Two mosquito strains were used in this study. Ae. albopictus Guangzhou strain was originally collected as larvae from Guangzhou city, Guangdong Province (GPS location: 23°07′ N and 113°16′ E), in 2019. Cx. pipiens pallens Beijing strain was originally collected as larvae from Beijing city (GPS location: 39°28′ N and 116°28′ E), in 2013. Both mosquito strains were reared under standard insectary conditions at 26 ± 1°C and 75 ± 5% relative humidity (RH) with a photoperiod of 14 h light: 10 h dark cycles. Prior to the infectious feed, adult mosquitoes were provided with 8% sucrose solution.

C6/36 (Ae. albopictus) and BHK-21 (baby hamster kidney) cells were maintained in our laboratory. C6/36 cells were cultured in RPMI 1640 medium (Gibco) supplemented with 10% fetal bovine serum (FBS) (Gibco) and 1% penicillin/streptomycin (Gibco) at 28°C in an incubator of 5% CO₂. BHK-21 cells were cultured in Dulbecco’s modified Eagle medium (DMEM) (Gibco) supplemented with 10% FBS and 1% penicillin/streptomycin at 37°C in an incubator of 5% CO₂.

The TAHV PJ01 strain isolate was originally isolated from Aedes sp. mosquitoes collected in Panjin city, Liaoning province (GPS location: 41°12′ N and 122°07′ E) in 2016, and used as a seed stock by passaging once in C6/36 cells and twice in BHK-21 cells. Then, the working stock was generated by inoculation into C6/36 cells, which was subsequently harvested and stored as individual 1.5 mL aliquots in freezing tubes at −80°C. The virus titers were 1.15 × 10⁷ plaque-forming unit (PFU)/mL as detected by plaque assay.

25–35 g female Kunming mice were used as a blood source and 4-day-old suckling Kunming mice were used as viral infection animal models in this study. All Kunming mice were purchased from Beijing VITAL RIVER Laboratory Animal Technology Co., LTD., and transferred to our laboratory and acclimated to the new environment for 24 h prior to the experiment to minimize the effects of transport.

Viral RNA of TAHV PJ01 strain was firstly extracted from virus stock by QIAamp Viral RNA Mini Kit (QIAGEN), and then reverse transcribed into cDNA using Eastep® RT Master Mix Kit (Promega) according to manufacturer’s instructions. The genome of TAHV consists of 3 segments: L, M and S. The middle region and the ends of segments were amplified by PCR and RACE (Rapid amplification of cDNA ends), respectively. The following reagents were used for PCR reactions: 2 μL of cDNA, 1 μL of forward primer, 1 μL of reverse primer, 10 μL of PrimeSTAR Buffer, 4 μL of dNTP Mixture, 0.5 μL of PrimeSTAR HS DNA Polymerase, and 31.5 μL of nuclease-free water to yield a 50 μL final reaction volume. Amplification reactions were performed in a PCR system (Biometra Tadvanced) programmed as follows: 30 cycles at 98°C for 10 s, 55°C for 5 s and 72°C for 60 s. To obtain the 3′ and 5′ ends sequence, a polyadenylated tail was added to the RNA genome by Poly(A) Polymerase (TaKaRa). Then, first-strand cDNA synthesis, RACE and In-Fusion cloning of RACE products were performed using SMARTer® RACE 5′/3’ Kit (TaKaRa) according to user manual. PCR and RACE products were sequenced at Shenzhen BGI Co., LTD (Shenzhen, China). Genome assembly was performed using SnapGene 2.3.2 (Insightful Science, USA). All primers used for amplification were listed in Supplementary Table 1.

The complete sequences of all three segments of 22 strains (except the MBV, which had only S segment sequence in the GenBank) belonging to the CSG in the genus Orthobunyavirus together with the representative members of the Bunyamwera serogroup (Bunyamwera virus) were used in the analysis (from GenBank as of 6 June 2022). Accession numbers of all the above strains are listed in Supplementary Table 2. Alignment of sequences was conducted by the ClustalW function in MEGA-X (Kumar et al., 2018). Phylogenetic analysis among nucleotide sequences was constructed by Neighbor-Joining method together with 1,000 replications bootstrap using MEGA-X. Pairwise distances were analyzed using SDTv1.2 (Muhire et al., 2014) and visualized by R package corrplot.

