Identification of MDV presence using open access sequencing data

Metagenomic next-generation sequencing (NGS) data for field mosquitoes in China were searched from the NCBI SRA database (https://www.ncbi.nlm.nih.gov/sra) and CNGB Sequence Archive database (https://db.cngb.org/cnsa/) using the keywords “mosquito OR Anopheles OR Aedes OR Culex”. Followed by removing the datasets with less than 1 million reads and keeping those derived from the field in China, a total of 29 published datasets from five provincial level administrative divisions (PLADs) were retrieved, including four from Guangdong (SRP188743), three from Yunnan (SRP148705), eleven from Ningxia, five from Gansu, and six from Shanxi (CNP0001261) (details in Additional file 1: Table S1).

Twenty-six mosquito densovirus (MDV) genomes were downloaded as the reference from GenBank using the search keywords “densovirus” AND “(mosquito OR aAnopheles OR aAedes OR Culex)”, with sequence length limited to longer than 3500 bp (see Additional file 2: Table S2 for the MDV accession number).

Adaptor and low-quality bases were removed with TrimGalore (v0.6.7) [19]. The remaining clean reads were aligned to the genomes of Ae. albopictus (AaloF1.2, GCA_001444175.1), Cx. quinquefasciatus (JHB2020, GCA_015732765.1), and An. sinensis (AsinS2.6, GCA_000472065.2) by bowtie2 (v2.4.4) [20]. All these genomes were downloaded from Vectorbase (http://www.vectorbase.org). Reads mapping to host genomes were removed to exclude interference from the host genome and possible insertion of viral sequences, and unmapped reads were aligned to the MDV reference using blatsn (v2.4.4, NIH, USA) with the parameter “evalue < 1e−10”. The mapped reads were retained as MDV-related reads. The abundance of MDV reads was divided by the library counts and multiplied by a million normalized to CPM.

Mosquito collection

Guangzhou is the third largest city in China and the largest in the southern part of the country. The population of Guangzhou is estimated to be over 13,900,000 in 2022. The region around Guangzhou is known for a massive influx of migrants, with up to 30 million additional migrants living in the area for at least six months out of every year. The average annual temperature in Guangzhou is 22.4 °C, and the rainfall is approximately 2123 mm per year. Therefore, the environmental and social factors in Guangzhou facilitate DENV transmission, and DENV epidemics have been reported in this region for more than 40 years [21]. To investigate the general prevalence of MDVs in Guangzhou natural Ae. albopictus populations, three sites were selected for mosquito collections, the residential district of Southern Medical University (SMU) (113°19′E, 23°11′N, 31 m above sea level (a.s.l.), population density of > 3000 people/km2), Liangtian (113°23′E, 23°21′N, 25 m a.s.l.; population density of approximately 1000 people/km2), and Dengcun (113°339′E, 23°309′N, 42 m a.s.l., 100 people/km2), representing three different types of ecological habitats: urban, suburban and rural. Mosquitoes were collected between June and July 2022 from three sites in Guangzhou (Fig. 1): SMU, Dengcun, and Liangtian. The mosquitoes were collected by using human-baited double-net (HDN) traps. After placing on an ice bath, only Ae. albopictus female mosquitoes based on morphological characteristics were retained for further MDV infection detection.

Fig. 1

Analysis of mosquito densovirus (MDV) positive rates in natural mosquito populations. A Stacked column chart showing the mosquito densovirus positive rates (red) and negative rates (blue) of reads in field mosquito metagenomic data from five provincial level administrative divisions. B MDV positive rates (red) and negative rates (blue) in natural mosquito populations collected from three sites in Guangzhou. SMU The residential district of Southern medical university

MDV presence in natural populations

DNA was extracted from individual female mosquitoes collected from natural populations from the 3 locations (n = 100 per site) and tested for MDV presence by traditional PCR (Additional file 3: Table S3). MDV-free samples from the laboratory colony were used as the negative control. A set of primers specific to MDVs was designed to amplify a 655 bp fragment (from position 899 to 1554 of the referenced AaeDV genome; GenBank accession number: M37899.1) located in the highly conserved NS1 open reading frame (ORF). This primer set was able to detect not only AaeDV, but also other related densoviruses, such as Aedes albopictus densovirus (AalDV), Anopheles gambiae densovirus (AgDV), Culex pipiens densovirus (CpDV), based on the high level of conservation in this region among MDVs (Fig. 1, Additional file 4: Fig. S1). Ribosomal protein S7 (Rps7) was used as an internal control and was amplified in the same test tube based on multiplex PCR (MPCR). See Additional file 3: Table S3 for primers and PCR protocols. All experiments were performed in a biosafety level 2 containment laboratory.

Cells and mosquitoes

Ae. albopictus C6/36 cells (ATCC, Manassas, USA, Cat# CRL-1660, RRID: CVCL_Z230) were grown at 28 °C in Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco BRL, NY, USA) supplemented with 10% fetal bovine serum (FBS; Gibco BRL, NY, USA) and maintained at 28 °C.

