Hong-Juan Peng

Genome sequence of the Asian Tiger mosquito, Aedes albopictus, reveals insights into its biology, genetics, and evolution

Significance Aedes albopictus is a highly adaptive species that thrives worldwide in tropical and temperate zones. From its origin in Asia, it has established itself on every continent except Antarctica. This expansion, coupled with its ability to vector the epidemic human diseases dengue and Chikungunya fevers, make it a significant global public health threat. A complete genome sequence and transcriptome data were obtained for the Ae. albopictus Foshan strain, a colony derived from mosquitoes from its historical origin. The large genome (1,967 Mb) comprises an abundance of repetitive DNA classes and expansions of the numbers of gene family members involved in insecticide resistance, diapause, sex determination, immunity, and olfaction. This large genome repertory and plasticity may contribute to its success as an invasive species. The Asian tiger mosquito, Aedes albopictus, is a highly successful invasive species that transmits a number of human viral diseases, including dengue and Chikungunya fevers. This species has a large genome with significant population-based size variation. The complete genome sequence was determined for the Foshan strain, an established laboratory colony derived from wild mosquitoes from southeastern China, a region within the historical range of the origin of the species. The genome comprises 1,967 Mb, the largest mosquito genome sequenced to date, and its size results principally from an abundance of repetitive DNA classes. In addition, expansions of the numbers of members in gene families involved in insecticide-resistance mechanisms, diapause, sex determination, immunity, and olfaction also contribute to the larger size. Portions of integrated flavivirus-like genomes support a shared evolutionary history of association of these viruses with their vector. The large genome repertory may contribute to the adaptability and success of Ae. albopictus as an invasive species.

Distribution of tick-borne diseases in China

As an important contributor to vector-borne diseases in China, in recent years, tick-borne diseases have attracted much attention because of their increasing incidence and consequent significant harm to livestock and human health. The most commonly observed human tick-borne diseases in China include Lyme borreliosis (known as Lyme disease in China), tick-borne encephalitis (known as Forest encephalitis in China), Crimean-Congo hemorrhagic fever (known as Xinjiang hemorrhagic fever in China), Q-fever, tularemia and North-Asia tick-borne spotted fever. In recent years, some emerging tick-borne diseases, such as human monocytic ehrlichiosis, human granulocytic anaplasmosis, and a novel bunyavirus infection, have been reported frequently in China. Other tick-borne diseases that are not as frequently reported in China include Colorado fever, oriental spotted fever and piroplasmosis. Detailed information regarding the history, characteristics, and current epidemic status of these human tick-borne diseases in China will be reviewed in this paper. It is clear that greater efforts in government management and research are required for the prevention, control, diagnosis, and treatment of tick-borne diseases, as well as for the control of ticks, in order to decrease the tick-borne disease burden in China.

Development of an Efficient Recombinant Mosquito Densovirus-Mediated RNA Interference System and Its Preliminary Application in Mosquito Control

The Aedes aegypti densovirus (AeDNV) has potential as a delivery vector for foreign nucleic acids into mosquito cells. In this study, we investigated the ability of plasmids containing recombinant viral transducing genome to induce RNA interference (RNAi) effects in C6/C36 cells. We then evaluated the efficiency of a recombinant AeDNV vector to induce RNAi in Aedes albopictus larvae. We found that the expression of V-ATPase was inhibited by up to 90% at 96 h post-transfection in transfected C6/C36 cells. In addition, the bioinsecticidal activities of various RNAi-expressing AeDNV vectors used to infect Ae. albopictus larvae were also tested. We found that when Ae. albopictus larvae were infected with recombinant AeDNV, expression of V-ATPase was downregulated by nearly 70% compared to controls. Furthermore, the median survival time bioassays demonstrated that recombinant AeDNV caused more serious pathogenic effects than the wild type virus. This is the first report showing that recombinant virus plasmid and corresponding recombinant AeDNV can be used as an effective in vitro and in vivo RNAi delivery system, respectively.

