Quantitative reverse transcription PCR assay to detect pyrethroid resistance in Culex mosquitoes

Pyrethroid insecticides are widely used to control mosquitoes that transmit diseases such as West Nile virus (WNV) to humans. A single nucleotide polymorphism (SNP) in the knockdown resistance locus (kdr) of the voltage gated sodium channel (Vgsc) gene of Culex mosquitoes confers knockdown resistance to pyrethroids. PCR-based assays that detect these SNPs in Culex species are currently available for Culex pipiens Linnaeus and Culex quinquefasciatus Say. RNAseq was employed to sequence the coding region of Vgsc for Culex tarsalis Coquillett and Culex erythrothorax Dyar, two WNV vectors. We utilized the cDNA sequence to develop a quantitative reverse transcriptase PCR assay that detects the L1014F mutation in the kdr of Vgsc. Because this locus is conserved, the assay successfully detected the SNPs in multiple Culex spp. vectors of WNV in the United States. The resulting Culex RTkdr assay was validated using quantitative PCR, CDC bottle bioassays, and sequencing of PCR products. Using sequencing, we determined the accuracy of the Culex RTkdr assay was 99%. Pyrethroid resistance was more common among Cx. pipiens than other Culex spp. and co-occured with agriculture. We anticipate that public health and vector control agencies may utilize the Culex RTkdr assay to map the distribution of pyrethroid resistance in Culex species to more efficiently control mosquitoes and the diseases they transmit.

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First Report of Peanut mottle virus in Forage Peanut (Arachis pintoi) in the United States

Plant Health Progress ◽

10.1094/php-12-17-0076-br ◽

2018 ◽

Vol 19 (1) ◽

pp. 13-14

Author(s):

K. K. Dey ◽

L. Hassell ◽

C. Li ◽

M. Elliott ◽

X. Sun

Keyword(s):

United States ◽

The United States ◽

Mottle Virus ◽

Arachis Glabrata ◽

First Report ◽

Arachis Pintoi ◽

Reverse Transcription Pcr ◽

The World ◽

Pcr Products ◽

The Many

Arachis pintoi is one of the many perennial peanuts grown in many tropical and subtropical countries around the world. Although Peanut mottle virus (PeMoV) was reported in Arachis glabrata from Georgia in 2007, there are no reports of PeMoV infecting A. pintoi in the United States. In June 2017, samples of A. pintoi that originated from Hardee County, FL, plants showed a variety of symptoms ranging from yellowing to dark islands, green vein banding, and mild mottling. They tested positive initially with broad-spectrum lateral flow antibody immunoassay and later were confirmed by sequencing the reverse-transcription PCR products. Detection of PeMoV in A. pintoi is significant because it is transmitted by aphids in a nonpersistent manner and is seed-borne in A. hypogea. It is not known if PeMoV is seed-borne in A. pintoi. However, A. pintoi is commonly vegetatively propagated using stolon cuttings. It is possible that PeMoV can spread to A. pintoi in Florida by all these means, making maintenance of virus-free propagation stock plants important. To our knowledge, this is the first report of PeMoV in A. pintoi the United States.

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Comparison of Automated Quantitative Reverse Transcription-PCR and Direct Fluorescent-Antibody Detection for Routine Rabies Diagnosis in the United States

Journal of Clinical Microbiology ◽

10.1128/jcm.01227-15 ◽

2015 ◽

Vol 53 (9) ◽

pp. 2983-2989 ◽

Cited By ~ 15

Author(s):

Michelle Dupuis ◽

Scott Brunt ◽

Kim Appler ◽

April Davis ◽

Robert Rudd

Keyword(s):

United States ◽

Reverse Transcription ◽

Gold Standard ◽

Fluorescent Antibody ◽

The United States ◽

Pcr Assay ◽

Qrt Pcr ◽

Reverse Transcription Pcr ◽

Direct Fluorescent Antibody ◽

Rabies Diagnosis

Rabies virus found worldwide and prevalent throughout the United States continues to be a public health concern. Direct-fluorescent antibody (DFA) detection remains the gold standard for rabies virus diagnostics. Assessing the utility of a high-throughput molecular platform such as the QIAsymphony SP/AS, in conjunction with quantitative reverse transcription-PCR (qRT-PCR), to augment or potentially replace the DFA test, was the focus of this project. Here we describe a triplex qRT-PCR assay, including assembly and evaluation for sensitivity, specificity, and ability to detect variants. Additionally, we compared the qRT-PCR assay to the gold standard direct fluorescent-antibody test. More than 1,000 specimens submitted for routine rabies diagnosis were tested to directly compare the two methods. All results were in agreement between the two methods, with one additional specimen detected by qRT-PCR below the limits of the DFA sensitivity. With the proper continued validation for variant detection, molecular methods have a place in routine rabies diagnostics within the United States.