Ae. albopictus mosquitoes of Foshan strain were used in this study. The mosquitoes were reared under standard insectary conditions at a constant 28 ± 2 °C, 70–80% relative humidity, and a 12:12-h light/dark photoperiod. Larvae were reared in 26.5 cm by 15.5 cm stainless steel rectangular pans and fed commercial finely ground turtle food (INCH-GOLD, Shenzhen, China). Colony adults were maintained in stainless steel cages [20 (length) × 20 (width) × 30 (height) cm, volume = 12,000 cm3] and supplied with a cotton wick soaked in 10% sugar solution. Adult females were fed defibrinate sheep blood (Solarbio Life Sciences, Beijing, China) using Hemotek membrane feeding systems (Hemotek Ltd., Blackburn, UK) three and four days after emergence. Every batch of mosquitoes was examined by conventional PCR to ensure that the experimental mosquitoes were free of MDVs.

To establish the AaeDV persistently infected cell line, C6/36 cells were infected with AaeDV at a final concentration of 1011 copies/ml; the infected cells were serially passaged every two days after three days of inoculation with AaeDV. The persistently infected C6/36 cells were serially passaged ten times after inoculation with AaeDV and used for further study.

Virus

pUCA is the infectious clone of AaeDV containing the full-length genomic DNA of AaeDV, which was kindly provided by Prof. Erica Suchman and Jonathan Carlson. The construction of plasmids has been previously described in detail [22]. The wildtype virus AaeDV was generated by transfecting pUCA into C6/36 cell lines according to a previously described method [22].

The dengue virus serotype 2 (DENV-2) New Guinea C strain (NGC) used throughout this study was maintained in the laboratory of the Guangdong Provincial Key Laboratory of Tropical Disease Research, School of Public Health, Southern Medical University. DENV-2 stocks were obtained by inoculating a nearly confluent monolayer of C6/36 cells in a 25 cm2 tissue culture flask, with the virus diluted to 1:6 in 3 ml RPMI-1640 medium containing 2% heat-inactivated fetal bovine serum (FBS; Gibco BRL, NY, USA). Supernatants from infected cells were collected three days after infection until an apparent cytopathic effect, centrifuged at 1500×g for 5 min, separated into 0.5 ml aliquots, and stored at − 80 °C. Viral titers are expressed as a 50% tissue culture infective dose (TCID50/ml), as based on the method described by Reed and Muench [23].

AaeDV and DENV-2 quantification

The AaeDV genome copy number was quantified using real-time quantitative PCR (qPCR). In brief, nonencapsidated genomic DNA was removed by treatment with TURBO DNase (Ambion, Austin, TX, USA) at 37 °C for 1 h. Total encapsidated genomic DNA was extracted using a Viral DNA Kit (Omega Bio-Tek, GA, USA). A standard curve was constructed using serial tenfold dilutions of a linear plasmid at known concentrations. The details of these procedures were described previously [24]. Virus genome copy numbers were determined using a SYBR green-based qPCR assay with SuperReal qPCR PreMix (Tiangen Biotech, Beijing, China). The primer sequences and reaction conditions used were as described previously [24].

DENV-2 RNA was quantified by real-time RT-PCR (qRT-PCR) using previously described specific primers according to published reaction conditions [25]. A 1995-bp DNA fragment, amplified using DENV2-F/DENV2-R primers, was ligated into pMD18-T plasmid after linearization with EcoRI restriction enzyme (Thermo Fisher Scientific, MA, USA) (Additional file 3: Table S3). A standard curve for DENV-2 was generated using a serial tenfold dilution of the linear plasmid containing a fragment of DENV-2 spanning nucleotides 9937–10,113 according to published procedures [25]. Each sample was analyzed in triplicate. After the specificity of PCR products was checked by the melt curve, viral genome copy numbers were determined by absolute quantification based on cycle threshold (Ct) values and standard curves.

Acute DENV-2 infection

AaeDV infected or uninfected C6/36 cells (106) were preseeded in a 12-well plate 1 day before DENV-2 infection. The C6/36 cell monolayers were washed with PBS and infected with DENV-2 at an MOI of 1. The plates were rocked gently for 15 min at room temperature for adsorption and incubated at 37 °C for 2 h. Then, individual wells of C6/36 cells were washed three times with PBS, and fresh medium was added. The infection was proceeded at 28 ℃ for 12, 24, 48, 72, 96, and 120 h, and was confirmed by immunofluorescence assay and qRT-PCR.

Antibodies and immunofluorescence assay

The details of the production of a rabbit polyclonal antibody against AaeDV NS1 have been described [6]. Mouse monoclonal anti-DENV virus E2 envelope glycoprotein antibodies were purchased from Abcam (Cambridge, MA, USA). The secondary antibodies used, donkey anti-rabbit IgG-Alexa Fluor-594 and goat anti-mouse IgG-Alexa Fluor-488, were purchased from Thermo Fisher Scientific (MA, USA).

