Surveillance of Chigger Mite Vectors for Tsutsugamushi Disease in the Hwaseong Area, Gyeonggi-do, Republic of Korea, 2015.

Owing to global climate change, the global resurgence of vector-borne infectious diseases and their potential to inflict widespread casualties among human populations has emerged as a pivotal burden on public health systems. Tsutsugamushi disease (scrub typhus) in the Republic of Korea is steadily increasing and was designated as a legal communicable disease in 1994. The disease is a mite-borne acute febrile disease most commonly contracted from October to December. In this study, we tried to determine the prevalence of tsutsugamushi disease transmitted by chigger mites living on rodents and investigated their target vector diversity, abundance, and distribution to enable the mapping of hotspots for this disease in 2015. A total of 5 species belonging to 4 genera (109 mites): Leptotrombidium scutellare 60.6%, L. pallidum 28.4% Neotrombicula tamiyai 9.2%, Euschoengastia koreaensis/0.9%), and Neoschoengastia asakawa 0.9% were collected using chigger mite collecting traps mimicking human skin odor and sticky chigger traps from April to November 2015. Chigger mites causing tsutsugamushi disease in wild rodents were also collected in Hwaseong for the zoonotic surveillance of the vector. A total of 77 rodents belonging to 3 genera: Apodemus agrarius (93.5%), Crocidura lasiura (5.2%), and Micromys minutus (1.3%) were collected in April, October, and November 2015. The most common mite was L. pallidum (46.9%), followed by L. scutellare (18.6%), and L. orientale (18.0%). However, any of the chigger mite pools collected from rodent hosts was tested positive for Orientia tsutsugamushi, the pathogen of tsutsugamushi disease, in this survey.

Geographical Genetic Variation and Sources of Korean Aedes albopictus (Diptera: Culicidae) Populations.

Aedes albopictus (Skuse, 1894) is a mosquito vector raising global health concerns owing to its transmission of dengue, Zika, and chikungunya viruses. This vector accounts for a large proportion of the Korean mosquito community; however, autochthonous clinical cases resulting from this species remain unreported in South Korea. This study aimed to examine the geographical genetic variations and sources of Ae. albopictus populations in South Korea exclusively on the basis of COI gene analysis from 292 samples collected from 37 localities in 2016-2018 and 290 reference sequences from GenBank. Thirty-eight haplotypes were identified among the 292 Ae. albopictus samples, with H1 (n = 190, 65.1%), H29 (n = 24, 8.22%), and H32 (n = 24, 8.22%) being the most common and widely distributed haplotypes in the mainland, southern coastal region, and Jeju Island, respectively. In general, high haplotype (≥0.5; 44.7%) and low nucleotide (≤0.00148 max.) diversity were observed in these populations. Based on eight regional groups, results of neutral tests and a mismatch analysis supported demographic expansions after bottlenecks. Furthermore, analysis of molecular variance, FST, and K2P distance showed that Gyeongsangnam-do, Jeju Island, and mainland groups were genetically differentiated. Haplotype network and phylogenetic analyses revealed that the sources of the main haplotypes are related to strains from other countries. The current findings need to be validated with additional sampling from heterogeneous habitats and different genetic markers. However, our results suggest that haplotype changes should be closely monitored for efficient vector surveillance and control.

Three-year surveillance of culicine mosquitoes (Diptera: Culicidae) for flavivirus infections in Incheon Metropolitan City and Hwaseong-si of Gyeonggi-do Province, Republic of Korea.

