Susceptibility and interactions between Aedes mosquitoes and Zika viruses.

Zika virus disease is caused by Zika virus infection, as transmitted by Aedes spp. mosquitoes. Many of the Zika virus strains isolated from patients display different pathogenicities toward humans. The vector mosquitoes for Zika virus are mainly of the Aedes genus, especially Aedes aegypti and Aedes albopictus. However, susceptibility and interactions between Aedes spp. mosquitoes and Zika viruses remain unclear. In this study, we chose two Zika virus strains (FSS13025 and PRVABC59) with different abilities to infect the primary vector mosquitoes Ae. aegypti and Ae. albopictus. The transcriptomes and small RNA profiles of infected and uninfected mosquitoes were comparatively analyzed, and differentially expressed genes were functionally examined using RNA interference. According to the results, the susceptibility of PRVABC59 was higher than that of FSS13025 in Aedes vector mosquitoes, and Ae. aegypti was more susceptible to Zika virus than was Ae. albopictus. For PRVABC59 infection, specific differential expression profiles correlated with Ae. aegypti and Ae. albopictus, and susceptibility was significantly affected when three targeted genes were successfully knocked down. Compared with PRVABC59, infection of Ae. albopictus with FSS13025 generated more 21-nt virus small interference RNA. It can be concluded that the susceptibility of vector Aedes spp. mosquitoes to Zika viruses varies and that the interactions between mosquitoes and Zika virus correlate with susceptibility.

Defervescent dengue patients might be a potential source of infection for vector mosquitoes.

BACKGROUND: Dengue is a re-emerging public health problem and mosquito-borne infectious disease that is transmitted mainly by Aedes aegypti and Ae. albopictus. Early diagnosis, isolation, and treatment of patients are critical steps for dengue epidemic control, especially to prevent secondary transmission of dengue virus (DENV). However, little is known about defervescent dengue patients as a source of infection. METHODS: This case study describes 1268 dengue patients hospitalized at Guangzhou Eighth People's Hospital from June 2013 to December 2014. The viral loads of each individual were measured using real-time reverse transcription-polymerase chain reaction. Ae. aegypti and Ae. albopictus were exposed to blood meal with gradated dengue viral loads to characterize the relationship between viremia in dengue patients and the vector competence of vector mosquitoes. RESULTS: The viral numbers in the blood were measured, ranging from 108 to 103 copies/ml from day 1 to day 12 after fever onset. Vector competence analysis of Ae. aegypti and Ae. albopictus indicated that viremia > 104 copies/ml can still infect vector mosquitoes, which implied that the defervescent dengue patients might be a source of infection. CONCLUSIONS: The results of this study indicate that some defervescent dengue patients still have sufficient viral load to infect vector mosquitoes. Therefore, the protection against mosquito biting for these people should be extended to prevent secondary transmission events.

Construction of an efficient genomic editing system with CRISPR/Cas9 in the vector mosquito Aedes albopictus.

Aedes (Stegomyia) albopictus, also known as the Asian tiger mosquito, is a mosquito which originated in Asia. In recent years, it has become increasingly rampant throughout the world. This mosquito can transmit several arboviruses, including dengue, Zika and chikungunya viruses, and is considered a public health threat. Despite the urgent need of genome engineering to analyze specific gene functions, progress in genetical manipulation of Ae. albopictus has been slow due to a lack of efficient methods and genetic markers. In the present study, we established targeted disruptions in two genes, kynurenine hydroxylase (kh) and dopachrome conversion enzyme (yellow), to analyze the feasibility of generating visible phenotypes with genome editing by the clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated protein 9 (Cas9) system in Ae. albopictus. Following Cas9 single guide RNA ribonucleoprotein injection into the posterior end of pre-blastoderm embryos, 30%-50% of fertile survivors produced alleles that failed to complement existing kh and yellow mutations. Complete eye and body pigmentation defects were readily observed in G1 pupae and adults, indicating successful generation of highly heritable mutations. We conclude that the CRISPR/Cas9-mediated gene editing system can be used in Ae. albopictus and that it can be adopted as an efficient tool for genome-scale analysis and biological study.

