The midgut is the first barrier to dengue virus (DENV) infections of mosquitoes and therefore is a major bottleneck for the subsequent development of vector competence. However, the molecular mechanisms responsible for this barrier are unknown. We constructed three small RNA libraries from the midguts of adult Aedes albopictus females that had been fed on either sugar solution, an uninfected blood meal, or a blood meal infected with DENV-2, and112 conserved microRNAs represented by 173 miRNA sequences were identified, with 34 novel microRNAs predicted by Mireap, RNAfold and Sfold software. In addition, the expression of aal-miR-1174, aal-miR-2951 and aal-miR-956 was confirmed via stem-loop quantitative real-time PCR (qRT-PCR). Compared with microRNA expression profiles of mosquitoes that had ingested a regular blood meal, 43 microRNAs were upregulated and 4were downregulated in mosquitoes that had ingested a DENV-2-infected blood meal. Among the differentially expressed microRNAs, miR-1767, miR-276-3p, miR-4448 and miR-4728-5p were verified via stem-loop qRT-PCR. Analyses indicated that the changing patterns in miRNA expression during DENV-2 infection were significant and varied at different time points post infection. Most miRNA were upregulated at 24 h but were downregulated at 48 h post DENV-2 intake. The aal-miR-4728-5p was chosen for an in vitro transient transfection assay, and the results show that this miRNA enhances DENV replication in C6/36 cells. This study provides the first information on microRNAs expressed in the midgut of Ae. albopictus and describes species-specific changes in their expression levels following infection by DENV-2.

The mosquito Aedes albopictus is an important vector for dengue virus (DENV) transmission. The midgut is the first barrier to mosquito infection by DENV, and this barrier is a critical factor affecting the vector competence of the mosquito. However, the molecular mechanism of the interaction between midgut and virus is unknown. Six small libraries of Ae. albopictus midgut RNAs were constructed, three of which from mosquitoes that were infected with DENV-2 after feeding on infected blood, and another three that remained uninfected with DENV-2 after feeding on same batch of infected blood. A total of 46 differentially expressed miRNAs were identified of which 17 significant differentially expressed miRNAs were selected. Compared to microRNA expression profiles of mosquitoes that were uninfected with DENV-2, 15 microRNAs were upregulated and two were downregulated in mosquitoes that were infected with DENV-2. Among these differentially expressed microRNAs, miR-1767, miR-276-3p, miR-4448 and miR-622 were verified by stem-loop qRT-PCR in samples from seven-day-infected and uninfected midguts and chosen for an in vitro transient transfection assay. miR-1767 and miR-276-3p enhanced dengue virus replication in C6/36 cells, and miR-4448 reduced dengue virus replication. To our knowledge, this study is the first to reveal differences in expression levels between mosquitoes infected and uninfected with DENV-2 after feeding on an infected blood meal. It provides useful information on microRNAs expressed in the midgut of Aedes albopictus after exposure to the virus.

As an important component of the causative agent of respiratory tract infections, enteric and eye infections, Human mastadenoviruses (HAdVs) species B spread easily in the crowd. In this study, we developed a recombinase polymerase amplification (RPA) assay for rapidly detecting HAdVs species B which was comprised of two different formats (real-time and lateral-flow device). This assay was confirmed to be able to detect 5 different HAdVs species B subtypes (HAdV-B3, HAdV-B7, HAdV-B11, HAdV-B14 and HAdV-B55) without cross-reactions with other subtypes and other respiratory tract pathogens. This RPA assay has not only highly sensitivity with low detection limit of 50 copies per reaction but also short reaction time (< 15 min per detection). Furthermore, the real-time RPA assay has excellent correlation with real-time PCR assay for detection of HAdVs species B presented in clinical samples. Thus, the RPA assay developed in this study provides an effective and portable approach for the rapid detection of HAdVs species B.

Pesticide resistance due to sodium channel point mutations has been well documented in many mosquito species. We tested the resistance of six, wild, Chinese populations of the mosquito Culex pipiens quinquefasciatus to deltamethrin and cyhalothrin. The full length of the sodium channel gene was cloned and sequenced from a pooled sample of mosquitoes from each population. Seven amino acid alterations were found (V250M, R436K, M943V, I973T, L1035F, L1035S and E1901D). Correlation between the frequencies of these mutations and the level of pesticide resistance (LC50) associated with them indicates that those at position L1035 (corresponding to position L1014F in the house fly, Musca domestica; GenBank Accession No.: X96668) are associated with resistance to deltamethrin and cyhalothrin. The frequency of the L1035F mutation was significantly correlated with resistance to deltamethrin (R2 = 0.536, P = 0.049) and cyhalothrin (R2 = 0.626, P = 0.030), and the combined frequency of the L1035F and L1035S mutations was significantly correlated with resistance to both deltamethrin (R2 = 0.661, P = 0.025), and cyhalothrin (R2 = 0.803, P = 0.008). None of the other mutations were correlated with either deltamethrin or cyhalothrin resistance. Interestingly, an HWE test indicated significant linkage between the M943V and I973T mutations (P < 0.01), but further research is required to determine the biological significance of this linkage. Identifying these mutations may be of practical benefit to the development of pesticide resistance management programs.