Virus-infected blood meals were prepared containing 1:1 mouse blood and TAHV PJ01 strain suspension supplemented with 3% FBS and 1% heparin sodium. Seven-day-old adult female mosquitoes that had been starved for 12 h were fed with this infected blood meal using a Hemotek membrane feeding system for Ae. albopictus and a saturated sponge for Cx. pipiens pallens. The infected blood meal was kept at 37°C during feeding. After 50 min of feeding, mosquitoes were cold-anesthetized, and blood-engorged mosquitoes were transferred and maintained in the standard conditions.

To evaluate the infection of the TAHV PJ01 strain in Ae. albopictus and Cx. pipiens pallens, 30 blood-engorged mosquitoes were, respectively, sampled at 2, 4, 6, 8, 10, 12, 16 and 18 day post exposure (dpe). The midguts, ovaries, salivary glands, and saliva of all sampled mosquitoes were dissected and collected carefully with sterile dissecting needles and individually transferred into 1.5-mL microtubes containing 1 mL of RNAiso Plus (TaKaRa). Then, organs were homogenized using 2.5 mm stainless steel grinding balls and an N▪9524R frozen grinder manufactured by Beijing HODER Technology Co., LTD. These samples were then used for RNA extraction and TAHV PJ01 strain detection via RT-qPCR.

To determine the ability of Ae. albopictus and Cx. pipiens pallens to horizontally transmit TAHV PJ01 strain, at 10 dpe, whole nests of 4-day-old suckling mice were placed in cages containing 50 mosquitoes that previously were fed with the virus-infected blood meals described above. 2 h after exposure, all the mosquitoes were removed from the cage and the whole bodies of newly blood-engorged mosquitoes were detected for virus infection by RT-qPCR. The suckling mice were also removed from the cage, counted red dots of mosquito bites and reared with their mother mouse. The suckling mice were then monitored daily. The healthy mice were sampled on days 4, 7 and 10 (2 mice per day) as routine sampling. The unhealthy mice were sampled immediately once found be sick or dead as additional sampling. For all of the above sampled mice, half of the brain was grinded in the DMEM and filtered to obtain supernatant for plaque assay to determine virus titer; The remaining portion of the brain was used for RNA extraction and TAHV PJ01 strain detection via RT-qPCR.

To determine the ability of Ae. albopictus to transovarial transmit TAHV PJ01 strain from parents to offspring, the eggs laid by infected mosquitoes during the first and second gonotrophic cycles were collected. In the first gonotrophic cycle, blood-engorged mosquitoes were allowed to oviposit at 6–10 dpe. The eggs were maintained in a desiccator at 26 ± 1°C and 90% RH with a photoperiod of 14 h light:10 h dark cycles for 7 days to permit embryonation, and were subsequently partially hatched. Eggs, hatched larvae, and emerged adults were tested for the presence of TAHV PJ01 strain by RT-qPCR. Then, parent female mosquitoes were fed with normal mouse blood at 10 dpe, and eggs from the second gonotrophic cycle were collected at 13–17 dpe. The eggs, larvae and adults of the second gonotrophic cycle were collected as above for TAHV PJ01 strain detection.

Total RNA from midguts, ovaries, salivary glands, saliva, eggs, hatched larvae and emerged adults was isolated using RNAiso Plus (TaKaRa), and from suckling mouse brain using RNeasy® Mini Kit (QIAGEN), according to the manufacturer’s instructions.

TAHV PJ01 strain was detected in these samples using GoTaq® Probe 1-Step RT-qPCR System (Promega). A pair of universal primers for CSG virus amplification (Lambert and Lanciotti, 2009) and a newly designed probe were used for detection and were listed in Supplementary Table 1. The following reagents were used for RT-qPCR reactions: 2 μL of RNA sample, 5 μL of GoTaq® Probe qPCR Master Mix, 0.2 μL of GoScript™ RT Mix, 0.5 μL forward primer, 0.5 μL reverse primer, 0.5 μL probe and 1.3 μL of nuclease-free water to yield a 10 μL final reaction volume. Amplification reactions were performed in a RT-qPCR system from LightCycler® 480 II (Roche) programmed as follows: 1 cycle at 45°C for 15 min, 95°C for 2 min, 40 cycles at 95°C for 15 s and 60°C for 30 s. Virus RNA (vRNA) copies were calculated by generating a standard curve using a recombinant plasmid containing virus segment insertion.

Virus titers in mouse brains were detected by plaque assay. The supernatants derived from the mouse brain virus suspensions were serially diluted and inoculated into BHK-21 cells cultured in a 6-well plate with 200 μL volumes. After 1 h incubation at 37°C of 5% CO₂, virus stocks were discarded and the cells were overlaid with 1:1 of 2% ultra-low gelling agarose (SIGMA) and high sugar-DMEM (2×) (GENOM). Cells were incubated at 37°C of 5% CO₂ and observed daily for cytopathic effect (CPE), after CPE emergence they were stained with a 2% crystal violet solution (2% crystal violet in 20% ethanol). The number of characteristic plaques was counted to represent the presence of the virus particles. The virus titers were expressed as PFU/mL.