Use of a Recombinant Mosquito Densovirus As a Gene Delivery Vector for the Functional Analysis of Genes in Mosquito Larvae.

In vivo microinjection is the most commonly used gene transfer technique for analyzing the gene functions in individual mosquitoes. However, this method requires a more technically demanding operation and involves complicated procedures, especially when used in larvae due to their small size, relatively thin and fragile cuticle, and high mortality, which limit its application. In contrast, viral vectors for gene delivery have been developed to surmount extracellular and intracellular barriers. These systems have the advantages of easy manipulation, high gene transduction efficiency, long-term maintenance of gene expression, and the ability to produce persistent effects in vivo. Mosquito densoviruses (MDVs) are mosquito-specific, small single-stranded DNA viruses that can effectively deliver foreign nucleic acids into mosquito cells; however, the replacement or insertion of foreign genes to create recombinant viruses typically causes a loss of packaging and/or replication abilities, which is a barrier to the development of these viruses as delivery vectors. Herein, we report using an artificial intronic small-RNA expression strategy to develop a non-defective recombinant Aedes aegypti densovirus (AaeDV) in vivo delivery system. Detailed procedures for the construction, packaging and quantitative analysis of the rAaeDV vectors, and for larval infection are described. This study demonstrates, for the first time, the feasibility of developing a non-defective recombinant MDV micro RNA (miRNA) expression system, and thus providing a powerful tool for the functional analysis of genes in mosquito and establishing a basis for the application of viral paratransgenesis for controlling mosquito-borne diseases. We demonstrated that Aedes albopictus 1st instar larvae could be easily and effectively infected by introducing the virus into the water body of the larvae breeding site and that the developed rAaeDVs could be used to overexpress or knock down the expression of a specific target gene in larvae, providing a tool for the functional analysis of mosquito genes.

[Preliminary application of a mosquito densovirus-mediated artificial intron in vitro and in vive of mosquito].

An artificial intron consisting of the 5'-donor site (from the first intron of the human beta-globin gene) and the 3'-acceptor site (from the intron of an immunoglobulin gene heavy chain variable region) was obtained with a splice overlap extension PCR and was then inserted in frame into the coding sequence of nostructural protein NS1 gene fused to GFP gene in a recombinant mosquito densovirus plasmid p7NS1-GFP. The constructed plasmid was named as p7NS1-Intron-GFP. The plasmid p7NS1-Intron-GFP was co transfected with the helper plasmid pUCA into C6/36 cells, then the packaged recombinant and wild type viruses were purified and recovered. The second-instars of Aedes albopictus larvae were exposed to recombinant and wild type virus mixed stock. The high level GFP expression in C6/36 cells and larvae was observed under fluorescence microscope, indicating that the inserted artificial intron exerted its normal function in self-splicing both in vitro and in vivo. This study laid a foundation for application of an artificial intron in insect cells and development of new strategy for genetic engineering technology of mosqtuito and its pathogens.

A recombinant AeDNA containing the insect-specific toxin, BmK IT1, displayed an increasing pathogenicity on Aedes albopictus.

The Aedes aegypti densovirus (AeDNV) has previously shown potential in mosquito control. To improve its efficacy as a biopesticide, the gene for an excitatory insect-specific toxin from Buthus martensii Karsch (BmK IT1) was inserted into the AeDNV genome and cloned into pUCA plasmid. The coding sequence for green fluorescent protein was ligated to the C-terminus of the BmK IT1 gene as a screening marker. Recombinant and helper plasmids were cotransfected into C6/36 cells; wild-type viruses were the controls. The recombinant viruses were identified and quantified by real-time polymerase chain reaction and exposed to Ae. albopictus larvae for the evaluation of its bioinsecticidal activity. LT(50) and LD(50) bioassays showed that the recombinant AeDNV had stronger and faster pathogenic effects on Ae. albopictus than the wild-type virus. This is the first report on the recombinant AeDNA containing the insect-specific toxin, BmK IT1, which may be used to develop a novel type of insecticide.

