Vector competence and immune response of Aedes aegypti for Ebinur Lake virus, a newly classified mosquito-borne orthobunyavirus.

The global impact of mosquito-borne diseases has increased significantly over recent decades. Ebinur Lake virus (EBIV), a newly classified orthobunyavirus, is reported to be highly pathogenic in adult mice. The evaluation of vector competence is essential for predicting the arbovirus transmission risk. Here, Aedes aegypti was applied to evaluate EBIV infection and dissemination in mosquitos. Our experiments indicated that Ae. aegypti had the possibility to spread EBIV (with a transmission rate of up to 11.8% at 14 days post-infection) through biting, with the highest viral dose in a single mosquito's saliva reaching 6.3 plaque-forming units. The highest infection, dissemination and ovary infection rates were 70%, 42.9%, and 29.4%, respectively. The high viral infection rates in Ae. aegypti ovaries imply the possibility of EBIV vertical transmission. Ae. aegypti was highly susceptible to intrathoracic infection and the saliva-positive rate reached 90% at 10 days post-infection. Transcriptomic analysis revealed Toll and Imd signaling pathways were implicated in the mosquito's defensive response to EBIV infection. Defensin C and chitinase 10 were continuously downregulated in mosquitoes infected via intrathoracic inoculation of EBIV. Comprehensive analysis of the vector competence of Ae. aegypti for EBIV in laboratory has indicated the potential risk of EBIV transmission through mosquitoes. Moreover, our findings support a complex interplay between EBIV and the immune system of mosquito, which could affect its vector competence.

1 Introduction

Mosquito-borne viruses (MBV), a group of heterogeneous RNA viruses, naturally replicate in both mosquitoes and vertebrate hosts and are the etiological agents of several human and animal diseases [1]. The clinically important MBVs are primarily distributed into four families: Flaviviridae (dengue viruses 1–4 (DENV), Zika virus (ZIKV), West Nile virus, Japanese encephalitis virus), Togaviridae (Chikungunya virus), Reoviridae (Yunnan orbivirus) and Peribunyavividae (Bunyamwera virus (BUNV)) [2-4]. Over the past few decades, the extensive global spread of arbovirus has been an issue of significant concern. In view of the dramatic emergence and unprecedented rapid spread of epidemic arboviral diseases, strong surveillance and risk assessments are necessary [5,6].

The genus Orthobunyavirus belonging to the family Peribunyaviridae (order Bunyavirales) contains numerous mosquito-borne bunyaviruses [7]. So far, orthobunyaviruses have been detected in various of mosquito species, including Ochlerotatus spp., Culex spp. and Aedes spp., and midges, such as Culicoides paraenesis, a vector of Oropouche virus (OROV) in South America [8-10]. A number of viruses in this genus cause severe human diseases, for instance, acute but self-limiting febrile illness (OROV), encephalitis (OROV and La Crosse virus (LACV)) and hemorrhagic fever (Ngari virus) [11,12]. Widespread epidemic distribution of orthobunyaviruses within the human population is reported, such as BUNV, which is endemic in many African countries as well as North America, and Batai virus (BATV) that is geographically prevalent in Europe [12]. In view of the global expansion of mosquito vectors, these viruses pose a considerable threat to human and animal health as well as food security.

Ebinur Lake virus (EBIV), a newly identified orthobunyavirus in China, was originally isolated from Culex modestus mosquito pools in Xinjiang Province [13]. The whole genome sequence of EBIV shares the greatest similarity with Germiston virus originating from South Africa [14]. EBIV has been shown to efficiently infect cell lines derived from rodent, avian, non-human primate, mosquito and human subjects [15]. Additionally, EBIV induces encephalopathy, hepatic and immunological system damage with a high mortality index in experimentally infected BALB/c mice [16]. While no confirmed human cases of EBIV have been recorded, IgM and/or IgG positive for EBIV were identified from several serum samples and two neutralizing antibody-positive cases detected via the plaque reduction neutralization test assay in an earlier study by Xia et al. [15].

Aedes aegypti is an important vector of MBVs [17]. Originally, Ae. aegypti was mainly distributed in Africa and Southeast Asia but has since colonized almost all other continents [18]. The distribution of Ae. aegypti is continuously expanding, with a further ~20% increase estimated by the end of this century [19]. Assessment of the vector competence of Ae. aegypti for transmission of newly emerging viruses is therefore critical for management of infection rates. And Ae. aegypti has been identified as a vector for many orthobunyaviruses, such as BUNV, LACV, and Cache Valley virus [12,20-22]. In the current study, we evaluated EBIV infection rates of Ae. Aegypti though both blood-feeding and intrathoracic injection routes. Transcriptome analysis of intrathoracically infected Ae. aegypti was additionally performed to explore the innate immune response of mosquito. The collective results provide valuable insights into the interactions between orthobunyaviruses and mosquito vectors that should aid in assessing the potential risk of EBIV.

2 Materials and methods

2.1 Viruses and cell lines

BHK cells were maintained in Dulbecco’s modified Eagle’s medium (Gibco) containing 10% fetal bovine serum (Bio-One) under 37°C and 5% CO2. Aedes albopictus C6/36 cells were grown in Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco) supplemented with 10% FBS (Gibco) at 28°C and 5% CO2.

EBIV (strain Cu20-XJ) was stored in our laboratory. The titer for the working stock of EBIV (propagated in BHK-21 cells) was 2.4 × 107 plaque-forming units mL−1 (PFU mL−1). Viral titers were determined using the plaque formation assay [23].

2.2 Mosquito rearing

Eggs of Ae. aegypti (Rockefeller strain) were acquired from the Laboratory of Tropical Veterinary Medicine and Vector Biology at Hainan University. Eggs and larvae of Ae. aegypti were maintained in a mosquito room under the following conditions: 28°C under a light:dark cycle of 12:12 h and relative humidity of 75% ± 5%. Emerged adult mosquitoes were fed an 8% glucose solution and maintained in mesh cages (30 × 30 × 30 cm) within incubators at 28 ± 1°C under relative humidity of 80% and light:dark cycle of 12:12 h. Mosquitoes were reared in an arthropod containment level 1 (ACL-1) laboratory.

2.3 Oral infection and intrathoracic inoculation

Prior to oral infection and intrathoracic inoculation, female mosquitoes (5 to 8 day-old) were starved for 24 h. The mosquito infection works were performed in an ACL-2 laboratory.

2.3.1 Infection via blood feeding

A virus-blood mixture (the defibrinate horse blood and virus ratio of 1:1) was used to feed female Ae. aegypti through an artificial mosquito feeding system (Hemotek). Fully engorged female mosquitoes (n ≥ 30) were transferred into new containers and maintained in an incubator at 28°C and humidity of 80% for 4 to 14 days until experimental use.

In a previous study by our group, following infection of adult female BALB/c mice with 10 PFU of EBIV, viremia reached 106 PFU ml-1 at 2 dpi [16]. Based on this finding, female mosquitoes fed a virus-blood mixture (final virus concentration of 3.7 × 106 PFU ml−1) were used to evaluate whether EBIV could effectively infect Ae. aegypti. Infected mosquitoes were subsequently collected at 4, 7, 10 and 14 dpi for viral RNA determination.

To establish the minimum concentration of EBIV in Ae. aegypti, female mosquitoes were fed different virus-blood concentrations (six serial titers ranging from 102 to 107 PFU ml−1). Infected mosquitoes were subsequently collected at 4 and 10 dpi for viral RNA determination.

To determine EBIV distribution in infected mosquitoes via artificial blood feeding (final viral titer of 6.4 × 106 PFU ml−1), the presence of viral RNA in saliva, head, gut, and ovary of mosquitoes at 4, 7, 10 and 14 dpi was examined. For collection of mosquito saliva, a previously reported protocol was employed [24]. Firstly, the wings and legs of mosquitoes were cut off, following which mouthparts were inserted into pipette tips filled with immersion oil. Mosquitoes secreted saliva into the oil for a 45–60 min period at room temperature. Different tissues examined under a dissecting microscope.