Japanese encephalitis virus (JEV) is a single stranded positive sense RNA virus of the genus Flavivirus that belongs to family Flaviviridae and emerged as one of the most pivotal form of viral encephalitis. The virus is transmitted to humans by mosquito vector and is an etiological agent of acute zoonotic infection. In this study, we investigated distribution and density over 3-year period in central regions of Korean peninsula. We selected two cities as mosquito-collecting locations and subdivided them into five collection sites; downtown Incheon Metropolitan City as a typical urban area, and the Hwaseong-si area as a rural area. A total of 35,445 female culicine mosquitoes were collected using black light traps or BG Sentinel™ traps from March to November 2016-2018. Aedes (Ae.) vexans nipponii was the most frequently collected specimens (48.91%), followed by Culex (Cx.) pipiens (32.05%), Ochlerotatus (Och.) dorsalis (13.58%), Och. koreicus (1.68%), and Cx. tritaeniorhynchus (1.49%). In the urban area, Cx. pipiens was the predominant species (92.21%) and the other species accounted for <5% of the total mosquitoes collected. However, in the rural area, Ae. vexans nipponii had the highest population (61.90%), followed by Och. dorsalis (17.10%), Cx. tritaeniorhynchus (1.84%) and Och, koreicus (1.78%). Culicine mosquitoes were identified at the species level, placed in pools of up to 30 mosquitoes each, and screened for flavivirus RNA using the SYBR Green-based RT-PCR. Three of the assayed 1092 pools were positive for Chaoyang virus from Ae. vexans nipponii and Japanese encephalitis virus from Cx. pipiens. The maximum likelihood estimations (the estimated number of virus-positive mosquitoes/1000 mosquitoes) for Ae. vexans nipponii positive for Chaoyang virus and Cx. pipiens for Japanese encephalitis virus were 3.095 and 0.20, respectively. The results of our study demonstrate that although mosquito-borne diseases were not detected in the potential vectors, enhanced monitoring and long-term surveillance of these vector viruses are of great public health importance.

Seasonal Occurrence of Haemaphysalis longicornis (Acari: Ixodidae) and Haemaphysalis flava, Vectors of Severe Fever With Thrombocytopenia Syndrome (SFTS) in South Korea.

The seasonal abundance of Haemaphysalis longicornis Neumann and H. flava Neumann (Acari: Ixodidae) was monitored from 2015 through 2017 in Gyeonggi-do, South Korea. The hard ticks were surveyed monthly using CO2 bait traps from April to November and flagging from July to September in a coniferous forest, a deciduous forest, shrubs, and a grassland vegetation types. Haemaphysalis longicornis and H. flava were most abundant in the grassland and shrubs, which yielded over 50% of the total number of ticks in all 3 yr. Furthermore, H. longicornis comprised up to 90% of the ticks collected. Generally, peaks of nymph, adult, and larva numbers were observed from April to June, from June to July, and from August to September, respectively. Half of the ticks were pooled and tested for the presence of the SFTS virus with negative results.

Four Year Surveillance of the Vector Hard Ticks for SFTS, Ganghwa-do, Republic of Korea.

The seasonal abundance of hard ticks that transmit severe fever with thrombocytopenia syndrome virus was monitored with a collection trap method every April to November during 2015-2018 and with a flagging method every July and August during 2015-2018 in Ganghwa-do (island) of Incheon Metropolitan City, Republic of Korea. This monitoring was performed in a copse, a short grass field, coniferous forest and broad-leaved forest. A total of 17,457 ticks (8,277 larvae, 4,137 nymphs, 3,389 females, and 1,654 males) of the ixodid ticks comprising 3 species (Haemaphysalis longicornis, H. flava, and Ixodes nipponensis) were collected with collection traps. Of the identified ticks, H. longicornis was the most frequently collected ticks (except larval ticks) (94.26%, 8,653/9,180 ticks (nymphs and adults)), followed by H. flava (5.71%, 524/9,180) and Ix. nipponensis (less than 0.04%, 3/9,180). The ticks collected with collecting traps were pooled and assayed for the presence of SFTS virus with negative results. In addition, for monitoring the prevalence of hard ticks, a total of 7,461 ticks (5,529 larvae, 1,272 nymphs, 469 females, and 191 males) of the ixodid ticks comprising 3 species (H. longicornis, H. flava, and Ix. nipponensis) were collected with flagging method. H. longicornis was the highest collected ticks (except larval ticks) (99.53%, 1,908/1,917 ticks (nymphs and adults)), followed by H. flava (1.15%, 22/1,917).

Annual Fluctuation in Chigger Mite Populations and Orientia Tsutsugamushi Infections in Scrub Typhus Endemic Regions of South Korea.

