed F0 An.coluzzii(82 from N'djamena and 65 from Massakory).High intensity pyrethroid resistance was observed with mortalities of<2%for permethrin,deltamethrin and etofenprox,and with<50%and<60%dead following exposure to 10x diagnostic doses of deltamethrin and permethrin,respectively.For both sites,<10%mortalities were observed with DDT.Synergist bioassays with piperonylbutoxide significantly recovered pyrethroid susceptibility in Massakory populations,implicating CYP450s(mortality=13.6%for permethrin,X^2=22.8,df=1,P=0.0006;mortality=13.0%for deltamethrin,x2:8.8,df=1,P<0.00031).Cone-bioassays established complete loss of efficacy of the pyrethroid-based LLINs;and a 100%recovery of susceptibility following exposure to the roof of PermaNet®3.0/containing piperonylbutoxide.Both populations were susceptible to malathion,but high bendiocarb resistance was observed in Massakory population.The absence o f a ce-1 mutation points to the role of metabolic resistance in the bendiocarb resistance.Both 1014F and 1014S mutations were found in both populations at around 60%and<20%respectively.Sequencing of intron-1 of the voltage-gated sodium channel revealed a low genetic diversity suggesting reduced polymorphism.Conclusions:Multiple resistance in An.coluzzii populations from Chad highlight challenges associated with deployment of LLINs and indoor residual spraying(IRS)in the Sahel of this country.The pyrethroid-synergists LLINs(e.g.PermaNet®3.0)and organophosphate-based IRS maybe the alternatives for malaria control in this region.

Comparative study of the effect of solvents on the efficacy of neonicotinoid insecticides against malaria vector populations across Africa

Background:New insecticides with a novel mode of action such as neonicotinoids have recently been recommended for public health by WHO.Resistance monitoring of such novel insecticides requires a robust protocol to monitor the development of resistance in natural populations.In this study,we comparatively used three different solvents to assess the susceptibility of malaria vectors to neonicotinoids across Africa.Methods:Mosquitoes were collected from May to July 2021 from three agricultural settings in Cameroon(Njombe-Penja,Nkolondom,and Mangoum),the Democratic Republic of Congo(Ndjili-Brasserie),Ghana(Obuasi),and Uganda(Mayuge).Using the CDC bottle test,we compared the effect of three different solvents(ethanol,acetone,MERO)on the efficacy of neonicotinoids againstAnopheles gambiae s.l.In addition,TaqMan assays were used to genotype key pyrethroid-resistant markers inAn.gambiae and odds ratio based on Fisher exact test were used to evaluate potential cross-resistance between pyrethroids and clothianidin.Results:Lower mortality was observed when using absolute ethanol or acetone alone as solvent for clothianidin(11.4-51.9% mortality in Nkolondom,31.7-48.2% in Mangoum,34.6-56.1% in Mayuge,39.4-45.6% in Obuasi,83.7-89.3% in Congo and 71.1-95.9% in Njombe pendja)compared to acetone+MERO for which 100% mortality were observed for all the populations.Similar observations were done for imidacloprid and acetamiprid.Synergist assays(PBO,DEM and DEF)with clothianidin revealed a significant increase of mortality suggesting that metabolic resistance mechanisms are contributing to the reduced susceptibility.A negative association was observed between the L1014F-kdr mutation and clothianidin resistance with a greater frequency of homozygote resistant mosquitoes among the dead than among survivors(OR=0.5;P=0.02).However,the I114T-GSTe2 was in contrast significantly associated with a greater ability to survive clothianidin with a higher frequency of homozygote resistant among survivors than other genotypes(OR=2.10;P=0.013).Conclusions:This study revealed a contrasted susceptibility pattern depending on the solvents with ethanol/acetone resulting to lower mortality,thus possibly overestimating resistance,whereas the MERO consistently showed a greater efficacy of neonicotinoids but it could prevent to detect early resistance development.Therefore,we recommend monitoring the susceptibility using both acetone alone and acetone+MERO(4μg/ml for clothianidin)to capture the accurate resistance profile of the mosquito populations.

First detection of F1534C knockdown resistance mutation in Aedes aegypti(Diptera:Culicidae)from Cameroon

Background: Aedes borne viral diseases,notably dengue,are increasingly reported in Cameroon with Aedes aegypti being a major vector.Data on insecticide resistance of this vector and underlying mechanisms needed for outbreak preparedness remain scarce in Cameroon.Here,we present the nationwide distribution of insecticide resistance in Ae.aegypti and investigate the potential resistance mechanisms involved.Methods:: Immature stages of Ae.aegypti were collected between March and July 2017 in 13 locations across Cameroon and reared until G1/G2/G3 generation.Larval,adult bioassays,and piperonyl butoxide(PBO)synergist assays were carried out according to World Health Organization guidelines.F1534C mutation was genotyped using allele specific polymerase chain reaction in field collected adults(Go)and the polymorphism of the sodium channel gene was assessed.Theχ2 test was used to compare the mortality rate between bioassays with insecticides only and bioassays after preexposure to PBO synergist.Results: Larval bioassay revealed that all the three populations tested with temephos were susceptible.Adult bioassays showed a good level of susceptibility toward both pyrethroids tested,0.25%permethrin and 0.05%deltamethrin,with six out of 10 populations susceptible.However,two populations(Douala and Edéa)were resistant(deltamethrin[73.2–92.5%mortality],permethrin[2.6–76.3%mortality]).The resistance to 4%dichlorodiphenyltrichloroethane was observed in four out of 10 populations tested(16.8–87.1%mortality).Resistance was also reported to carbamates including 0.1%propoxur(60.8–87.1%mortality)and to 0.1%bendiocarb(82.9%mortality).All populations tested were fully susceptible to 1%fenitrothion.A partial recovery of susceptibility was observed in the pyrethroid resistant population of Douala after pre-exposed to PBO suggesting the implication of cytochrome P450 monoxygenases permethrin resistance.Genotyping and sequencing detected the F1534C kdr mutation in the two pyrethroid resistant locations of Edéa and Douala,with allelic frequency of 3.3%and 33.3%respectively.However,the high genetic diversity of the sodium channel gene supports the recent introduction of this mutation in Cameroon.Conclusions: This study revealed the contrasting resistance profiles to insecticides of Ae.aegypti populations in Cameroon suggesting that,instead of a unique nationwide control approach,a regionally adapted strategy will be needed to control this vector.The localised distribution of the F1534C kdr mutation supports this region-specific control strategy.