[Antibacterial activity of recombinant thanatin expressed by E.coli ER2566].

OBJECTIVE: To express antibacterial peptide thanatin in the prokaryotic expression system and test its antibacterial activity. METHODS: The DNA sequence coding for the 21 peptides of thanatin was synthesized using the preferred genetic codes of E. coli, cloned into pTYB11 plasmid, and transformed into E.coli ER2566. The expression of thanatin fused with intein was induced by IPTG in E.coli, and intein-thanatin specifically bound to the column through intein tag was cleaved overnight at 4 degrees celsius; in DTT/cysteine buffer. RESULTS: The cleaved thanatin was eluted with a protein concentration of 245 microg/ml in the first 4 ml. The purified thanatin had showed strong antibacterial activities against G- bacteria such as Shigella flexneri, Klebsiella pneumoniae, Shigella snnei, Escherichia coli O157, toxin producing Escherichia coli, Pseudomonas aeruginosa, and fungi such as Candida albicans, with especial potency in killing drug-resistant Klebsiella pneumoniae, Pseudomonas aeruginosa, and extended-spectrum beta-lactamases (ESBL)-producing E.coli. Eighty strains of drug-resistant (ESBL-producing) and 30 strains of sensitive E. coli were used for anti-bacterial assay, and no significant differences in the antibacterial activity of thanatin were found between the sensitive and drug-resistant E. coli (P>0.05). CONCLUSION: The recombinant thanatin obtained shows strong antibacterial activity against drug-resistant and sensitive bacteria, and can be a potential substitute for routine antibiotics in the treatment of G- bacterial infections.

[Isolation, identification and analysis of the expression profile of miRNAs in Aedes albopictus].

OBJECTIVE: To verify the miRNA in Aedes albopictus and characterize the expression profile of several miRNAs across all the life stages of Aedes albopictus. METHODS: Based on the published miRNA sequences of Anopheles gambiae and Aedes aegypti, 6 DIG-labeled antisense probes were synthesized. The total RNAs from Aedes albopictus in 6 developmental stages (embryo, early larvae, late larvae, pupa, male and female adults) were extracted with a mirVana miRNA isolation kit, loaded onto 15% denaturing polyacrylamide gel and hybridized with the appropriate DIG-labeled probes. RESULTS: Northern blotting detected 5 miRNAs in Aedes albopictus, of which mir-9a was mainly expressed in embryo and larva stages, let-7 in pupa and adult stages, miR-184 in all life stages, mir-M1 only in the embryos and miR-1175 in all the life stages except for embryos. The expression profiles of these miRNAs in Aedes albopictus were similar to those in D. melanogaster and An.stepheni. miR-1174 was not detected in any of the developmental stages of Aedes albopictus. CONCLUSION: These results present the first direct experimental evidence of miRNA in Aedes albopictus. The expression profiles of the analyzed miRNAs in Aedes albopictus showed stage specificity and conservation with other mosquitoes. Further studies on the functions of these miRNAs may offer new insights in mosquito biology and may lead to novel approaches to the development of insecticides.

[Effect of cinobufagin on nuclear factor-kappaB pathway in HepG2 cells].