OBJECTIVES: Chigger mites are vectors for scrub typhus. This study evaluated the annual fluctuations in chigger mite populations and Orientia tsutsugamushi infections in South Korea. METHODS: During 2006 and 2007, chigger mites were collected monthly from wild rodents in 4 scrub typhus endemic regions of South Korea. The chigger mites were classified based on morphological characteristics, and analyzed using nested PCR for the detection of Orientia tsutsugamushi. RESULTS: During the surveillance period, the overall trapping rate for wild rodents was 10.8%. In total, 17,457 chigger mites (representing 5 genera and 15 species) were collected, and the average chigger index (representing the number of chigger mites per rodent), was 31.7. The monthly chigger index was consistently high (> 30) in Spring (March to April) and Autumn (October to November). The mite species included Leptotrombidium pallidum (43.5%), L. orientale (18.9%), L. scutellare (18.1%), L. palpale (10.6%), and L. zetum (3.6%). L. scutellare and L. palpale populations, were relatively higher in Autumn. Monthly O. tsutsugamushi infection rates in wild rodents (average: 4.8%) and chigger mites (average: 0.7%) peaked in Spring and Autumn. CONCLUSION: The findings demonstrated a bimodal pattern of the incidence of O. tsutsugamushi infections. Higher infection rates were observed in both wild rodents and chigger mites, in Spring and Autumn. However, this did not reflect the unimodal incidence of scrub typhus in Autumn. Further studies are needed to identify factors, such as human behavior and harvesting in Autumn that may explain this discordance.

Investigation and research on ticks carrying spotted fever group rickettsia in the border area of Tumen River Basin / 中华预防医学杂志

Objective@#To understand the situation and genotype distribution of spotted fever group rickettsia (SFGR) in the border area of Tumen River Basin in free ticks in Yanbian Korean Autonomous Prefecture (Yanbian Prefecture), Jilin Province. @*Methods@#From April to September, 2017, ticks were collected using flagging method from Hunchun, Tumen, Helong and Longjing cities in the Tumen River basin of Yanbian Prefecture. Outer membrane protein A (ompA) was detected by Polymerase Chain Reaction (PCR), then, the species were identified by gene sequencing and analyzed systematically. The positive rate of pools and MIR(minimum infection rate per 100 ticks,MIR) of SFGR were calculated, and the difference of positive rate of pools among ticks with different characteristics was compared by Chi-square test. @*Results@#A total of 3 079 ticks were collected and divided into 536 pools. The positive rate of pools of SFGR nucleic acid was 39.7% (213 pools). The MIR of SFGR was 6.9%.The positive rate of pools of SFGR in Dermacentor silvarum, Haemaphysalis concinna, Haemaphysalis japonica, Haemaphysalis longicornis and Ixodes persulcatus were 80.4% (41/51), 14.0% (25/179), 20.2% (18/89), 78.9% (101/128) and 25.9% (21/81), and the difference was statistically significant (P<0.001). There was statistical difference in the positive rate of pools of SFGR in developmental stages of ticks (P<0.001); the positive rate of pools of female adults, male adults, nymph and larvae were 36.4% (95/261), 34.2% (67/196), 56.3% (40/71) and 7/8, and the MIR was 7.9%, 7.7%, 4.9% and 3.5%. The five genotype was detected which was Candidatus Rickettsia longicornii, Rickettsia raoultii, Rickettsia heilongjiangensis, Candidatus Rickettsia tarasevichiae,Rickettsia monacensis and have 98%-100% homology with known gene sequences. Candidatus Rickettsia longicornii, Rickettsia raoultii, Rickettsia heilongjiangensis and Candidatus Rickettsia tarasevichiae showed close evolutionary relationship with known specie (have 98%-100% homology with known gene sequences); Rickettsia monacensis showed Far from evolutionary relationship with known species (have 98% homology with known gene sequences). @*Conclusion@#SFGR infection of ticks is common in the border areas of the Tumen River Basin. There was high diversity in SFGR species and tick species in the areas surveyed.

Distribution of malaria vectors and incidence of vivax malaria at Korean army installations near the demilitarized zone, Republic of Korea.