Four parameters were calculated, specifically, infection rate (%) = 100 × (number of mosquitoes with virus-positive bodies or midguts/number of total mosquitoes), dissemination rate (%) = 100 × (number of mosquitoes with virus-positive heads/number of mosquitoes with virus-positive bodies or midguts), transmission rate (%) = 100 × (number of mosquitoes with virus-positive saliva/number of mosquitoes with virus-positive bodies or midguts) and ovary infection rate (%) = 100 × (number of mosquitoes with virus-positive ovaries/number of mosquitoes with virus-positive midguts).

2.3.2 Infection via intrathoracic injection

Intrathoracic injection was performed as follows: mosquitoes were anesthetized at -20°C for 1 min. Female mosquitoes were selected and placed on an ice plate. Under the dissecting microscope, a loaded needle (filled with EBIV) was inserted into the mosquito thorax using a Nanoject III auto-nanoliter injector (Drummond). Each mosquito was administered 100 nL EBIV by pressing the INJECT button. Injected mosquitoes (n = 30) were transferred into new containers and maintained in the incubator at 28°C and humidity of 80% for 2 to 14 days.

To evaluate the effects of the different barriers of Ae. aegypti after EBIV infection, female mosquitoes were injected with 340 PFU virus and subsequently collected at 2, 4, 7, 10 and 14 dpi for viral RNA determination.

To establish the minimum concentration of EBIV infection in Ae. aegypti, female mosquitoes were injected with different doses of virus (three serial concentrations ranging from 0.34 to 340 PFU) and infected mosquitoes collected at 7 dpi for viral RNA determination.

To assess EBIV distribution in infected mosquitoes subjected to intrathoracic injection (at a viral dose of 340 PFU), viral RNAs in saliva, head, gut, and ovary of mosquitoes at 2, 4, 7 and 10 dpi were determined and tissue/saliva collection was performed as above.

Four parameters were calculated, specifically, gut infection rate (%) = 100 × (number of mosquitoes with virus-positive guts/number of total mosquitoes), head infection rate (%) = 100 × (number of mosquitoes with virus-positive heads/number of total mosquitoes), saliva-positive rate (%) = 100 × (number of mosquitoes with virus-positive saliva/number of total mosquitoes) and ovary infection rate (%) = 100 × (number of mosquitoes with virus-positive ovaries/number of total mosquitoes).

2.4 qRT-PCR analysis of viral RNA

To detect the virus load in whole female mosquitoes or tissue/saliva samples from female mosquitoes, each mosquito or tissue/saliva sample was placed in 200–300 μL RPMI 1640 and stored at -80°C until experimental use. All samples were initially homogenized using a Low Temperature Tissue Homogenizer Grinding Machine (Servicebio) (operating frequency = 60 Hz, operation time = 15 s, pause time = 10 s, cycles = 2, and setting temperature = 4°C), followed by centrifugation for 10 min at 12, 000 × g min and 4°C. Total RNA of each sample was extracted using an automated nucleic acid extraction system following the manufacturer’s instructions (NanoMagBio).

Using the CFX96 Touch Real-Time PCR Detection System (Bio-Rad) and One Step TB Green PrimeScript PLUS RT-PCR Kit (Takara), viral RNA copies of each sample were quantified. The primers used are presented in S1 Table. The cutoff for EBIV-positive samples determined via qRT-PCR was Ct < 35. The positive cutoff value was evaluated by comparing paired serial ten-fold dilutions either inoculated on cells or assayed via qRT-PCR (S2 Table) [25]. The equation for the standard curve was y = −3.5566x + 37.887 [x = lg (titer of EBIV), y = Ct value and R = 0.9979], which was generated using 10-fold serial dilutions of virus (1.3 × 106 PFU ml-1) and used to calculate the EBIV titer in each sample [26].

2.5 Immunohistochemical detection of EBIV antigen

Midguts and ovaries were dissected from orally infected female mosquitoes (n = 30) at 14 dpi and intrathoracically infected female mosquitoes (n = 15) at 7 dpi, respectively. Tissues were fixed using 4% paraformaldehyde for 1 h and washed with PBS containing 0.3% Triton X-100 (PBST) five times. Next, tissues were placed in blocking solution (PBS containing 5% goat serum and 0.3% Triton X-100) for 1 h and incubated with primary mouse anti-EBIV-NP antibody (derived from mice serum, diluted 1:200 in PBST containing 5% goat serum) for 24 h, followed by secondary Alexa Fluor 549-conjugated goat anti-mouse IgG (H+L) (diluted 1:250 in PBST containing 5% goat serum; Invitrogen) overnight. The actin cytoskeleton was stained with Alexa Fluor 488 Phalloidin (Invitrogen) for 1 h. After each step, tissues were washed at least five times in 0.3% PBST to avoid effects of reagents on subsequent affecting following operations. Finally, tissues were mounted onto slides using SlowFade Diamond Antifade Mountant (Invitrogen) and images recorded with the aid of a Leica SP8 confocal microscope (filter information TD 458/514/561; Leica, Germany). Using LAS X software (Leica), z-stack images were merged and scale bars added. PowerPoint 2019 was utilized for image grouping. All samples were analyzed under the same microscope and software settings.

2.6 Visualization of EBIV particles via transmission electron microscopy

Ovaries and midguts were obtained from female mosquitoes (n = 20) infected via intrathoracic inoculation at 7 dpi with the aid of a dissection microscope and tissues fixed in 2.5% glutaraldehyde until experimental use. Fixed samples were handled in the Center for Instrumental Analysis and Metrology (Wuhan Institute of Virology, China), sectioned using an ultramicrotome and visualized under a Tecnai G20 TWIN transmission electron microscope (FEI, United States). PowerPoint 2019 was used for image grouping.

2.7 Transcriptomic analysis

Female mosquitoes infected via intrathoracic inoculation and mock-infected mosquitoes were collected at 2 and 7 dpi. Each pool (comprising ten mosquitoes) was subjected to total RNA extraction using TRIzol reagent (Invitrogen). Three independent biological replicates were prepared for each sample. Next, samples were delivered to Wuhan Benagen Tech Solutions Company for commercial RNA-seq and data analysis. RNA degradation and contamination were monitored via agarose gel electrophoresis on 1% gels. RNA purity was assessed using the NanoPhotometer spectrophotometer (IMPLEN, CA, USA) and RNA integrity determined using the RNA Nano 6000 Assay kit of the Bioanalyzer 2100 system (Agilent Technologies, CA, USA). Sequencing libraries were generated using NEBNext Ultra RNA Library Prep Kit for Illumina (NEB, USA) following the manufacturer’s recommendations and index codes added to attribute sequences to each sample. Clustering of index-coded samples was performed on a cBot Cluster Generation System using the TruSeq PE Cluster Kit v3-cBot-HS (Illumina) according to the manufacturer’s instructions. After cluster generation, library preparations were sequenced on an Illumina Novaseq platform and 150 bp paired-end reads generated. Clean reads were obtained by removing reads containing adapter, reads containing ploy-N and low-quality reads from raw data and subsequently used for de novo assembly using Trinity program (http://trinityrnaseq.sourceforge.net/) and mapped to the Ae. aegypti genome database (RefSeq: GCF_002204515.2). The Unigene sequences of samples were searched against the Nr, KEGG and GO databases (E-value ≤ 1E-5) using BLASTX to retrieve protein functional annotations based on sequence similarity. The fragments per kilobase of exon per million mapped fragments values were directly applied to compare gene expression differences between samples. The DESeq package was employed to obtain the “base mean” value for identification of differentially expressed genes (DEGs). Absolute value of log2 ratio ≥ 1 and p ≤ 0.05 were set as the thresholds for significance of gene expression differences between two samples. Scatter diagrams and bubble diagrams were generated with GraphPad Prism statistical software 9.1.0.