A Meta Analysis on Risks of Adverse Pregnancy Outcomes in Toxoplasma gondii Infection

Objective Quantified risks of congenital Toxoplasma gondii infection and abnormal pregnancy outcomes following primary maternal infection were evaluated with meta- analysis based on published studies. Methods The related literatures were searched in multiple literature databases regardless of languages. Odds ratio (OR) and 95% confidence interval (CI) were used to evaluate the risks of vertical transmission of Toxoplasma gondii and abnormal pregnancy outcomes following primary maternal infection with meta-analysis. Results 53 of the 2632 searched literatures were included in our analysis. The incidence of abnormal pregnancy outcomes in T. gondii infected pregnant women (infected group) was significantly higher than that in the uninfected pregnant women (control group) (OR = 5.10; 95% CI, 3.85–6.75). Toxoplasma gondii infection rate in the abnormal-pregnancy-outcome group was significantly higher than in the normal-pregnancy group (OR = 3.71; 95% CI, 3.31–4.15). The pooled rate of vertical transmission was 20% (95% CI, 15%–26%) in maternal infection of T. gondii. The incidences of vertical transmission in women who were infected in the first, second or third trimester of pregnancy were 5% (95%CI, 2%–16%), 13% (95%CI, 7%–23%), and 32% (95%CI, 24%–41%), respectively. The rates of vertical transmission in women who were treated with spiramycin-only, PSF (pyrimethamine + sulfadiazine + folinic acid) or PS (pyrimethamine + sulfadiazine) combined with spiramycin, or other untypical treatments were 13% (95%CI, 7%–22%), 13%(95%CI, 7%–25%), and 24%(95%CI, 18%–32%), respectively. Conclusions Toxoplasma gondii infection can result in adverse pregnancy outcomes in pregnant women. The pooled rate of vertical transmission was 20% in maternal infection and the incidences of vertical transmission increased in the first, second or third trimester of pregnancy. The pooled rates of transmission in groups treated with spiramycin-only, PSF or PS combined with spiramycin, or other untypical treatments were not significantly different.

Predictive Symptoms and Signs of Severe Dengue Disease for Patients with Dengue Fever: A Meta-Analysis

The aim of the meta-analysis was to provide more solid evidence for the reliability of the new classification. A systematic literature search was performed using PubMed, Armed Forces Pest Management Board Literature Retrieval System, and Google Scholar up to August 2012. A pooled odds ratio (OR) was calculated using either a random-effect or a fixed-effect model. A total of 16 papers were identified. Among the 11 factors studied, five symptoms demonstrated an increased risk for SDD, including bleeding [OR: 13.617; 95% confidence interval (CI): 3.281, 56.508], vomiting/nausea (OR: 1.692; 95% CI: 1.256, 2.280), abdominal pain (OR: 2.278; 95% CI: 1.631, 3.182), skin rashes (OR: 2.031; 95% CI: 1.269, 3.250), and hepatomegaly (OR: 4.751; 95% CI: 1.769, 12.570). Among the four bleeding-related symptoms including hematemesis, melena, gum bleeding, and epistaxis, only hematemesis (OR: 6.174; 95% CI: 2.66, 14.334; P < 0.001) and melena (OR: 10.351; 95% CI: 3.065, 34.956; P < 0.001) were significantly associated with SDD. No significant associations with SDD were found for gender, lethargy, retroorbital pain, diarrhea, or tourniquet test, whereas headache appeared protective (OR: 0.555; 95% CI: 0.455, 0.676). The meta-analysis suggests that bleeding (hematemesis/melena), vomiting/nausea, abdominal pain, skin rashes, and hepatomegaly may predict the development of SDD in patients with DF, while headache may predict otherwise.