BACKGROUND: As a result of the reintroduction of malaria in the Republic of Korea (ROK) in 1993 and the threat to military and civilian populations, the Korea Military National Defense (MND) increased emphasis on vector control in 2012 at ROK Army (ROKA) installations located near the DMZ, while decreasing chemoprophylaxis, fearing potential drug resistance. Mosquito surveillance demonstrated a need for continuous monitoring of disease patterns among ROKA soldiers and vector malaria infection rates to ensure positive outcomes. METHODS: Anopheles spp. were collected from May-October at three ROKA installations in three locations near the DMZ. Each of the areas included one installation <2 km and two installations 11-12 km from the DMZ in Paju and Yeoncheon counties, Gyeonggi Province. Anopheles spp. were identified by polymerase chain reaction (PCR) techniques and then assayed for the presence of vivax malaria sporozoites. The ROK MND reported vivax malaria patients monthly to Korea Centers for Disease Control and Prevention. Correlations for the incidence of Plasmodium vivax patients and infected Anopheles species were analysed using the Wilcoxon rank sum test, Pearson correlation test and liner regression analysis. RESULTS: A total of 4282 Anopheles spp. were collected. Anopheles kleini (69.5 %) was the most commonly collected, followed by Anopheles pullus (17.3 %), Anopheles belenrae (4.5 %), Anopheles sineroides (4.2 %), Anopheles sinensis sensu stricto (2.7 %), and Anopheles lesteri (1.9 %). Overall, 21 malaria patients were reported by the ROK MND. The monthly incidence of the malaria patients correlated with the monthly number of Plasmodium vivax sporozoite positive Anopheles spp. The monthly numbers of An. kleini demonstrated the highest correlations to the numbers of ROKA malaria patients throughout the mosquito season (P < 0.01). Anopheles spp. positive for P. vivax and malaria patients at ROKA installations located <2 km from the DMZ were higher than for ROKA installations located 11-12 km from DMZ. CONCLUSION: The number of Anopheles spp. positive for P. vivax sporozoites correlated with the monthly number of malaria cases and exposure of ROKA soldiers from May-October to P. vivax malaria infections. Malaria vector surveillance and vector control is warranted as part of an effective malaria management program at ROKA installations located near DMZ.

First Isolation of Severe Fever with Thrombocytopenia Syndrome Virus from Haemaphysalis longicornis Ticks Collected in Severe Fever with Thrombocytopenia Syndrome Outbreak Areas in the Republic of Korea.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging tick-borne infectious disease that is endemic to China, Japan, and the Republic of Korea (ROK). In this study, 8313 ticks collected from SFTS outbreak areas in the ROK in 2013 were used to detect the SFTS virus (SFTSV). A single SFTSV was isolated in cell culture from one pool of Haemaphysalis longicornis ticks collected from Samcheok-si, Gangwon Province, in the ROK. Phylogenetic analysis showed that the SFTSV isolate was clustered with the SFTSV strain from Japan, which was isolated from humans. To the best of our knowledge, this is the first isolation in the world of SFTSV in ticks collected from vegetation.

Detection of Japanese encephalitis virus genotype V in Culex orientalis and Culex pipiens (Diptera: Culicidae) in Korea.

Japanese encephalitis virus (JEV) causes significant viral encephalitis and is distributed throughout the Asian countries. The virus is known to be transmitted by Culex tritaeniorhynchus, which mainly breeds in rice paddies in Korea. In this study, we investigated the presence of other mosquito species that can transmit JEV as a second or regional vector. We selected five cities where patients have experienced JE in the last 5 years as mosquito-collecting locations and subdivided them into four collection sites according to the mosquito habitats (cowshed, downtown area, forest, and swamp). Mosquitoes were caught using the BG-Sentinel trap, CDC black-light trap, Fay-Prince trap, and Gravid trap. A total of 993 pools from 22,774 mosquitoes were prepared according to their species, collection date, and site. We performed a SYBR Green 1-based real-time RT-PCR assay to detect JEV from the mosquito pools. A total of six JEV-positive pools were detected from Culex orientalis and Culex pipiens caught in the Gangwon-do and Gyeonngi-do provinces. All the detected JEVs were revealed as genotype V by phylogenetic analysis of the envelope gene. Our findings confirm that a new genotype of JEV was introduced in Korea and suggest that two mosquito species may play a role in JEV transmission.