C6/36 cells were grown on glass coverslips, washed with phosphate-buffered saline (PBS), fixed using an acetone/absolute alcohol (3:2) mixture, blocked with 1% BSA + 0.05% PBST solution for 2 h at 37 °C, and washed with PBST. Following incubation with the primary antibodies, the coverslips were washed with PBS and stained with Alexa Fluor 594-conjugated donkey anti-rabbit IgG and/or Alexa Fluor 488 Hoechst-conjugated goat anti-mouse stain for 1 h at room temperature. DAPI was used for nuclear staining. Images were visualized under a 100 × oil objective using a FluoView 1000 confocal microscope (Olympus, Tokyo, Japan). Analysis of images and calculation of positive signals of immunofluorescence were performed using ImageJ software (1.49v, NIH, USA) [26].

Mosquito injections with AaeDV

Two-day-old Ae. albopictus female adults were infected with AaeDV via intrathoracic injection. Briefly, adults were anesthetized on ice, and approximately 69 nl of AaeDV stock (1011 GE/ml) was injected into the thorax under a microscope, as described previously [27, 28]. After injection, the adult mosquitoes were immediately transferred to small plastic cups (1000 ml, 11 cm diameter at the top), fed a 10% glucose solution through soaked cotton wicks and allowed to recover. Mosquitos were injected with RPMI 1640 medium only as mock-infected controls. Three independent biological replicates were included for each treatment (n = 30 per replicate).

Superinfection interference in vitro and in vivo

To examine interference between DENV-2 and AaeDV in Aedes mosquito cells, stable and persistent AaeDV-infected C6/36 cells were superinfected with DENV at an MOI of 1. Genome copies of DENV-2 and AaeDV were measured in cell lysates and supernatant by qPCR, and the percentage of DENV-2- and AaeDV-positive/negative cells was calculated at 12, 24, 48, 72, 96, and 120 h post superinfection (hpi). To determine the effect of superinfection on the percentage of virus-positive C6/36 cells, we performed immunofluorescence microscopy in monoinfected and superinfected C6/36 cells, and quantified the fluorescence intensity using ImageJ software (1.49v, NIH, USA) [26].

Injected mosquitoes were allowed to recover under standard rearing conditions. Four days after injection, Ae. albopictus female mosquitoes were exposed for 2 h to infectious blood meals using a blood-soaked pledget technique in three independent replicates [29]. Each blood meal comprised fresh virus diluted in commercially available defibrinated sheep blood and 1% sucrose to provide a final viral titer of approximately 107 TCID50/ml of DENV-2. After feeding, fully engorged females were selected and returned to standard rearing. To evaluate the influences of AaeDV infection on the susceptibility of Ae. albopictus to DENV infection, adult female mosquitoes were first infected with AaeDV [106 genome equivalents (geq)/per mosquito] via thoracic injection and then artificially fed with a mixture of blood with approximately 109 RNA copies/ml of DENV, as described in the methods section. The IR, DR, and SGIR were evaluated, and a monoinfected adult was used as a control. The workflow is shown in Fig. 5A and B.

Vector susceptibility experiments

At 0, 5, 10, and 15 days post DENV-2 infection (dpi), surviving mosquitoes were anesthetized with CO2 and dissected on a precooled glass slide for vector susceptibility assays. Disposable insect microneedles were used to separate the midgut, ovaries, and salivary glands of each mosquito under a dissecting microscope. The samples were washed three times in PBS and then homogenized by using a Motor-Driven Tissue Grinder prior to TRIzol addition, and viral nucleic acid was extracted using MiniBEST Viral RNA/DNA Extraction Kit Ver. 5.0 (TaKaRa, Japan) following the manufacturer's protocol. First-strand cDNA synthesis and RT-PCR amplification were performed according to methods that have been described in detail previously [30]. The primer sequences and PCR conditions used are listed in Additional file 3: Table S3.

Vector susceptibility of Ae. albopictus to DENV-2 was evaluated by calculating the midgut infection rate (MIR; the number of infected midguts/the number of tested midguts), dissemination rate (DR; the number of infected ovaries/the number of infected midguts), and salivary gland infection rate (SGIR; the number of infected salivary glands/the number of tested mosquitoes) [31, 32]. Genome copies of AaeDV were quantified in all midguts, ovaries, and salivary gland and genome copies were further determined in DENV2-positive tissues by absolute qRT-PCR.

Data analysis

In this study, logistic regression was used to examine the vector competence of Ae. albopictus for DENV-2. IR, DR, and SGIR were compared separately at different time points, and the P value was corrected by Bonferroni adjustments. Chi-square (and Fisher’s exact) tests were used to determine the difference in vector competence between the AaeDV-infected group and the negative group. Tukey's LSD tests of variance were performed post hoc on the DENV-2 levels in tissues for each group after log transformation. Student's t test was used at each time point to compare DENV-2 RNA copies between the AaeDV-infected and negative groups. A P value < 0.05 was considered statistically significant (*), with a P value < 0.01 representing strong statistical significance (**). Statistical analyses were conducted using SPSS 20.0 (IBM, Chicago, IL, United States).