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Management of Insecticide-Resistant Soybean Aphids in the Upper Midwest of the United States

Journal of Integrated Pest Management ◽

10.1093/jipm/pmy014 ◽

2018 ◽

Vol 9 (1) ◽

Cited By ~ 15

Author(s):

Robert L Koch ◽

Erin W Hodgson ◽

Janet J Knodel ◽

Adam J Varenhorst ◽

Bruce D Potter

Keyword(s):

United States ◽

Pyrethroid Resistance ◽

Management Strategies ◽

The United States ◽

Soybean Aphid ◽

Upper Midwest ◽

Pyrethroid Insecticides ◽

Soybean Aphids ◽

Development Of Resistance ◽

Laboratory Confirmation

Abstract Since the first observation of soybean aphid, Aphis glycines Matsumura (Hemiptera: Aphididae), in North America in 2000, it has become the most economically damaging insect of soybean in the Upper Midwest of the United States. For the last 17 yr, soybean aphid management has relied almost entirely on the use of foliar-applied broad-spectrum insecticides. However, in 2015 in Minnesota, failures of foliar-applied pyrethroid insecticides were reported and pyrethroid resistance was confirmed with laboratory bioassays using lambda-cyhalothrin and bifenthrin. In 2016 and 2017, further reports of failures of pyrethroid insecticides and/or laboratory confirmation of resistance occurred in Iowa, North Dakota, South Dakota, and Manitoba. In response to the challenge posed by insecticide-resistant soybean aphids, we recommend several management strategies for minimizing further development of resistance and subsequent pest-induced crop losses: 1) scout and use the economic threshold to determine when to apply insecticides, 2) apply the insecticides properly, 3) assess efficacy 3–5 d after application, and 4) alternate to a different insecticide group if another application is required. In the long term, soybean aphid management must move beyond insecticide-based management to true integrated pest management by incorporating multiple tactics.

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Comparison of the knockdown resistance locus (kdr) in Anopheles stephensi, An. arabiensis, and Culex pipiens s.l. suggests differing mechanisms of pyrethroid resistance in east Ethiopia

10.1101/2020.05.13.093898 ◽

2020 ◽

Author(s):

Tamar E. Carter ◽

Araya Gebresilassie ◽

Shantoy Hansel ◽

Lambodhar Damodaran ◽

Callum Montgomery ◽

...

Keyword(s):

Insecticide Resistance ◽

Culex Pipiens ◽

Anopheles Stephensi ◽

Pyrethroid Resistance ◽

Resistance Mechanism ◽

Target Site ◽

Resistance Locus ◽

Knockdown Resistance ◽

Resistance Mutations ◽

Vector Species

AbstractThe malaria vector, Anopheles stephensi, which is typically restricted to South Asia and the Middle East, was recently detected in the Horn of Africa. Controlling the spread of this vector could involve integrated vector control that considers the status of insecticide resistance of multiple vector species in the region. Previous reports indicate that the knockdown resistance mutations (kdr) in the voltage-gated sodium channel (vgsc) are absent in both pyrethroid resistant and sensitive variants of An. stephensi in east Ethiopia but similar information on other vector species in the same areas is limited. In this study, kdr and the neighboring intron was analyzed in An. stephensi, An. arabiensis, and Culex pipiens s. l. collected in east Ethiopia between 2016 and 2017. Sequence analysis revealed that all of Cx. pipiens s.l. (n = 42) and 71.6% of the An. arabiensis (n=67) carried kdr L1014F known to confer target-site pyrethroid resistance. Intronic variation was only observed in An. stephensi (segregating sites = 6, haplotypes = 3) previously shown to have no kdr mutations. In addition, no evidence of non-neutral evolutionary processes was detected at the An. stephensi kdr intron which further supports target-site mechanism not being a major resistance mechanism in this An. stephensi population. Overall, these results suggest differences in evolved mechanisms of pyrethroid/DDT resistance in populations of vector species from the same region. Variation in insecticide resistance mechanisms in East Ethiopian mosquito vectors highlight possible species or population specific biological factors and distinct environmental exposures that shape their evolution.