2.8 Statistical analysis

All data were analyzed with GraphPad Prism statistical software 9.1.0. Differences in continuous variables and mosquito infection, dissemination, transmission and ovary infection rates were analyzed using the non-parametric Kruskal-Wallis test for multiple comparisons and Fisher’s exact test where appropriate, as specified in the figure legends. P values ≤ 0.05 were considered statistically significant.

3. Results

3.1 EBIV is disseminated from midgut to saliva in Ae. aegypti through blood feeding

The mean viral titers of EBIV-positive mosquitoes at the four time-points were not significantly different and all values were above 102 PFU ml-1. Notably, viral levels in some infected mosquitoes were remarkably higher, with the highest viral titer recorded as 106.4 PFU ml-1 (Fig 1A). The infection rates at the four time-points ranged from 40.3% to 70.7%. The infection rate at 4 dpi was significantly higher than that at 7 (p = 0.0086) and 14 dpi (p = 0.0005) and comparable with that at 10 dpi (Fig 1B).

Fig 1

EBIV infection rates of Ae. aegypti through oral feeding.

EBIV titers (A) and infection rates (B) of mosquitoes at 4, 7, 10 and 14 days after feeding on blood meal containing 3.7 × 106 PFU ml−1 EBIV. EBIV titers (C) and infection rates (D) of mosquitoes from six serial viral titer groups at 4 and 10 days after feeding on blood meal containing 102 to 107 PFU ml−1 EBIV. EBIV titers in gut (E), head (F), saliva (G) and ovary (H) samples of mosquitoes at 4, 7, 10 and 14 days after feeding on blood meal containing 6.4 × 106 PFU ml−1 EBIV. Infection rates (I), dissemination rates (J), transmission rates (K) and ovary infection rates (L) of mosquitoes at 4, 7, 10 and 14 days after feeding on blood meal containing 6.4 × 106 PFU ml−1 EBIV. Each dot represents an individual mosquito. The same letters indicate no significant differences (multiple comparisons using non-parametric Kruskal-Wallis analysis). Differences in the rates were analyzed with Fisher’s exact test (*: p ≤ 0.05, **: p ≤ 0.01, ***: p ≤ 0.005 and ****: p ≤ 0.001).

With increasing levels of EBIV in blood meals, the proportion of infected mosquitoes gradually increased and mean viral titers at 4 dpi and 10 dpi in mosquitoes fed the same dose of EBIV showed no significant differences (Fig 1C). The infection rates of mosquitoes fed 106 and 107 PFU ml-1 EBIV at 4 dpi (30.3% and 62.2%) and 10 dpi (48.6% and 52.4%) were significantly higher relative to the other groups while differences between these groups were not marked (Fig 1D).

The mean titers in EBIV-positive guts at 4 (102.7 PFU mL-1), 7 (102.9 PFU mL-1), and 10 (102.8 PFU mL-1) dpi were higher than those in heads, saliva and ovaries (Fig 1E). At 14 dpi, the mean viral titer in heads (103.5 PFU mL-1) was comparable to that in guts (103.2 PFU mL-1) (Fig 1F). Notably, the mean viral titer in saliva was highest at 14 dpi (102.5 PFU mL-1) (Fig 1G) and at this time-point, the highest mean virus titer was also detected in the ovary (103.6 PFU mL-1) (Fig 1H). The infection rates at the four time-points ranged from 50%-70% and were not significantly different (Fig 1I). The dissemination rates ranged from 13.3% to 42.9% (Fig 1J). The transmission rates ranged from 0% to 11.8%, with the highest rate (11.8%) recorded at 14 dpi (Fig 1K). The ovary infection rates at 4, 10 and 14 dpi were 23.8%, 28.6% and 29.4%, respectively. All ovary samples at 7 dpi did not contain detectable levels of virus (Fig 1L).

3.2 Ae. aegypti is highly susceptible to EBIV infection through intrathoracic inoculation

After injection of 340 PFU EBIV, mean viral titer of EBIV-positive mosquitoes at 2 dpi (105.5 PFU mL−1, H = 48.01, p < 0.0001) was significantly higher than that at 4, 7 and 10 dpi. The mean viral titer at 14 dpi (105.3 PFU ml−1) was the second highest among all five time-points (Fig 2A). Infection rates at the five time-points were 100% (Fig 2B).

Fig 2

EBIV titers and infection rates of Ae. aegypti through intrathoracic inoculation.

EBIV titers (A) and infection rates (B) of mosquitoes at 4, 7, 10 and 14 days after injection with 340 PFU EBIV. EBIV titers (C) and infection rates (D) of mosquitoes injected with 0.34 to 340 PFU EBIV at 7 dpi. EBIV titers in gut (E), head (F), saliva (G) and ovary (H) samples of mosquitoes injected with 34 PFU EBIV at 2, 4, 7 and 10 dpi. Gut (I), head (J), saliva (K) and ovary (L) infection rates of mosquitoes injected with 34 PFU EBIV at 2, 4, 7 and 10 dpi. Each dot represents an individual mosquito. The same letters indicate no significant differences (multiple comparisons using non-parametric Kruskal-Wallis analysis). Differences in rates were analyzed with Fisher’s exact test (*: p ≤ 0.05, **: p ≤ 0.01, ***: p ≤ 0.005 and ****: p ≤ 0.001).

The mean viral titer was the lowest in EBIV-positive mosquitoes injected with 0.34 PFU EBIV (104.3 PFU ml−1). Mean viral titer values of EBIV-positive mosquitoes injected with 3.4, 34 and 340 PFU EBIV were 105.1 PFU mL−1, 105.8 PFU mL−1 and 105.9 PFU mL−1, respectively, which were not significantly different (Fig 2C). The infection rates of all groups of mosquitoes were 100%, except that injected with 0.34 PFU EBIV, which had an infection rate of only 46% (Fig 2D).

Mean titers in EBIV-positive guts at 4 dpi (105.0 PFU ml−1) and 10 dpi (105.1 PFU mL−1) were significantly higher than that at 2 dpi (104.1 PFU ml−1, H = 13.70, p = 0.0033) (Fig 2E). The mean titers in EBIV-positive head samples were gradually increased at the four time-points, with the highest value of 105.7 PFU ml−1 observed at 7 dpi (H = 63.88, p < 0.0001) (Fig 2F). Notably, viral titers in saliva at the four time-points were not significantly different and the highest recorded value was 102.2 PFU ml−1 at 4 dpi (H = 20.27 and p = 0.0001) (Fig 2G). Mean viral titers in ovary samples were also high at 4 dpi (104.9 PFU ml−1), 7 dpi (105.0 PFU ml−1) and 10 dpi (104.9 PFU ml−1) relative to that at 2 dpi (104.0 PFU ml−1) (Fig 2H). Gut, head and ovary infection rates were 100% (Fig 2I, 2J and 2L). With increasing days after infection, saliva infection rates were slightly increased, ranging from 34.5% to 90%. The saliva infection rate was highest (up to 90%) at 10 dpi (p < 0.0001, comparison between 2 and 10 dpi) (Fig 2K).

3.3 Presence of EBIV antigens and viral particles in infected mosquitoes

Immunohistochemical analysis confirmed the presence of EBIV antigen in the midgut region of orally infected mosquitoes at 14 dpi (Fig 3A) and midgut and ovary samples of mosquitoes infected via intrathoracic inoculation at 7 dpi (Fig 3B and 3C). The EBIV antigen accumulated around the cytoskeleton in midgut endothelial cells (Fig 3A and 3B). In ovarioles, viral signals accumulated around the nucleus in nurse cells but not follicle cells (Fig 3C). The EBIV antigen was not detected in tissues from uninfected mosquitoes (Fig 3D–3F).

Fig 3

Immunohistochemical visualization and electron micrographs of EBIV in Ae. aegypti midgut and ovary.