The vector competence of Ae. albopictus and Cx. pipiens pallens mosquito populations were characterized via the following indicators: positive rates and vRNA copies of midguts, ovaries, salivary glands and saliva; Infection rate (IR), transmission rate (TR), dissemination rate (DR) and dissemination efficiency (DE); Infection and morbidity of suckling mice; Infection of offspring in the first and second gonotrophic cycle.

GraphPad Prism 8.0 (GraphPad Software, San Diego, CA, USA) was used for data processing and visualization. SPSS 26 software (IBM, Armonk, NY, USA) was used for statistical analysis. Fisher’s exact test was used to compare rates. The Least Significant Difference (LSD) and the Tamhane test were used to compare the means ± standard errors (SEs) of RNA copy across multiple groups when passing and failing the homogeneity of variance test, respectively. An independent sample t-test was used to compare the means ± SEs of RNA copy between the two groups. p < 0.05 was considered statistically significant.

The virus was originally isolated from a pool of Aedes sp. mosquitoes and its genome was sequenced via the next-generation sequencing technology. The complete sequences of the virus have been submitted to the GenBank database (Accession No.: Segments S: OP727994.1; Segments M: OP727995.1; Segments L: OP727996.1). Analysis of the assembled sequences via the BLAST tool revealed that the newly isolated virus was the most correlated to TAHV, which belongs to the CSG of the genus Orthobunyavirus, and it was subsequently named TAHV PJ01 strain in this study.

Together with the other members in the CSG of the genus Orthobunyavirus, the TAHV PJ01 strain was analyzed the phylogenetic relationship between the CSG based on all three segments, except the MBV, which only had complete CDSs for the S segments in GenBank. Bunyamwera virus, belonging to the Bunyamwera serogroup was used as the outgroup (Supplementary Table 2). The phylogenetic results of all three segments showed that the TAHV PJ01 strain belongs to the genus Orthobunyavirus and clusters with the members of CSG (Figures 1A–C). The overall topology of the phylogenetic trees obtained from the S and L segment sequences were similar, as the TAHV PJ01 strain was independent of the clade of TAHV isolated from Xinjiang Uygur Autonomous Region (highlighted in a light blue color in Figures 1A,C). However, for the M segment, the TAHV PJ01 strain was localized within the above clade (highlighted in a light blue color in Figure 1B).

The nucleotide identity analysis matrix of the selected viruses indicated the highest identity of the S segment whereas the lowest identity of the M segment in the CSG. The S, M, and L segments of TAHV PJ01 strain shared the highest sequence similarity with those of TAHV strain XJ0625 and XJ0710 (93.40%), TAHV strain XJ0625 (82.50%), and TAHV strain XJ0708 and XJ0710 (82.90%), respectively (Figures 1D–F).

Virus replication in the midguts, ovaries, salivary glands, and saliva of Ae. albopictus and Cx. pipiens pallens were, respectively, tested at 2, 4, 6, 8, 10, 12, 16 and 18 dpe. For Ae. albopictus, from 2 dpe onwards, TAHV PJ01 strain could be detected in the midguts, ovaries and salivary glands on every sampling day (Figures 2A–E), and could be detected in saliva on all sampling days except 4 and 8 dpe (Figures 2A,D,F). Positive rate of midguts and ovaries remained high throughout the sampling days and peaked at 96.7 and 90.0% at 6 dpe and 6/16 dpe, respectively. Positive rate of salivary glands and saliva were generally maintained in a fluctuating upward trend, reaching 60.0 and 30.0% at 16/18 dpe and 18 dpe (Figure 2A). The vRNA copies of positive samples were calculated and showed a pattern of: ovary > midgut > salivary gland > saliva, with a maximum average vRNA copy of 5.91 ± 0.30 lg RNA copies/μL at 6 dpe, 5.40 ± 0.25 lg RNA copies/μL at 6 dpe, 4.71 ± 0.28 lg RNA copies/μL at 12 dpe and 3.75 ± 0.25 lg RNA copies/μL at 18 dpe, respectively (Figures 2B–F).