Coincidence between geographical distribution of Leptotrombidium scutellare and scrub typhus incidence in South Korea.

To clarify the geographical distribution of scrub typhus vectors in Korea, a survey of larval trombiculid mites was conducted from 2005 to 2007 by collecting wild small mammals twice a year (spring and autumn) at 24 sites nationwide. A total of 67,325 mites representing 4 genera and 14 species were collected from 783 trapped rodents, corresponding to a chigger index (number of chigger mites per rodent) of 86.0. The predominant mite species were Leptotrombidium pallidum (52.6%), Leptotrombiduim scutellare (27.1%), Leptotrombidium palpale (8.2%), Leptotrombidium orientale (5.6%), and Neotrombicula tamiyai (1.7%). However, the proportions of L. scutellare in southern areas, including endemic provinces such as Jeollabuk-Do (34.3%), Jeollanam-Do (49.0%), and Gyeongsangnam-Do (88%), were relatively higher than in central Korean regions where L. pallidum was predominant. In autumn, the ratio of L. scutellare increased to 42% while the ratio of L. pallidum decreased. The geographical distribution map of the L. scutellare chigger index was identical to the incidence pattern of scrub typhus, whereas those of overall mites and L. pallidum showed no relationship with case incidence patterns. Distribution mapping analysis shows an identical geographical distribution of L. scutellare and epidemic incidence of scrub typhus in South Korea. L. pallidum could be another vector at all other parts of the Korean peninsula, including the eastern and northern regions that have a low level of scrub typhus incidence.

Prediction Forecast for Culex tritaeniorhynchus Populations in Korea.

OBJECTIVES: Japanese encephalitis is considered as a secondary legal infectious disease in Korea and is transmitted by mosquitoes in the summer season. The purpose of this study was to predict the ratio of Culex tritaeniorhynchus to all the species of mosquitoes present in the study regions. METHODS: From 1999 to 2012, black light traps were installed in 10 regions in Korea (Busan, Gyeonggi, Gangwon, Chungbuk, Chungnam, Jeonbuk, Jeonnam, Gyeongbuk, Gyeongnam, and Jeju) to capture mosquitoes for identification and classification under a dissecting microscope. The number of mosquitoes captured/week was used to calculate its daily occurrence (mosquitoes/trap/night). To predict the characteristics of the mosquito population, an autoregressive model of order p (AR(p)) was used to execute the out-of-sample prediction and the in-sample estimation after presumption. RESULTS: Compared with the out-of-sample method, the sample-weighted regression method's case was relatively superior for prediction, and this method predicted a decrease in the frequency of Cx. tritaeniorhynchus for 2013. However, the actual frequency of this species showed an increase in frequency. By contrast, the frequency rate of all the mosquitoes including Cx. tritaeniorhynchus gradually decreased. CONCLUSION: The number of patients with Japanese encephalitis has been strongly associated with the occurrence and density of vector mosquitoes, and the importance of this infectious disease has been highlighted since 2010. The 2013 prediction indicated an increase after an initial decrease, although the ratio of the two mosquito species decreased. The increase in vector density may be due to changes in temperature and the environment. Thus, continuous prevalence prediction is warranted.

Estimation of the genome sizes of the chigger mites Leptotrombidium pallidum and Leptotrombidium scutellare based on quantitative PCR and k-mer analysis.