Immunolocalization of EBIV antigen in midguts of mosquitoes with oral infection at 14 dpi (A) and intrathoracic inoculation at 7 dpi (B). Immunolocalization of EBIV antigen in ovaries of mosquitoes infected via intrathoracic inoculation at 7 dpi (C). Immunolocalization of EBIV antigen in midguts of mosquitoes fed blood without EBIV at 14 dpi (D) and mosquitoes injected with 100 nl RPMI 1640 at 7 dpi (E). Immunolocalization of EBIV antigen in ovaries from mock-injected mosquitoes at 7 dpi (F). F-actin was stained with phalloidin (green). The cell nucleus was stained with DAPI (blue). NC: nurse cells, FC: follicle cells. (G-J) Virions observed in gut are indicated by red arrows on electron micrographs. H and J are the enlarged insets of the boxes in G and I, respectively. (K-N) Virions observed in ovary are indicated by red arrows on electron micrographs. L and N are the enlarged insets of the boxes in K and M, respectively. EBIV virion clusters were detected using a mouse anti-EBIV polyclonal antibody and goat anti-mouse IgG labeled with red fluorescent secondary antibody.

To confirm the presence of EBIV viral particles in infected mosquitoes, sections of the digestive tract and ovaries from mosquitoes infected via intrathoracic inoculation were prepared and examined via transmission electron microscopy. Notably, spherically shaped virus-like particles (VLPs) 78–140 nm in diameter were stacked in intracellular vesicles and detectable in cells of the digestive tract (Fig 3G–3J). Similarly, VLPs 60–100 nm in diameter were present in nurse cells of the ovary (Fig 3K–3N). As expected, VLPs were not detectable in uninfected mosquitoes.

3.4 Immune and metabolic responses of Ae. aegypti to EBIV infection

Transcriptome experiments showed significant upregulation of 17 genes and downregulation of 28 genes in mosquitoes subjected to intrathoracic inoculation of EBIV at 2 dpi while significant upregulation of 45 genes and downregulation of 65 genes was observed in infected mosquitoes at 7 dpi (p value ≤ 0.05, |log2foldchange| ≥ 1) (Fig 4A and 4B).

Fig 4

EBIV affects specific gene expression patterns in intrathoracically infected mosquitoes.

Significantly upregulated (orange) and downregulated (blue) genes in EBIV-infected compared with mock-infected mosquitoes at 2 dpi (A) and 7 dpi (B). (C) Genes displaying differential responses in EBIV-infected mosquitoes at 2 and 7 dpi were determined via a scatter plot of expression changes. KEGG pathway analyses of DEGs at 2 dpi and 7 dpi are presented in (D) and (E), respectively. Enriched GO categories associated with DEGs at 2 dpi and 7 dpi are shown in (F) and (G), respectively.

Limited genes were upregulated in mosquitoes infected via intrathoracic inoculation at 2 dpi, most of which were undefined (S3 Table). The top three upregulated genes encoded macrophage erythroblast attacher-like (E3 Ubiquitin Ligase), zinc finger protein 37 homolog isoform X1/X2 and beta-galactoside-binding lectin-like (Fig 4A). Among the upregulated genes at this time-point, adult-specific cuticular protein 20 (ACP-20) showed the highest expression (S3 Table). Moreover, the number of downregulated genes was similar with that of upregulated genes. The most of proteins encoded by these genes were related to cellular immunity and apoptosis, including antimicrobial peptides (AAEL000625-PA, cecropin A precursor and defensin-C), toll-like receptor 13, cell death abnormality protein 1 isoform X2 and putative lysozyme-like protein. In addition, three odorant binding genes (gene-LOC5577119, gene-LOC5571968 and gene-LOC5567749) and three serine protease easter genes (gene-LOC5579410, gene-LOC5568135 and gene-LOC5569890) were significantly downregulated (S3 Table). The top four proteins encoded by downregulated genes were fizzy-related protein homolog involved in the protein ubiquitination pathway, AAEL000625-PA, angiopoietin-2-like (a growth factor whose activities are mediated through tyrosine kinase receptors [27]) and cecropin A precursor (Fig 4A). Among the downregulated genes at this time-point, defensin-C gene showed the greatest decrease expression (S3 Table).

Nearly half the upregulated genes in mosquitoes infected via intrathoracic inoculation at 7 dpi were undefined (S4 Table). Moreover, the top three proteins encoded by upregulated genes were uncharacterized (Fig 4B). Among the downregulated genes at this time point, gene-LOC110676601 encoding the voltage-dependent anion-selective channel, a pore-forming protein located in the outer mitochondrial membrane, showed the greatest decrease expression (S4 Table). Among the four genes showing the most significant downregulation, the protein encoded by gene-LOC110680747 was identified as zinc finger protein 827-like while the other three were unknown (Fig 4B). In addition, four venom allergen 5 genes (gene-LOC5575393, gene-LOC5575399, gene-LOC5575400 and gene-LOC5575401) were significantly downregulated. Previous findings suggest that venom allergen 5 is associated with deltamethrin resistance in mosquitoes [28] and Ae. aegypti venom allergen-1 promotes DENV and ZIKV transmission through activating autophagy in host immune cells [29]. Statistical comparison of differentially expressed genes between mosquitoes infected via intrathoracic inoculation at 2 dpi and 7 dpi revealed significant differences among three genes, specifically, defensin C, chitinase 10 (gene-LOC5570579) and gene-LOC5569955 (Fig 4C).

KEGG pathway and GO enrichment analyses were performed to identify the biological functions and pathways activated in EBIV-infected Ae. aegypti at 2 and 7 dpi. KEGG results revealed significantly enriched genes in four pathways at 2 dpi, including Toll and Imd signaling and endocytosis (Fig 4D). However, the enrichment pathways at 7 dpi were mainly related to biosynthesis and metabolism, including steroid hormone biosynthesis, N-glycan biosynthesis, ribosome, RNA degradation, and steroid biosynthesis at 7 dpi (Fig 4E). Similarly, GO analysis showed significant enrichment of immune-related processes at 2 dpi and metabolism-related processes at 7 dpi (Fig 4F and 4G).

4. Discussion

This study explored the vector competence of Ae. aegypti for EBIV in view of the susceptibility of this mosquito species to other orthobunyaviruses [22,24]. For instance, Cx. quinquefasciatus is reported to be refractory to BUNV, but not Ae. aegypti [21]. Further studies on additional species and geographic strains of mosquitoes, such as Ae. albopictus, Cx. quinquefasciatus, and Cx. tritaeniorhynchus, should be conducted to identify the primary mosquito vectors for EBIV.

Generally, adult mice show resistance to orthobunyavirus infections while three-week-old or younger mice are more susceptible [16,30-32]. However, a previous study by our group showed that > 90% adult BALB/c mice succumbed to death upon intraperitoneal infection with an extremely low dose of EBIV (1–10 PFU), indicating greater pathogenicity of EBIV to mice compared to other orthobunyavirus [16]. Here, the mean viral titer of EBIV-positive saliva was > 101.5 PFU ml−1 (the average dose of EBIV per mosquito was > 6.3 PFU) at 14 dpi in mosquitoes infected via blood feeding, highlighting the potential risk of EBIV transmission to vertebrate hosts by mosquito. However, the issue of whether EBIV-positive mosquitoes are capable of transmitting viruses to naïve vertebrate hosts via biting is yet to be established and further studies on the EBIV-mosquito-vertebrate transmission cycle are warranted.