OBJECTIVE: To investigate the effect of cinobufagin on nuclear factor-kappaB (NF-kappaB) pathway of liver cancer cell line HepG2. METHODS: Dual-luciferase cis-reporting system was used to detect the relative value of pNF-kappaB-TA-luc upon tumor necrosis factor-alpha (TNF-alpha) stimulation of NF-kappaB pathway. Western blotting was used to detect the protein level of NF-kappaB p65, and RT-PCR was used to detect the gene transcription level of intercellular adhesion molecule-1 (ICAM-1), a target downstream gene of NF-kappaB. RESULTS: At the concentration of 0.25 and 0.5 microg/ml, cinobufagin significantly lowered the relative value of luciferase (P<0.05). The results of Western blotting showed that cinobufagin significantly suppressed the protein expression of NF-kappaB p65. The transcription level of ICAM-1 was reduced by different doses of cinobufagin. CONCLUSION: The anti-cancer effect of cinobufagin may be related to its activity in inhibiting the activation of NF-kappaB pathway.

[Bioinformatics analysis of mosquito densovirus nostructure protein NS1].

OBJECTIVE: To analyze and predict the structure and function of mosquito densovirus (MDV) nostructual protein1 (NS1). METHODS: Using different bioinformatics software, the EXPASY pmtparam tool, ClustalX1.83, Bioedit, MEGA3.1, ScanProsite, and Motifscan, respectively to comparatively analyze and predict the physic-chemical parameters, homology, evolutionary relation, secondary structure and main functional motifs of NS1. RESULTS: MDV NS1 protein was a unstable hydrophilic protein and the amino acid sequence was highly conserved which had a relatively closer evolutionary distance with infectious hypodermal and hematopoietic necrosis virus (IHHNV). MDV NS1 has a specific domain of superfamily 3 helicase of small DNA viruses. This domain contains the NTP-binding region with a metal ion-dependent ATPase activity. A virus replication roller rolling-circle replication(RCR) initiation domain was found near the N terminal of this protein. This protien has the biological function of single stranded incision enzyme. CONCLUSION: The bioinformatics prediction results suggest that MDV NS1 protein plays a key role in viral replication, packaging, and the other stages of viral life.

A severe eosinophilic meningoencephalitis caused by infection of Angiostrongylus cantonensis.

This paper reports a severe case of eosinophilic meningoencephalitis after infection with Angiostrongylus cantonensis.

[Construction of the life cycle of Angiostrongylus cantonensis in laboratory].

OBJECTIVE: To construct the life cycle of Angiostrongylus cantonensis (A.cantonensis) in laboratory condition. METHODS: SD rats were infected orally with the third-stage larvae of A.cantonensis collected from Jiangmen, Guangdong province. Six weeks after infection, the first-stage larvae were isolated from fresh feces of the rats by using Baermann funnel to infect 25 second-generation white jade snails raised in laboratory at the daily dose of 300 000 for 3 consecutive days. Three weeks later, the snails were dissected for counting the third-staged larvae of A.cantonensis, and those positive for A.cantonensis infection were fed directly to 10 fasting rats. The serum samples of the rats were then collected 2 weeks later for examination of specific antibodies using ELISA. The feces of the infected rats were examined microscopically after 6 weeks, and the brain, heart and lungs of the infected rats were dissected to observe the larvae at 3, 5, and 8 weeks, respectively. RESULTS: The 3-stage larvae of A.cantonensis were found in the second-generation snails 3 weeks after infection. The positivity rate of serum specific antibodies was 100% in the 10 rats 2 weeks after feeding of the infected snails. The 1-stage larvae were detected in the feces of the rats 6 weeks after infection, and the fourth-stage larvae were found in the brain of the rats at 3 weeks, while adult worm and eggs were found in the heart and lungs of the infected rats at 5 and 8 weeks. CONCLUSION: The successful establishment of human colon carcinoma cell line with PRL-3 gene knock-down provide a basis for investigation of the role of PRL-3 gene in the metastasis of human colorectal carcinoma.

[Improved preparation of pure alive eggs of Schistosoma japonicum].