BACKGROUND: Leptotrombidium pallidum and Leptotrombidium scutellare are the major vector mites for Orientia tsutsugamushi, the causative agent of scrub typhus. Before these organisms can be subjected to whole-genome sequencing, it is necessary to estimate their genome sizes to obtain basic information for establishing the strategies that should be used for genome sequencing and assembly. METHOD: The genome sizes of L. pallidum and L. scutellare were estimated by a method based on quantitative real-time PCR. In addition, a k-mer analysis of the whole-genome sequences obtained through Illumina sequencing was conducted to verify the mutual compatibility and reliability of the results. RESULTS: The genome sizes estimated using qPCR were 191 ± 7 Mb for L. pallidum and 262 ± 13 Mb for L. scutellare. The k-mer analysis-based genome lengths were estimated to be 175 Mb for L. pallidum and 286 Mb for L. scutellare. The estimates from these two independent methods were mutually complementary and within a similar range to those of other Acariform mites. CONCLUSIONS: The estimation method based on qPCR appears to be a useful alternative when the standard methods, such as flow cytometry, are impractical. The relatively small estimated genome sizes should facilitate whole-genome analysis, which could contribute to our understanding of Arachnida genome evolution and provide key information for scrub typhus prevention and mite vector competence.

Transovarial transmission of Orientia tsutsugamushi in Leptotrombidium palpale (Acari: Trombiculidae).

Transovarial transmission of Orientia tsutsugamushi in colonies of Leptotrombidium palpale was studied in the parent and F1 and F2 generations. Both transovarial transmission and filial infection rates were 100% in the parent and F1 generations of Leptotrombidium palpale. The filial infection rate in the F1 generation was 100%, but it declined to 94.3% in the F2 progeny. The sex ratio of the F1 generation from infected L. palpale was 1∶0.8 (male:female) and the proportion of males was relatively high. This study is the first to report on the transovarial transmission of O. tsutsugamushi in L. palpale. High transovarial transmission rates in L. palpale suggest that this species might be one of the major vectors of tsutsugamushi disease in Korea.

Functional characterization of Autographa californica multiple nucleopolyhedrovirus ORF43 and phenotypic changes of ORF43-knockout mutant.

ORF43 (ac43) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is a highly conserved baculovirus gene of unknown function. To investigate the role of ac43 in the baculovirus lifecycle, we constructed an ac43-deleted mutant AcMNPV, Ac43KO. After transfection into Spodoptera frugiperda cells, Ac43KO produced polyhedra much larger in size than those of wild-type AcMNPV. Interestingly, some of the nucleocapsids were singly enveloped in the polyhedrin matrix while the nucleocapsids of AcMNPV are known to be multiply enveloped. Furthermore, Ac43KO led to a defect in the transcription and expression of polyhedrin, which resulted in reduced occlusion body production. However, Ac43KO did not affect production of budded virus as there was no remarkable difference in budded virus titer. These results suggest that ac43 plays an important role in the expression of polyhedrin, the morphogenesis of occlusion body, and the assembly of virions occluded in occlusion bodies.

Existence of lysogenic bacteriophages in Bacillus thuringiensis type strains.

We screened the existence of bacteriophages in 67 Bacillus thuringiensis type strains by phage DNA extraction and PCR using phage terminase small subunit (TerS)-specific primers to the supernatants and the precipitated pellets of Bt cultures, and by transmission electron microscopy. The various bacteriophages were observed from the supernatants of 22 type strains. Ten type strains showed the extracted phage DNAs and the amplified fragment by TerS PCR but 12 type strains showed only the phage DNAs. Their morphological characteristic suggests that they belong to Family Siphoviridae which had a long tail and symmetrical head.

NeuroBactrus, a novel, highly effective, and environmentally friendly recombinant baculovirus insecticide.