The highest saliva-positive rate in intrathoracic-injected mosquitoes reached 90%, compared to mosquitoes subjected to oral feeding (11.8%). Notably, after oral infection, a few EBIV-positive saliva samples of infected mosquitoes with viral gut loads > 104 PFU mL−1 were detected (Fig 1G), suggesting that EBIV can enter saliva when virus titer in the gut reaches a threshold of 104 PFU ml−1. Following intrathoracic injection, EBIV bypassed the midgut barrier and was able to rapidly infect the whole mosquito body, including saliva and ovary (Fig 2G–2H). The distinct results obtained with the two routes indicate that the midgut of Ae. aegypti is the main barrier to EBIV transmission. In view of the characteristics of arbovirus, which need to maintain their life cycle by switching between vertebrate hosts and invertebrate vectors, a strong possibility of evolutionary adaptation during this process is suggested [33,34]. For instance, a single amino acid substitution could increase susceptibility of Ae. albopictus and lead to virus dissemination more rapidly from the midgut to secondary tissues [35]. A similar situation was observed with ZIKV, whereby the mutation enhanced ZIKV infectivity in mosquitoes [36]. Therefore, once the virus evolves to increase ease of cross through the midgut barrier, it may present an emerging threat to human or animal health. In addition, it is not uncommon for orthobunyaviruses to spread during the immature life stages of mosquitoes through a mode of vertical transmission [37,38]. The high EBIV infection rates in ovary observed in the current study support the possibility of vertical transmission (Figs 1H and 2H).

During the process of invasion, arboviruses need to cope with innate immune responses and overcome several barriers of mosquito vectors. To date, four major barriers have been identified, specifically, the midgut infection barrier, midgut escape barrier (MEB), salivary gland infection barrier, and salivary gland escape barrier [39]. Ae. aegypti was moderately susceptible to EBIV after oral feeding, with 70.0% mosquitoes showing midgut infection at 10 dpi, whereas the dissemination rate was significantly lower than the infection rate (38.1%) at this time-point (p = 0.0432). Notably, the proportion of EBIV-positive saliva samples was 90.0% at 10 days post intrathoracic injection. The combined results of blood feeding and intrathoracic injection experiments suggest that MEB presents the primary barrier to systematic EBIV infection of Ae. aegypti. In the presence of an efficient MEB, virus replication is limited to the midgut or inefficient dissemination occurs [40]. The basal lamina of the midgut may exert effects on effective dissemination [41]. Several studies have reported an inverse relationship between LACV dissemination rates and thickness of basal lamina of Ae. triseriatus [42,43]. An alternative reason for ineffective dissemination could be that EBIV is unable to overcome or evade antiviral immune responses in midgut. Electron microscopy of the midgut section of mosquitoes infected via intrathoracic injection revealed the presence of a large number of autophagosomes and lysosomes, suggesting the involvement of autophagy in EBIV infection. Recent studies on arbovirus infection have demonstrated that autophagy serves as an antiviral defense mechanism [44-46]. Similarly, replication of Rift Valley fever virus belonging to the family Phenuivridae is reported to be limited by activation of autophagy in Drosophila [47]. However, other studies suggest that autophagy is beneficial for flavivirus replication and transmission in Ae. aegypti [29,48]. Additionally, evidence supporting dose-dependent competence of midgut infection has been obtained [39,49,50], which indicates that with increasing viral titers in host blood, the risk of EBIV transmission to vertebrates is bigger.

Based on transcriptome analysis of intrathoracic-infected Ae. aegypti, Toll and Imd signaling pathways are implicated in the protective response of mosquito to EBIV infection [22]. Endocytosis plays an important role in host entry of several virus types, such as flaviviruses and bunyaviruses [51,52]. Several immune-related genes have been identified in EBIV-infected Ae. aegypti, such as E3 ubiquitin ligase related to autophagy [53], zinc finger protein 37 homolog isoform X1/X2 (a transcription factor) and beta-galactoside-binding lectin-like involved in the process of virus infection [54] (Fig 4A). Moreover, five zinc-finger protein-encoding genes showed differential expression patterns (gene-LOC110680747, gene-LOC5571062, gene-LOC5576563 and gene-LOC110680309 were downregulated while gene-LOC5573104 was upregulated) (S4 Table). Zinc-finger proteins are one of the most abundant groups of proteins with a wide range of molecular functions. Recently, the zinc-finger protein ZFP36L1 was shown to enhance host anti-viral defense against influenza A virus [55], supporting a role of these proteins in response to viral infection.

In conclusion, Ae. aegypti can be infected by EBIV and presents a potential risk of virus transmission. Further research on the vector competence of various mosquito species for this newly classified orthobunyavirus and epidemiological studies are essential to provide valuable insights that could aid in management of potential future outbreaks.

Primer sequences of genes used for qRT-PCR.

(DOCX)

Click here for additional data file.

Correlations between the EBIV-induced cytopathic effect in BHK-21 cells and CT values of virus RNA determined via qRT-PCR.

(DOCX)

Click here for additional data file.

Differentially expressed genes in mosquitoes infected via intrathoracic inoculation compared to mock-infected mosquitoes at 2 dpi.

(XLSX)

Click here for additional data file.

Differentially expressed genes in mosquitoes infected via intrathoracic inoculation compared to mock-infected mosquitoes at 7 dpi.

(XLSX)

Click here for additional data file.

29 Mar 2022

Dear Dr. Xia,

Thank you very much for submitting your manuscript "Vector competence and transcriptional response of Aedes aegypti for Ebinur Lake virus, a newly classified mosquito-borne orthobunyavirus" for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. In light of the reviews (below this email), we would like to invite the resubmission of a significantly-revised version that takes into account the reviewers' comments.

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***********************

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: -Are the objectives of the study clearly articulated with a clear testable hypothesis stated? Yes

-Is the study design appropriate to address the stated objectives? Yes

-Is the population clearly described and appropriate for the hypothesis being tested? Yes

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested? Yes

-Were correct statistical analysis used to support conclusions? Yes

Reviewer #2: Methods section has a poor english. Should be entirely reviewed.

If the objective of the study is additionally to show vector competence, than transmission to a host (animal model) should be included.

Reviewer #3: I have only a few major points for the authors to consider/clarify and a number of minor points that are easy to fix. Please see the "Summary and General Comments" section.

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Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: -Does the analysis presented match the analysis plan? Yes

-Are the results clearly and completely presented? Yes

-Are the figures (Tables, Images) of sufficient quality for clarity? Yes

Reviewer #2: results either needs a extensive review.

Reviewer #3: I have only a few major points for the authors to consider/clarify and a number of minor points that are easy to fix. Please see the "Summary and General Comments" section.

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Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: -Are the conclusions supported by the data presented? Yes

-Are the limitations of analysis clearly described? No

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study? Yes

-Is public health relevance addressed? Yes

Reviewer #2: transmission is not an adequate term to use in this manuscript, reasons pointed above. Mainly, no host was exposed to these experimentally infected mosquitoes.

Therefore, I strongly suggest that the term "vector competence" should be changed by "vector capacity" in the manuscript in the current format. Competence should be proved by transmission experiments. Here, the virus was detected to be replicating in mosquito tissues, and viral titers were demonstrated in their saliva, but transmission experiments are lacking.

Reviewer #3: I have only a few major points for the authors to consider/clarify and a number of minor points that are easy to fix. Please see the "Summary and General Comments" section.

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Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: (No Response)

Reviewer #2: This manuscript describes the experimental infection of aedes aegypti colonies by oral route and after intrathoracic administration of EBIV, a novel orthobunyavirus discovered on China. It did not report any transmission experiment, so results should be carefully reviewed since mice were not exposed to these experimentally infected mosquitoes. Despite the low viral titers detected in orally infected mosquitoes, the presence of the virus in the ovaries is of biological importance, because it can mean that this virus potentially can be transmitted vertically. To assess this information, male/female prole originated from these experimentally infected mosquitoes should be tested, since more than 20% infection rate is likely to represent this possibility of vertical transmission. Figures have na excelent quality, except for figure 4, which in this format is not possible to read (font size is to small and lacking resolution quality). The most interesting findings however, are the transvcriptome results, since studies evaluating mosquito innate response (related to apoptosis induction, antimicrobial response and ubiquitin pathway) are lacking in literature. Writting of this parto f results, and discussion section, is a better english than the rest of the manuscript.