Compared with Ae. albopictus, Cx. pipiens pallens showed significant differences in TAHV PJ01 strain infection, with general downward trends of virus positive rates and all of them were less than 20.0% (Figure 2G). No virus was present in saliva on all sampling days, and by 16 dpe, no sample was virus-positive except 3.3% of the midguts (Figure 2G). Maximum vRNA copy of the few infected mosquitoes was 5.44 lg RNA copies/μL in ovaries at 14 dpe, 4.92 lg RNA copies/μL in midguts at 14 dpe, and 4.56 lg RNA copies/μL in salivary glands at 10 dpe (Figures 2H–L). The IR, TR, DR and DE of Ae. albopictus and Cx. pipiens pallens were also evaluated and were shown in Supplementary Table 3.

Horizontal transmission experiments of the TAHV PJ01 strain from mosquitoes to mice were performed. Positive results of virus detection in the mice brain means that the virus breaks through the blood–brain barrier to replicate in the suckling mice brain. In this study, more than one red blotch was present on the skin of all tested suckling mice after removal from the cage, which indicated that all tested suckling mice were bitten by at least one mosquito. Among the 50 mosquitoes used in each group, 37 Ae. albopictus were blood-engorged and positive with a vRNA copy of 6.11 ± 0.30 lg RNA copies/μL, 35 Cx. pipiens pallens were blood-engorged and 29 of them were positive with vRNA copy of 3.00 ± 0.24 lg RNA copies/μL.

There are significant differences between the two mosquito species in their ability to horizontally transmit TAHV PJ01 strain. Among 11 sucking mice that were bitten by Ae. albopictus, three (27.3%) developed obvious neurological symptoms, including inactivity, hind-leg paralysis and difficulty turning over, at day 5 (Figure 3C). One of these three (33.3%) diseased mice died within 24 h. Those 3 mice were sampled on day 5 and the other 8 were routinely sampled on days 4, 7 and 10 post-bite (2 mice per day and 2 additional on day 10). vRNA copies and virus titer in the brain of all the above mice were, respectively, detected via RT-qPCR and plaque assay. Results showed that nine mice, including those 2 diseased and 1 dead, 9 of the eleven (81.8%) suckling mice had vRNA in their brains within 10 days of observation (Figure 3A). Six viral RNA positive mice that exhibited no symptoms had an average vRNA copies of 3.83 ± 0.20 lg RNA copies/μL and no live virus, however there was no significant difference in the average vRNA copy among the different sampling days (p = 0.634). Two diseased mice had an average vRNA copies of 8.64 ± 0.19 lg RNA copies/μL, and their virus titers were 1.70 × 10⁵ PFU/mL and 2.20 × 10⁵ PFU/mL, respectively. The dead mouse had the highest vRNA copies of 9.09 lg RNA copies/μL accompanied by the highest virus titers of 1.10 × 10⁶ PFU/mL (Figures 3A,B). However, after being bitten by Cx. pipiens pallens, only one suckling mouse (12.5%) routinely sampled on day 10 had vRNA in its brain at a copy concentration of 2.32 lg RNA copies/μL (Figure 3A). No live virus was present and no illness or death were observed in this mouse within 10 days of observation.

As Ae. albopictus exhibited more susceptibility to the TAHV than Cx. pipiens pallens, offspring of the infected Ae. albopictus mosquitoes in the different development stages (eggs, larvae and adults) were detected to demonstrate vertical transmission. A total of 1,500 F1 progeny in 90 pools in the first gonotrophic cycle and 750 F1 progeny in 48 pools in the second gonotrophic cycle were tested (Table 1). In the first gonotrophic cycle, the IR of eggs was 96.7% with a vRNA copy of 3.12 ± 0.17 lg RNA copies/μL. From the egg stage to the larvae stage, both IR and vRNA copies decreased significantly (P<0.001), reaching 30.0% and 1.52 ± 0.05 lg RNA copies/μL, and then steadily decreased to 13.3% and 1.48 ± 0.03 lg RNA copies/μL at the adult stage. In the second gonotrophic cycle, IR, MIR (minimum infection rate) and RNA copy all showed decreasing trends from the egg to the adult stage similar to those in the first gonotrophic cycle. At the adult stage, the IR was 13.3%, the MIR was 1.3%, and the vRNA copy was 1.43 ± 0.04 lg RNA copies/μL. However, there were no significant differences in the same stage between different gonotrophic cycles, except that vRNA copies of positive eggs and larvae in the second gonotrophic cycle were significantly higher than those in the first gonotrophic cycle (P<0.01). The fact that the TAHV PJ01 strain can be detected in adults of F1 progeny demonstrated that the TAHV PJ01 strain can be vertically transmitted from parents to offspring in Ae. albopictus.