A novel recombinant baculovirus, NeuroBactrus, was constructed to develop an improved baculovirus insecticide with additional beneficial properties, such as a higher insecticidal activity and improved recovery, compared to wild-type baculovirus. For the construction of NeuroBactrus, the Bacillus thuringiensis crystal protein gene (here termed cry1-5) was introduced into the Autographa californica nucleopolyhedrovirus (AcMNPV) genome by fusion of the polyhedrin-cry1-5-polyhedrin genes under the control of the polyhedrin promoter. In the opposite direction, an insect-specific neurotoxin gene, AaIT, from Androctonus australis was introduced under the control of an early promoter from Cotesia plutellae bracovirus by fusion of a partial fragment of orf603. The polyhedrin-Cry1-5-polyhedrin fusion protein expressed by the NeuroBactrus was not only occluded into the polyhedra, but it was also activated by treatment with trypsin, resulting in an ∼65-kDa active toxin. In addition, quantitative PCR revealed that the neurotoxin was expressed from the early phase of infection. NeuroBactrus showed a high level of insecticidal activity against Plutella xylostella larvae and a significant reduction in the median lethal time against Spodoptera exigua larvae compared to those of wild-type AcMNPV. Rerecombinant mutants derived from NeuroBactrus in which AaIT and/or cry1-5 were deleted were generated by serial passages in vitro. Expression of the foreign proteins (B. thuringiensis toxin and AaIT) was continuously reduced during the serial passage of the NeuroBactrus. Moreover, polyhedra collected from S. exigua larvae infected with the serially passaged NeuroBactrus showed insecticidal activity similar to that of wild-type AcMNPV. These results suggested that NeuroBactrus could be recovered to wild-type AcMNPV through serial passaging.

Construction and characterisation of an antifungal recombinant Bacillus thuringiensis with an expanded host spectrum.

A novel antifungal Bacillus thuringiensis strain 19-22, ssp. kurstaki (H3a3b3c), was characterised. This strain included cry1Aa, cry1Ab, cry1Ac, and cry1D, which have high insecticidal activities against lepidopteran larvae other than Spodoptera exigua. To expand the host spectrum, a cry1E gene whose product is active against S. exigua was introduced into the isolate. The transformant successfully expressed the Cry1E protein without any loss of its original antifungal activities. These results indicate that this recombinant strain exhibits dual activities and may be used as an integrated control agent to control plant diseases and insect pests.

Fast and efficient generation of recombinant baculoviruses by in vitro transposition.

A novel recombinant bacmid, bEasyBac, that enables the easy and fast generation of pure recombinant baculovirus without any purification step was constructed. In bEasyBac, attR recombination sites were introduced to facilitate the generation of a recombinant viral genome by in vitro transposition. Moreover, the extracellular RNase gene from Bacillus amyloliquefaciens, barnase, was expressed under the control of the Cotesia plutellae bracovirus early promoter to negatively select against the non-recombinant background. The bEasyBac bacmid could only replicate in host insect cells when the barnase gene was replaced with the gene of interest by in vitro transposition. When bEasyBac was transposed with pDualBac-EGFP, the resulting recombinant virus, AcEasy-EGFP, showed comparable levels of EGFP expression efficiency to the plaque-purified recombinant virus AcEGFP, which was constructed using the bAcGOZA system. In addition, no non-recombinant backgrounds were detected in unpurified AcEasy-EGFP stocks. Based on these results, a high-throughput system for the generation of multiple recombinant viruses at a time was established.

Analysis of genes expression of Spodoptera exigua larvae upon AcMNPV infection.

BACKGROUND: The impact of Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) infection on host gene expression in Spodoptera exigua 4th instar larvae was investigated through the use of 454 sequencing-based RNA-seq of cDNA libraries developed from insects challenged with active AcMNPV or heat-inactivated AcMNPV. METHODOLOGY/PRINCIPAL FINDINGS: By comparing the two cDNA libraries, we show that 201 host genes are significantly up-regulated and 234 genes are significantly down-regulated by active AcMNPV infection. Down-regulated host genes included genes encoding antimicrobial peptides, namely three gloverin isoforms and an attacin, indicating that the viral infection actively repressed the expression of a portion of the host immune gene repertoire. Another interesting group of down-regulated host genes included genes encoding two juvenile hormone binding proteins and a hexamerin, all of which are involved in juvenile hormone regulation. The expression of these genes was enhanced by the topical application of Juvenile Hormone III (JHIII) in the insects challenged with heat-inactivated AcMNPV. However, infection with the active virus strongly suppresses the expression of these three genes, regardless of the absence or presence of JHIII. CONCLUSIONS/SIGNIFICANCE: Using RNA-seq, we have identified groups of immune-regulated and juvenile hormone-regulated genes that are suppressed by infection with active AcMNPV. This information and further studies on the regulation of host gene expression by AcMNPV will provide the tools needed to enhance the utility of the virus as an effective protein expression system and as an insecticide.