The entire manuscript needs a english review by a english native speaker, several sentences in the abstract, introduction, material and methods and results, present gramar mistakes (plural/singular mistakes, verb conjugated in the present or future, were it should be presented in the past tense, for instance). Introduction should be more direct and concise to reflect the article content solely. Sentences are in general disconected from each other, they need a conection to let the text more “fluid”, direct, concise. Paragraphs are to big, descriptions should be re-written. Overall, the manuscript should be reviewed to remove non necessary and repetitive descriptions as well.

Abstract:

“Ebinur Lake virus (EBIV) has been verified with highly virulent pathogenic to adult laboratory mice, and antibodies against EBIV have been detected in humans.” – please verify gramar mistakes, redundancy ...(EBIV) has been shown to be highly pathogenic...

Does these antibodies were veirified by PRNT, to rule out cross reactions? I sugest to keep it simple and remove this part from abstract (...and antibodies against EBIV have been detected in humans).

It was showed that EBIV can be transmitted – how? Does the mosquito secrete virus particles in their saliva? How much in viral titers? Viral load? Did the authors performed experimental infection in mice with these experimentally infected mosquitoes? These points should be reflected in the abstract

Intrathoracic infection is not the natural infection route for vectors. Since mosquitoes feed on infected blood, oral feeding is the most likely route to reproduce what hapens in nature.

Introduction: the entire topic should be reformulated to be strait to the point.

Line 50: are primarily distributed to three families and one order: Flaviviridae, Togaviridae and Reoviridae belong to orders Amarillovirales, Martellivirales and Reovirales. Sugestion only: use either the four orders and discriminate families, or use the names of the families within Bunyavirales were arboviruses are classified instead.

Please use the current ICTV classification of viral families.

This part o f the introduction should be removed:

Lines 56-60: “For example, the most notable flavivirus DENV, the pathogen of dengue 56 fever, has increased dramatically within the past 20 years, and more than 3.9 billion people in 128 countries are reported to be at risk of dengue infection [4]. After ZIKV and WNV Zika virus are introduced into the Western hemisphere, the viruses have a rapid geographical spread, and cause a large number of infections in the population [5,6].”

Introduction should be designed to what is really necessary to understand the importance of the study and the description of novel characteristics of this novel Peribunyaviridae.

Lines 64-66: “So far, the orthobunyaviruses can be discovered in mosquitoes of the different species, such as Ochlerotatus spp., Culex spp. and Aedes spp. [8-10].” Verify gramar mistakes, please: example: “So far, orthobunyaviruses have been descovered to infect different mosquito species...”

Line 67: Oropouche virus cause self-limiting acute febrile disease and, in 5% of infected people, mainly children, can cause asseptic meningitis.

Overall, this paragraph seems fragmented, needs a revision to be direct, concise, linked to the rest o f the introduction, considerations about flaviviruses, viruses circulating in Europe, should be entirely removed.

It is necessary to explain what is known about this novel virus, and the importance of studying and discovering new viruses before they became a public health problem.

Observe verb conjugation in this sentence: “Ebinur Lake virus (EBIV), a newly identified orthobunyavirus in China, is isolated from Culex modestus mosquito pools in Xinjiang Province” – replace “is” for “was” or “has been”.

Line 87-89: “EBIV can efficiently infect cells derived from rodent, avian, non-human primate, mosquito and human.” Were these tests performed in cell lines or primary cells? Please discriminate in the sentence.

Lines 89-90: “After the EBIV infection, BALB/c mice show the encephalopathy, hepatic damage and immunological system damages with high mortality” please verify gramar:

Sugestion - EBIV induced encephalopathy, hepatic and immunological system damages with high mortality index in experimentally infected BALB-c mice”

Lines 90-91: “Even though there is no report about confirmed human cases of EBIV, the serological proofs for EBIV infection has been detected”

Considering cross-reactions, please indicate wether this study was performed by PRNT or other serological test and introduce this possibility here.

Xia H, Liu R, Zhao L, Sun X, Zheng Z, Atoni E, et al. Characterization of Ebinur Lake Virus

636 and Its Human Seroprevalence at the China-Kazakhstan Border. Front Microbiol. 2019;10:3111: “Of the 211 tested serum samples, 17 were identified as IgM positive (1:4), 26 as IgG positive (1:10), and 4 as both IgM and IgG positive for EBIV by IFA (Supplementary Figure 3). In the participant with fever, higher IgM (10.37% vs. 0) and IgG (14.63 vs. 4.26%) positive rates were observed. Female participants had a much higher IgM-positive rate than male participants (13.95 vs. 4.00%), but no obvious difference was observed for IgG (13.95 vs. 9.6%). Study participants belonging to the >60-year age group had the highest positive rate of both IgM (16.67%) and IgG (12.69%) among all age groups. Furthermore, based on the occupational groups, the proportion of positive samples for IgM and IgG was highest among retirees (20%), followed closely by farmers and factory workers (17.28%). In addition, the neutralizing antibody prevalence of EBIV was 0.95% (2/211) (Table 1). For the two neutralizing antibody-positive cases, one male participant aged 50 years had a 1:8 PRNT90 titer, and the second case was from a female participant aged 50 years with a 1:16 PRNT90 titer. Both of these two study participants were from the Fifth Division of Xinjiang Production and Construction Corps region.”

Does the PRNT was performed only with EBIV? Did the test included other possible circulating orthobunyavirus to compare titers?

Line 92: Due to the health risk of EBIV: is there any clinical description in humans? If not, remove this sentence.

This virus was described in a Culex species. Culex sp. are more frequently associated to orthobunyaviruses than Aedes sp. Why the authors choosed Aedes aegypti instead of Culex mosquitoes colonies to perform this experimental study?

Lines 99-101: which is beneficial to better prepare for and respond to potential outbreak of EBIV, and to understand the transmission mechanism of orthobunyavriuses. – I strongly sugest this sentence to be removed, since by the results of previous studies, is not enoguh evidence of human infection. I also recommend that animals from the region should be tested for the virus.

Material and methods:

Line 111: “The EBIV isolate Cu20-XJ is obtained from Cx. modestus mosquitoes in 2013” – please verify verb conjugation (here is necessary to use past not present time).

Line 117: “Eggs of Ae. aegypti (Rockefeller 117 strain) was obtained from Laboratory of...” eggs – plural, was – singular.

Lines 120-121: ...”and a relative humidity of 75 ± 5% humidity, approximately.” Humidity is repeated.

Lines 125-126: ...”Then put the cups into the mesh cages (30 x 30 x 30 cm), which were kept in the insect incubator with the condition...” ...Cups were kept into the mesh....

Lines 132-133: Before oral-infection, five to eight days old, adult mosquitoes were collected by using the mosquito absorbing machine and placed into plastic cups (24oz). And wrap the cup with a cut mosquito net mesh and then cover it with a lid with a hole in the middle.

... “and placed into plastic cups (24oz), wrapped with a mosquito net mesh and covered with a lid containing a oppening in the center”...

Line 165: future tense should me avoided in manuscripts. Item 2.4 seems to be a “copy and paste” from Project arquive. Really hard to read.

Line 211: “Ten mosquitoes as a pool were used to extract total RNA using TRIzol reagent (Invitrogen).” ¬sugestion: “Each pool (n=10 especimens) was subjected to total RNA extraction using...”.

Line 226: “The clean reads were performed de novo assembly” did you mean: “Clean reads were used for de novo asembly?”

Results:

EBIV can be transmitted by Ae. aegypti through oral feeding – actually, in this manuscript transmission to a vertebrate was not evaluated. The most corrected presentation of this data is as the presence of EBIV titers in aedes aegypti saliva, or that EBIV infect several mosquito tissues.

In order to assess transmission rates, non infected mice should be exposed to these mosquitoes, and their tissues tested for the presence of the virus after the incubation period.