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First Report of Hosta virus X Infecting Hosta Plants in China

Plant Disease ◽

10.1094/pdis-09-12-0810-pdn ◽

2013 ◽

Vol 97 (3) ◽

pp. 429-429 ◽

Cited By ~ 4

Author(s):

M. S. Wei ◽

Y. J. Zhang ◽

G. F. Li ◽

J. Ma ◽

M. Li

Keyword(s):

Plant Protection ◽

The United States ◽

Coding Region ◽

Potential Threat ◽

Sequence Comparisons ◽

First Report ◽

Reverse Transcription Pcr ◽

Viral Particles ◽

Leaf Deformation ◽

Beijing China

Hosta (Hosta spp.) plants showing leaf deformation, puckering, and ink-bleed symptoms were collected in July 2012 from a park at Dongcheng district, Beijing, China. Three out of six samples tested positive for Hosta virus X (HVX) by immunostrip and double-antibody sandwich (DAS)-ELISA with HVX-specific serological reagents from Agdia Inc. (Elkhart, IN, USA). Filamentous viral particles were trapped and observed from the infected hosta leaf sap by immuno-serological electron microscopy (ISEM) (antibodies from Agdia). To confirm HVX infection, three ELISA-positive samples were analyzed by reverse transcription-PCR assay, using virus-specific primers HVXf (5′-ATCCGTTATCTACAGGGGACCAG-3′) and HVXr (5′-TAAGTTAGTGGAACGGTTAGCCCGAT-3′) that amplified a 1,067-bp fragment including the coat protein (CP) coding region. The CP nucleotide sequence comparisons showed 99% to 100% homology among the three isolates named HVXBJ4, HVXBJ5, and HVXBJ6 (GenBank Accession No. JX535292, JX535293, and JX535294) and with the HVX sequences previously reported in GenBank. HVX has been reported from the United States, Korea, the Netherlands, Poland, France, the Czech Republic, and New Zealand (1,2). To our knowledge, this is the first report of HVX infecting hosta plants in China. As an ornamental and medicinal plant, hosta has been cultivated in China for more than 2,000 years. The presence of HVX in Beijing is a potential threat to the landscape in the city. HVX can be spread by vegetative propagation material or mechanical contact (3). Hence, to cultivate HVX-free hosta and restrict the movement of HVX-infected hosta is vitally important in the future. HVX has become economically important in the world more recently. Globalization of trade in hosta plants has increased the risk of movement of HVX. The national plant protection organization should establish effective quarantine strategy and the growers take proper planting measures to avoid further spreading of this virus. References: (1) S. Currier et al. Plant Dis. 80:1040, 1996. (2) M. H. Park et al. Arch. Virol. 148:2039, 2003. (3) K. H. Ryu et al. Acta Hortic. 722:91, 2006.

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Insecticide susceptibility status and knockdown resistance (kdr) mutation in Aedes albopictus in China

Parasites & Vectors ◽

10.1186/s13071-021-05095-5 ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Yong Wei ◽

Xueli Zheng ◽

Song He ◽

Xuli Xin ◽

Jiachun Zhang ◽

...

Keyword(s):

Mortality Rate ◽

High Resistance ◽

Pyrethroid Resistance ◽

Management Strategies ◽

Resistance Management ◽

World Health ◽

Knockdown Resistance ◽

Phenotypic Resistance ◽

Pcr Products ◽

Health Organization

Abstract Background Aedes (Stegomyia) albopictus (Skuse, 1894) is the main vector of dengue virus in China. The resistance to insecticides is a huge obstacle for the control of this species, and determining its resistance status and mechanisms in China is essential for the implementation of vector management strategies. Methods We have investigated the larval and adult resistance status of Ae. albopictus to deltamethrin in eight field populations in China. Mutations at the voltage-gated sodium channel gene, related to the knockdown resistance (kdr) effect, were detected by sequencing of PCR products. The eight field populations were examined for pyrethroid resistance using the World Health Organization standard bioassays, and the association between the mutations and phenotypic resistance was tested. Results The eight field populations of larvae of Ae. albopictus in China exhibited high resistance to deltamethrin; the RR50 values ranged from 12 (ZJ) to 44 (GZ). Adult bioassay revealed that Ae. albopictus populations were resistant to deltamethrin (mortality rate < 90%), except ZJ population (probably resistant, mortality rate = 93.5%). Long knockdown time in the field populations was consistent with low mortality rates in adult bioassay. F1534S mutation showed increased protection against deltamethrin in all populations except BJ and SJZ populations, whereas I1532T mutation showed increased protection against deltamethrin in only BJ population. Conclusion There were different degrees of resistance to deltamethrin in field Ae. albopictus populations in China. The longest knockdown time and lowest mortality rate observed in Ae. albopictus population in Guangzhou indicate the severity of high resistance to deltamethrin. The patchy distribution of deltamethrin resistance and kdr mutations in Ae. albopictus mosquitoes suggests the necessity for resistance management and developing counter measures to mitigate the spread of resistance. Graphical Abstract