To prepare a large amount of pure alive Schistosoma japonicum eggs, rabbit was infected with 2000 cercariae and its liver was taken aseptically 38-45 days after infection and homogenized. The homogenate was screened through different sieves(60, 120, 200, 300, 360 meshes per inch respectively), and washed with 1.2% NaCl. The eggs and leftover were then digested with 0.25% trypsin for 2 hours, sieved over 360 meshes per inch and washed with RPMI 1640 medium. The collected eggs reached to (95.1 +/- 6.4)% of live eggs, with a high efficiency.

[Construction of a yeast model for screening Aedes albopictus ecdysone agonist pesticides].

OBJECTIVE: To reconstitute a transactivation system in yeast (yeast model) for screening the pesticides acting on ecdysone metabolism route and eventually influencing the process of ecdysis. METHODS: The fragment of 5 times repeated EcRE from Drosophila melanogaster was synthesized and the HSP27 promoter from D. melanogaster genome was amplified with PCR. The two sequences were connected and followed by a reporting gene--green fluorescence protein (GFP) gene. The EcRE-HSP27 promoter-GFP fragment was inserted into the expression plasmid pPIC3.5 and integrated into the yeast chromosome to construct yeast A. EcR and USP coding sequences of Aedes albopictus were synthesized, and these two fragments were inserted into Pichia pastoris expression plasmid pGAPZ as two respective reading frames. The two reading frames were integrated into Pichia pastoris chromosome in another recombinant site (pGAPZ and pPIC3.5k share different recombinant sites while being integrated into Pichia pastoris yeast chromosome). EcR and USP were constituted and expressed in the yeast. This recombinant yeast was called yeast B. The model yeast was thus constructed. A known ecdysone agonist-tebufenozide was used to test the yeast model. The effect of tebufenozide on the model yeast was observed under fluorescent microscope. Semi-quantitative RT-PCR was used to test the transcription level of GFP in the tebufenozide affected yeast and the control. RESULTS: In the model yeast, the intracellular expressed EcR and USP constituted EcR/USP heterodimer interacting with EcRE, the expression of GFP was activated, and green fluorescence was observed in model yeast under fluorescent microscope. Tebufenozide affected model yeast showed less fluorescence in comparison to the control model yeast, indicating that the transcription of GFP was suppressed by tebufenozide. Yeast housekeeping gene Actin-1 was used as inner control, semi-quantitative RT-PCR was operated and the result was scanned. The ratio of the brightness of GFP to Actin-1 was calculated automatically, and that of tebufenozide added yeast and the control yeast was 0.614 and 1.134 respectively. This result showed a low transcription level of GFP in tebufenozide affected model yeast, comparing to that of the control. CONCLUSION: The ecdysone-related transacting system in yeast has been constructed, and the model yeast can be used to screen the ecdysone agonists which can act on the ecdysone metabolic route.

[Screening and identification of therapeutic effect evaluation antigens of angiostrongyliasis].

OBJECTIVE: To identify antigens which may help evaluate the therapeutic effect of angiostrongyliasis from adult worm antigen of Angiostrongylus cantonensis. METHODS: The adult worm antigens of A. cantonensis were analyzed by Western blotting with the sera of rats infected with A. cantonensis before and after treatment. The sera of rats were tested by enzyme-linked immunosorbent assay (ELISA). RESULTS: The antigens with relative molecular mass between 38,000 and 78,000 reacted not only with the sera of rats before treatment, but also with that after treatment. The antigens with M(r) between 190,000 and 17,000 reacted with the sera of rats before treatment but not with that after treatment; those with M(r) between 32,000 and 24,000 antigens strongly reacted with the former, but the reaction became much weakened with the latter. The AC32-IgG antibody appeared earlier than the AC-IgG, and disappeared rapidly after treatment. Six of the 10 treated rats became negative for AC-IgG as found by ELISA. CONCLUSION: The antigens of adult worm antigen of A. cantonensis with M(r) of 190,000, 32,000, 24,000, 17,000 and 16,000 may serve as candidate antigens for therapeutic effect evaluation of angiostrongyliasis.