271-276: “The mean titers in the EBIV-positive guts at 4 (102.7 PFU ml-1), 7 (102.9 PFU ml-1), and 10 (102.8 PFU 272 ml-1) dpi were higher than those in heads, saliva and ovaries (Fig. 1E). At 14 dpi the viral titer in heads (103.5 PFU ml-1) was similar with the guts (103.2 PFU ml-1) at the same point (Fig. 1F). It was worth noting that the viral titer in saliva could get a highest virus titer at 14 dpi (102.5 PFU ml-1) (Fig. 1G) and at this time point ovaries also get the highest mean virus titer (103.6 PFU ml-1)” – By these titers, is not possible to afirm that aedes aegypti transmit the virus. Is necessary to expose a experimental model to these infected mosquitoes to prove that.

Line 297: EBIV was highly susceptible in Ae. aegypti through intrathoracic inoculation – this objective should be more clear. By this title, it was inferred that the virus could not pass by mosquito gut barriers. However, Aedes aegypti was susceptible to virus infection, not otherwise, and by this route of inoculation, viral titers were higher in mosquito tissues than by oral route.

It seems initially that intrathoracic route was used after oral route because viral titers or infection was not that efficient by the most similar route of infection in nature (oral feeding). In other places, it seems that this inoculation was used to evaluate mosquito innate response. Please review the results and abstract section to define these contents more properly.

“The mean titers in the EBIV-positive guts at 4 dpi (105.0 PFU ml−1) and 10 dpi (105.1 PFU ml−1) were significantly higher than that at 2 dpi (104.1 PFU ml−1, H = 13.70 and p = 0.0033) (Fig. 1E).” – and higher than in orally infected mosquitoes at these time points.

Lines 338-339: “could get very high values” – I do not agree these are very hogh titers. These are medium values representing that the viruses efectively replicated in these tissues.

Lines 341-343: “With the increase of days after infection, the transmission rates were also slightly increased, ranging from 34.5-90%. The highest transmission rate...”

I do not think transmission rate is adequate to these results. Secretion/excretion rate, since transmission means actually the infection to be successful in a host exposed to these mosquitoes.

Discussion: overall, this is the most well written parto f the manuscript. Results are properly explored. I would like to know what the authors have to say about Ae. Aegypti been just a model, as the natural vector or reservoir might be a Culex species. Also, if the authors intend to perform mosquito experimental infection and test their transmission potential to experimental models (mice). Oral infection is the natural route of vector infection. Intrathoracic inoculation is not a natural route of infection. What is the explanation for the discrepant results between these two routes?

Reviewer #3: I have only a few major points for the authors to consider/clarify and a number of minor points that are easy to fix. Please see the "Summary and General Comments" section.

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Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: Line 47 - Mosquito-borne viruses (MBVs), as a group of heterogeneous RNA viruses, naturally survive in both mosquitoes and vertebrate hosts, and are the aetiological agents of many human diseases.

"Mosquito-borne viruses" may be confusing because of the insect-specific viruses that do not replicate in vertebrate cells. It seems that Ebinur Lake virus has demonstrated capacity to infect vertebrate and invertebrate cell lines so it can be categorized as an arbovirus, which is a term well-established. Also, viruses replicate rather than survive.

I suggest changing to:

Arthopod-borne viruses (arbovirus), as a group of heterogeneous RNA viruses, naturally replicate in both mosquitoes and vertebrate hosts, and are the aetiological agents of many human diseases.

Line 49 - The medically important MBVs are primarily distributed to three families and one order: the Flaviviridae family [dengue viruses 1-4 (DENV), Zika virus (ZIKV), West Nile virus (WNV), Japanese encephalitis virus (JEV), etc], the Togaviridae family [Chikungunya virus (CHIKV), etc], the Reoviridae order [(Banna virus (BAV), etc) and Bunyavirales order [Rift Valley fever virus (RVFV), etc] [2, 3].

I suggest changing to:

Line 49 - The medically important arboviruses are primarily distributed to four families: Flaviviridae [dengue viruses 1-4 (DENV), Zika virus (ZIKV), West Nile virus (WNV), Japanese encephalitis virus (JEV), etc], Togaviridae [Chikungunya virus (CHIKV), Mayaro virus (MAYV) etc], Reoviridae [Banna virus (BAV), etc], and Peribunyaviridae [Oropouche orthobunyavirus (OROV) etc] [2, 3].

Line 57 - After ZIKV and WNV Zika virus are introduced into the Western hemisphere, the viruses have a rapid geographical spread, and cause a large number of infections in the population

I suggest changing to:

After ZIKV and WNV were detected in the Western hemisphere, they rapidly spread causing large number of neurological disorders mainly in Brazil and United States, respectively.

Line 60 - change "mosquito-borne" by "arbovirus", and add the proper references.

Line 65 - change "Ochlerotatus spp., Culex spp. and Aedes spp. [8-10]" by "Ochlerotatus spp., Culex spp. and Aedes spp. and also midges, as Culicoides paraensis, which is a vector of OROV in South America [8-10]"

From line 92 to 95 - Authors should indicate here why not using Culex spp for the experimental infection, or at least describe if any experimental work has been done with Culex spp. EBIV was detected in Culex modestus, so it would make sense evaluate the vector capacity and competence of the most common Culex spp. in China as well. This topic also needs to be discussed in the discussion section.

Line 117 - Authors need to explain and discuss at some point in the manuscript the advantages and disadvantages of using the Rockefeller strain of Ae. aegypti for these experiments. We know that different populations of Ae. aegypti can have different vectorial capacity for other arboviruses, so it would be helpful for the readers to know why not using a wild population of Ae. aegypti reared from eggs collected in the field in China. Wild populations would reflect a more realistic vectorial capacity due ecological and biological characteristics that could interfere with EBIV replication, as the infection by insect-specific viruses etc. Perhaps this can be considered a first step for the vector competence analysis, but it needs t be clear for the reader the reasons for the chosen approach.

Line 136 - Mosquitoes were fed with supernatant of EBIV-infected BHK cells

Line 143 - Under the dissecting microscope, insert the loaded needle (filled with supernatant of EBIV-infected BHK cells)

I noticed the virus concentration used for inoculation and blood meal is described in the results section, but I wonder if it would not be easier for the reader having that information here at the material and methods.

Line 147 - "nl of virus" My understanding is that authors did not concentrate virus for inoculation, so here "virus" needs to be replaced by supernatant of EBIV-infected BHK cells.

Line 186 - "mice anti-EBIV-NP" Was this primary antibody prepared by the authors in previous study? If so, cite here. Is this mouse hyper immune ascitic fluid? If so, provide that information here.

Line 248 to 252 could be transferred to the material and methods section.

Line 252 to 256 - It is not very clear if titration was done by the equation described on line 177 and 178 or plaque assay. If by the described equation, please explain it better, citing references that support the calculation used. If plaque assay was used, describe the method at some point at material and methods.

Line 381-384 - "we compared the differences of gene expression between the intrathoracic inoculation and

mock-injected mosquitoes at the same time point". Please, make clear here, or later in line 470-473 in the discussion section, the reason for not using orally-infected mosquitoes to evaluate down-regulation of immune-related genes.

Lines 468-469 and 482-484 describe the same potential for vertical transmission.

Discussion section - Overall, I believe the discussion section can be improved. I missed in the discussion section the limitations of this study, which could enrich the discussion of this laborious study. Authors could discuss the potential different responses using different field Ae. aegypti populations, the experimental infection with Culex spp, etc. Also, the increasing followed by reduction in dissemination and infection rates observed at 10 dpi etc. Discussion of down-regulation of genes, for instance, is restricted to a couple of lines and should be better explored.

Reviewer #2: Overall, the main goal of this study is to demonstrate that viral replication alters gene expression in an experimental vector model. Therefore, after an extensive review, i recommend this article to be evaluated again by reviewers.

Reviewer #3: This manuscript describes the EBIV- Aedes aegypti interactions at the vector competence level. Two infection ways possess divergent infection, dissemination rate, and ovary infection rates at different dpi. The authors also present the morphology of EBIV in different organs via IFA and EM. Finally, the transcriptome data provide the relevant immune/ metabolism genes expression profile, potentially associated with the vector transmission capability. The work is well-executed, with an adequate number of replicates and controls. The study has generated novel results of interest and increased our knowledge on this newly classified mosquito-borne orthobunyavirus and the vector. I have only a few major points for the authors to consider/clarify and a number of minor points that are easy to fix.