Functional characterization of three MicroRNAs of the Asian Tiger Mosquito, Aedes albopictus

BackgroundTemporal and stage specific expression of microRNAs (miRNAs) in embryos, larvae, pupae and adults of Aedes albopictus showed differential expression levels across the four developmental stages, indicating their potential regulatory roles in mosquito development. The functional characterization of these miRNAs was not known. Accordingly our study evaluated the functional characterization of three miRNAs, which are temporally up-regulated in the various developmental stages of Ae. albopictus mosquitoes.MethodsmiRNA mimics, inhibitors and negative controls were designed and their knock-in and knock-down efficiency were analyzed by qRT-PCR after transfecting the mosquito cell lines C6/36, and also by injecting in their specific developmental stages. The functional role of each individual miRNA was analyzed with various parameters of development such as, hatching rate and hatching time in embryos, eclosion rate in larvae, longevity and fecundity in the adult mosquitoes.ResultsThe knock-in with the specifically designed miRNA mimics showed increased levels of expression of miRNA compared with their normal controls. We confirmed these findings using qRT-PCR, both by in vitro expression in C6/36 mosquito cell lines after transfection as well as in in vivo expression in developmental stages of mosquitoes by microinjection. The knock-down of expression with the corresponding inhibitors showed a considerable decrease in the expression levels of these miRNAs and obvious functional effects in Ae. albopictus development, detected by a decrease in the hatching rate of embryos and eclosion rate in larvae and a marked reduction in longevity and fecundity in adults.ConclusionThis study carried out by knock-in and knock-down of specifically and temporally expressed miRNAs in Ae. albopictus by microinjection is a novel study to delineate the importance of the miRNA expression in regulating mosquito development. The knock-down and loss of function of endogenously expressed miRNAs by the miRNA inhibitors in specific developmental stages had considerable effects on development, but enhancement of their gain of function was not observed on knock-in of these specific miRNAs. Hence, our study indicates that an optimal level of endogenous expression of miRNA is indispensable for the normal development and maintenance of the vectorial population density and pathogen transmissibility of this mosquito vector.

Juvenile hormone-activated phospholipase C pathway enhances transcriptional activation by the methoprene-tolerant protein

Significance Juvenile hormone (JH) controls many key processes during insect life cycles. Some of the effects of JH are mediated by a membrane-associated mechanism. In other circumstances, an intracellular JH receptor, methoprene-tolerant protein (MET), is activated upon binding of JH and directly regulates the expression of JH target genes. Here we use adult female mosquitoes as an example to demonstrate that both mechanisms are interconnected to coordinate hormonal responses. Our results indicate that the phospholipase C pathway is activated shortly after hormone exposure. Activation of this pathway induces phosphorylation of MET and profoundly increases the ability of MET to bind to JH target. This study establishes the link between the membrane-initiated JH signaling and the MET-mediated genomic action of JH. Juvenile hormone (JH) is a key regulator of a wide diversity of developmental and physiological events in insects. Although the intracellular JH receptor methoprene-tolerant protein (MET) functions in the nucleus as a transcriptional activator for specific JH-regulated genes, some JH responses are mediated by signaling pathways that are initiated by proteins associated with plasma membrane. It is unknown whether the JH-regulated gene expression depends on the membrane-mediated signal transduction. In Aedes aegypti mosquitoes, we found that JH activated the phospholipase C (PLC) pathway and quickly increased the levels of inositol 1,4,5-trisphosphate, diacylglycerol, and intracellular calcium, leading to activation and autophosphorylation of calcium/calmodulin-dependent protein kinase II (CaMKII). When abdomens from newly emerged mosquitoes were cultured in vitro, the JH-activated gene expression was repressed substantially if specific inhibitors of PLC or CaMKII were added to the medium together with JH. In newly emerged female mosquitoes, RNAi-mediated depletion of PLC or CaMKII considerably reduced the expression of JH-responsive genes, including the Krüppel homolog 1 gene (AaKr-h1) and the early trypsin gene (AaET). JH-induced loading of MET to the promoters of AaKr-h1 and AaET was weakened drastically when either PLC or CaMKII was inactivated in the cultured tissues. Therefore, the results suggest that the membrane-initiated signaling pathway modifies the DNA-binding activity of MET via phosphorylation and thus facilitates the genomic responses to JH. In summary, this study reveals an interplay of genomic and nongenomic signaling mechanisms of JH.