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A Novel Allele Specific Polymerase Chain Reaction (AS-PCR) Assay to Detect the V1016G Knockdown Resistance Mutation Confirms Its Widespread Presence in Aedes albopictus Populations from Italy

Insects ◽

10.3390/insects12010079 ◽

2021 ◽

Vol 12 (1) ◽

pp. 79

Author(s):

Verena Pichler ◽

Emiliano Mancini ◽

Martina Micocci ◽

Maria Calzetta ◽

Daniele Arnoldi ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Aedes Albopictus ◽

Pyrethroid Resistance ◽

Resistance Mutation ◽

Chain Reaction ◽

Pcr Assay ◽

Pyrethroid Insecticides ◽

Allele Specific ◽

Polymerase Chain ◽

Novel Allele

Polymerase chain reaction (PCR)-based genotyping of mutations in the voltage-sensitive sodium channel (vssc) associated with resistance to pyrethroid insecticides is widely used and represents a potential early warning and monitoring system for insecticide resistance arising in mosquito populations, which are vectors of different human pathogens. In the secondary vector Aedes albopictus—an Asian species that has invaded and colonized the whole world, including temperate regions—sequencing of domain II of the vssc gene is still needed to detect the V1016G mutation associated with pyrethroid resistance. In this study we developed and tested a novel allele-specific PCR (AS-PCR) assay to genotype the V1016G mutation in this species and applied it to the analysis of wild populations from Italy. The results confirm the high accuracy of the novel AS-PCR and highlight frequencies of the V1016G allele as >5% in most sampling sites, with peaks of 20–45% in coastal touristic sites where pyrethroid treatments are extensively implemented, mostly for mosquito nuisance reduction. The high frequency of this mutation observed in Italian Ae. albopictus populations should serve as a warning bell, advocating for increased monitoring and management of a phenomenon which risks neutralizing the only weapon today available to counteract (risks of) arbovirus outbreaks.

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Decade Long Upsurge In Target Site Pyrethroid Resistance In Bed Bug Populations In The United States

10.21203/rs.3.rs-887494/v1 ◽

2021 ◽

Author(s):

Cari D. Lewis ◽

Brenna. A. Levine ◽

Coby Schal ◽

Edward L. Vargo ◽

Warren Booth

Keyword(s):

United States ◽

Pyrethroid Resistance ◽

Rapid Evolution ◽

The United States ◽

Global Scale ◽

Adaptive Traits ◽

Short Term ◽

Pyrethroid Insecticides ◽

Bed Bug ◽

The Past

Abstract Over the past three decades, the bed bug Cimex lectularius has resurged as a prominent indoor pest on a global scale. Knockdown-associated insecticide resistance (kdr) involving the voltage-gated sodium channel, targeted by organochlorine and pyrethroid insecticides, was first reported in C. lectularius within a few years of the widespread use of Dichlorodiphenyltrichloroethane (DDT) and has been implicated as a significant factor contributing to the species recent resurgence. Since then, selection with pyrethroid insecticides has intensified, yet little is known regarding its short-term impacts on the frequency of kdr-associated mutations. Here, we report temporal changes in the frequencies of three kdr-associated mutations in C. lectularius populations collected across the United States from two time periods, sampled approximately a decade apart. Results reveal a significant increase in the frequencies of kdr-associated mutations over this period, and absence of the insecticide-susceptible genotype in recent collections. Furthermore, a significant transition towards infestations possessing multiple kdr-associated mutations was observed. These results suggest that the persistent use of pyrethroid insecticides over the past decade continues to impose strong selection pressure on C. lectularius populations, driving the proliferation of kdr-associated mutations. They demonstrate that, if unabated, strong anthropogenic selection can drive the rapid evolution of adaptive traits.