A Systematic Review and Meta-Analysis of the Efficacy of Anti-Toxoplasma gondii Medicines in Humans

No effective drug and definitive “gold standard” treatment for Toxoplasma gondii (T. gondii) infection has been available so far, though some medicines have been commonly used in the treatment of T. gondii infection, such as spiramycin, azithromycin, traditional Chinese medicine (TCM), pyrimethamine- sulfadiazine (P-S), trimethoprim-sulfamethoxazole (TMP-SMX), and pyrimethamine-clindamycin (P-C). A systematic review and meta-analysis were performed to compare the efficacies of these conventional medicines in the treatment. Cohort studies for the treatment of acute T. gondii infection were searched from PubMed, Google Scholar, ect. All the cases number for different group extracted from each included literature were input to meta-analysis 3.13 software to calculate the pooled negative conversion rate (NCR), cure rate (CR) or vertical transmission rate based on their sample size and weight. The pooled NCR with 95% confidence intervals (CI) was used to evaluate the overall rate of a diagnosis positive result conversion to a negative result after treatment, which of spiramycin, azithromycin and TCM were 83.4% (95%CI, 72.1%-90.8%), 82.5% (95%CI, 75.9%-87.6%), and 85.5% (95%CI, 71.3%-93.3%) respectively, with no statistical difference between them. The pooled CR with 95% CI was used to evaluate the overall rate of complete disappearance of clinical symptoms for toxoplasmic encephalitis after therapy, which of P-S, TMP-SMX, and P-C were 49.8% (95%CI, 38. 8% -60.8%), 59.9% (95%CI, 48.9%-70.0%), and 47.6% (95%CI, 24.8%-71.4%) respectively, with no statistical difference between them. Primary T. gondii infection in pregnancy was treated mainly with spiramycin alone or combined with other drugs, and the pooled rate of vertical transmission was about 9.9% (95%CI, 5.9%-16.2%) after therapy. Toxoplasmic encephalitis in AIDS patients was usually treated with sulfonamides combined with other drugs and the pooled CR was 49.4% (95%CI, 37.9%-60.9%).

Association between Liver Fluke Infection and Hepatobiliary Pathological Changes: A Systematic Review and Meta-Analysis

Objective To provide information about the role of liver fluke infection as a risk factor for hepatobiliary pathological changes and promote awareness among the people living in endemic areas, a systematic review and meta-analysis based on published studies was conducted to examine the association between liver fluke infection and hepatobiliary pathological changes. Methods Relevant original literature was searched in multiple literature databases, including PubMed, Cochrane, Clinical Evidence, Trip Database, Clinical Trials, Current Controlled Trials, Web of Science, the China National Knowledge Infrastructure (CNKI) database, and the Wanfang academic journal full-text database. Studies were selected based on strict screening with inclusion and exclusion criteria. Tests of heterogeneity, sensitivity and publication bias were performed with the Review Manager software, version 5.3, and meta-regression analyses were performed with the Stata software, version 11.0 (Stata Corporation, College Station, TX, USA). Pooled risk ratios (RRs) and odds ratios (ORs) with their 95% confidence intervals (95% CIs) were calculated and used to evaluate the risk of hepatobiliary pathological changes resulting from liver fluke infection. Linear trend analyses were conducted to determine the dose-response relationship using IBM SPSS Statistics 20.0. Result A total of 36 studies were included in the meta-analysis. Significant associations were found between liver fluke infection and cholangitis or cholecystitis (RR: 7.80, P<0.001; OR: 15.98, P<0.001), cholelithiasis (RR: 2.42, P = 0.03; OR: 4.96, P = 0.03), hepatocellular carcinoma (OR: 4.69, P<0.001) and cholangiocarcinoma (RR: 10.43, P<0.001; OR: 4.37, P<0.001). In addition, heavier infection was significantly associated with higher incidence of hepatobiliary pathological changes (P<0.05). However, cirrhosis was not significantly associated with liver fluke infection (RR: 3.50, P = 0.06; OR: 5.79, P = 0.08). The statistical heterogeneity was significant, no significant difference was observed in the sensitivity analysis, and no publication bias was found. Conclusion The meta-analysis found that liver fluke infection was significantly associated with cholangitis, cholecystitis, cholelithiasis, hepatocellular carcinoma and cholangiocarcinoma and that more severe infection was associated with higher incidence. However, the association between liver fluke infection and cirrhosis was not significant.

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.