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Discriminating Between Isolates of PSbMV Using Nucleotide Sequence Polymorphisms in the HC-Pro Coding Region

Plant Disease ◽

10.1094/pdis-91-5-0490 ◽

2007 ◽

Vol 91 (5) ◽

pp. 490-496 ◽

Cited By ~ 6

Author(s):

V. A. Torok ◽

J. W. Randles

Keyword(s):

The United States ◽

Molecular Diagnostic ◽

Coding Region ◽

Rt Pcr ◽

Reference Isolate ◽

Length Polymorphisms ◽

Pcr Rflp ◽

Pcr Products ◽

Polymerase Chain ◽

Pea Seed

The molecular diversity of 14 isolates of Pea seed-borne mosaic virus (PSbMV) from southern Australia, 13 previously described isolates from Pakistan, and a reference isolate from the United States have been studied to determine whether a relatively simple molecular diagnostic assay and classification scheme could be developed for this virus. The Australian isolates were placed into either pathotype P1 or pathotype P4 by bioassay on differential genotypes of Pisum sativum. The Pakistani isolates represented pathotypes P1, P4, U1, and U2, and an undetermined pathotype. The reference US isolate was pathotype P1. A reverse transcription-polymerase chain reaction (RT-PCR) assay based on an amplicon from the variable HC-Pro coding region of potyviruses was shown to distinguish PSbMV from seven other legume infecting potyviruses. Restriction fragment length polymorphisms (RFLPs) generated from the HC-Pro RT-PCR products of all 28 isolates using seven restriction endonucleases placed them into eight groups. A phylogenetic tree based on a Bray-Curtis similarity comparison placed the groups into three clusters. The groups and clusters had no clear association with either pathotype or geographic source. It is concluded that within the range of viruses and isolates tested, the RT-PCR-RFLP method will both specifically identify PSbMV and provide a simple, qualitative, and rapid means for placing PSbMV isolates into groups. Applications could include mapping and tracking isolates in space and time.

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Insecticide resistance status of Aedes aegypti in Bangladesh

Parasites & Vectors ◽

10.1186/s13071-020-04503-6 ◽

2020 ◽

Vol 13 (1) ◽

Author(s):

Hasan Mohammad Al-Amin ◽

Fatema Tuj Johora ◽

Seth R. Irish ◽

Muhammad Riadul Haque Hossainey ◽

Lucrecia Vizcaino ◽

...

Keyword(s):

Aedes Aegypti ◽

Insecticide Resistance ◽

Pyrethroid Resistance ◽

Control Strategies ◽

Resistance Mechanisms ◽

Capital City ◽

Detoxification Enzymes ◽

Knockdown Resistance ◽

Metabolic Resistance ◽

Pyrethroid Insecticides

Abstract Background Arboviral diseases, including dengue and chikungunya, are major public health concerns in Bangladesh where there have been unprecedented levels of transmission reported in recent years. The primary approach to control these diseases is to control the vector Aedes aegypti using pyrethroid insecticides. Although chemical control has long been practiced, no comprehensive analysis of Ae. aegypti susceptibility to insecticides has been conducted to date. The aim of this study was to determine the insecticide resistance status of Ae. aegypti in Bangladesh and investigate the role of detoxification enzymes and altered target site sensitivity as resistance mechanisms. Methods Eggs of Aedes mosquitoes were collected using ovitraps from five districts across Bangladesh and in eight neighborhoods of the capital city Dhaka, from August to November 2017. CDC bottle bioassays were conducted for permethrin, deltamethrin, malathion, and bendiocarb using 3- to 5-day-old F0–F2 non-blood-fed female mosquitoes. Biochemical assays were conducted to detect metabolic resistance mechanisms, and real-time PCR was performed to determine the frequencies of the knockdown resistance (kdr) mutations Gly1016, Cys1534, and Leu410. Results High levels of resistance to permethrin were detected in all Ae. aegypti populations, with mortality ranging from 0 to 14.8% at the diagnostic dose. Substantial resistance continued to be detected against higher (2×) doses of permethrin (5.1–44.4% mortality). Susceptibility to deltamethrin and malathion varied between populations while complete susceptibility to bendiocarb was observed in all populations. Significantly higher levels of esterase and oxidase activity were detected in most of the test populations as compared to the susceptible reference Rockefeller strain. A significant association was detected between permethrin resistance and the presence of Gly1016 and Cys1534 homozygotes. The frequency of kdr (knockdown resistance) alleles varied across the Dhaka Aedes populations. Leu410 was not detected in any of the tested populations. Conclusions The detection of widespread pyrethroid resistance and multiple resistance mechanisms highlights the urgency for implementing alternate Ae. aegypti control strategies. In addition, implementing routine monitoring of insecticide resistance in Ae. aegypti in Bangladesh will lead to a greater understanding of susceptibility trends over space and time, thereby enabling the development of improved control strategies.

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