1-I wonder if the title should be more specific to better reflect the results obtained in this work.

2- The English needs to be clarified so that the reader clearly understands what is being said. This must be done throughout the document, but here are some examples:

L21. ... diseases has been increasing in the last...

L25. ... it is necessary to assess mosquitoes' vector capacity for EBIV to predict its risk to...

L27... was shown that EBIV could be...

L33. Delete “all”

L33. …demonstrated EBIV could alter…

L34. processes

3-I have some comments on the methods. The authors need to be clarified.

a. L112, please provide the information for mice, strain, age.

b. L114, a reference, needs to cite to describe this plaque assay.

c. L113, adult female?

d. It would be better to provide the numbers of mosquitoes in each experiment, such as viral detection, IFC, and EM.

e. a reference need to cite for the rates used in this manuscript.

f. filter information for confocal.

4- I have the feeling that this 3.7 of the Results section is some kind of last minute add-on. I would suggest adding more words in the relevant pathways, especially for the functions in the modulation of virus infection.

5- I favor future work, and possible points would be investigated in the Discussion section, but some words in the discussion need to be said for the transcriptome data. I have some suggestions for the authors to consider. For example, the function of the up-regulation genes in immune response; how the metabolism pathways work on the virus infection?

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Reviewer #3: Yes: Xin-Ru Wang

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6 May 2022

Submitted filename: Cover Letter and Response to Comments.docx

Click here for additional data file.

21 Jun 2022

Dear Dr. Xia,

Thank you very much for submitting your manuscript "Vector competence and immune response of Aedes aegypti for Ebinur Lake virus, a newly classified mosquito-borne orthobunyavirus" for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. The reviewers appreciated the attention to an important topic. Based on the reviews, we are likely to accept this manuscript for publication, providing that you modify the manuscript according to the review recommendations.

Please prepare and submit your revised manuscript within 30 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to all review comments, and a description of the changes you have made in the manuscript.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Thank you again for your submission to our journal. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Adly M.M. Abd-Alla, Prof asso.

Associate Editor

PLOS Neglected Tropical Diseases

Eric Dumonteil

Deputy Editor

PLOS Neglected Tropical Diseases

***********************

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #2: objectives are clearly stated, study design seems to be firstly made to prove oral natural infection occur in this species and virus reach salivary glands. In a second moment, the authors choosed intrathoracic inoculation to overcome gut barrier of virus dissemination to stablish persistent salivary gland infection and study which genes are modulated by virus replication in mosquitoes (Ae aegypti). I am not convinced by the results that Ae aegypti is a good choice, either that this species could sustain virus transmission of this virus.

sample size is ok

ethical or regulatory requirements are ok too.

Reviewer #3: (No Response)

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Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #2: Current version is a lot more clear and in a better fashion.

-The analysis presented match the analysis plan? Abstract contains few affirmations not sustained by the presented results that should be reviewed again. Ovary infection rates and saliva titers after intrathoracic infection, and gene expression modulation by viral infection are important results of the study.

-Are the results clearly and completely presented? See comments above.

-Are the figures (Tables, Images) of sufficient quality for clarity? Yes. Figures are very clear.

Reviewer #3: (No Response)

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Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #2: -Are the conclusions supported by the data presented? See considerations about abstract. Discussion is a lot improved.

-Are the limitations of analysis clearly described? It is a lot improved in this version.

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study? Yes.

-Is public health relevance addressed? These studies do not implicate public health directly. Few data relate this pathogen to humans in literature. This study contributes to understand Ae aegypti replication of the virus. Main findings are related to mosquito model, mosquito immune response activation, importance of gut barrier to limit virus dissemination in mosquito body to reach salivary glands, possible mosquito vertical transmission since the virus reached ovaries. Biological importance in an anthropophilic mosquito species is well demonstrated.

Reviewer #3: (No Response)

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Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #2: (No Response)

Reviewer #3: (No Response)

--------------------

Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #2: Main consideration:

This version of manuscript is a lot more uniform, direct and concise. Importance of invertebrate model studies is clearly demonstrated by the findings, since EBIV infected ovary tissues (reflecting it possibly is transmitted to offsprings) and viral titers are present in saliva. Also, demonstrate gut barrier limited virus dissemination after natural infection and, virus replication leaded to gene expression related to mosquito immune response. These findings are well demonstrated and should be evidenced in the abstract. Therefore, abstract should be reformulated to properly define results and findings of the study.

Public health importance should be minimized since i. human transmission was not accessed in this manuscript; ii. the study did not show a high frequency of positivity in the saliva of Ae aegypti when infection is made with high viral titer through oral route, only when non-natural, intrathoracic route is used. Gut barrier probably limited the virus dissemination to salivary glands, leading to few mosquitoes with virus in their saliva after oral infection, when considering the total number of mosquitoes included in the experiment/evaluated in each time period (figure 1G). Therefore, this observations should be tacking into consideration to complete review of the manuscript.

By the results, is not possible to say this species could spread the virus, since a high number of mosquitoes infected through oral route did not show virus titers in saliva.

Should reflect these observations:

“Following intrathoracic injection, EBIV bypassed the midgut barrier and was able to rapidly infect the whole mosquito body, including saliva and ovary (Fig. 2G to H). The distinct results obtained with the two routes indicate that the midgut of Ae. aegypti is the main barrier to EBIV transmission.”

In my opinion, oral infection does not seem to be very efficient in this species of mosquito, next studies should consider to use other mosquito population.

Seems gut barrier limits the establishment of persistent infection in salivary glands of Ae aegypti even when a high viral load is inoculated. Only a few mosquitoes of this species are infected through natural vector infection route, considering the number of mosquitoes included in the experiment.

Therefore, implications to public health at this point, should not be made.

Author summary is a lot better focused in the biological importance of the main findings of the study. Abstract should be reformulated to continuous that line of thinking.

Lines 94-95: “it poses a considerable risk to public health and may offer a new threat to human or animal health and economic prosperity” –is not enough data to affirm this virus is a public health threat. In my opinion, the study should exclude these affirmations and reflect the important findings and conclusions related to mosquitoes. As far as is possible to comprehend, this virus is important to animal health, not yet proved with sufficient data that it can cause an outbreak involving humans.

Discussion section should be reflected on abstract writing.

Reviewer #3: (No Response)

--------------------

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Reviewer #2: No

Reviewer #3: No

Figure Files:

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org.

Data Requirements:

Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. For an example see here: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.

Reproducibility:

To enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

References

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article's retracted status in the References list and also include a citation and full reference for the retraction notice.

30 Jun 2022

Submitted filename: Response to Comments.docx

Click here for additional data file.

8 Jul 2022

Dear Dr. Xia,

We are pleased to inform you that your manuscript 'Vector competence and immune response of Aedes aegypti for Ebinur Lake virus, a newly classified mosquito-borne orthobunyavirus' has been provisionally accepted for publication in PLOS Neglected Tropical Diseases.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.

IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.

Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us now if you or your institution is planning to press release the article. All press must be co-ordinated with PLOS.

Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Adly M.M. Abd-Alla, Prof asso.

Associate Editor

PLOS Neglected Tropical Diseases

Eric Dumonteil

Deputy Editor

PLOS Neglected Tropical Diseases

***********************************************************

14 Jul 2022

Dear Dr. Xia,

We are delighted to inform you that your manuscript, "Vector competence and immune response of Aedes aegypti for Ebinur Lake virus, a newly classified mosquito-borne orthobunyavirus ," has been formally accepted for publication in PLOS Neglected Tropical Diseases.

We have now passed your article onto the PLOS Production Department who will complete the rest of the publication process. All authors will receive a confirmation email upon publication.

The corresponding author will soon be receiving a typeset proof for review, to ensure errors have not been introduced during production. Please review the PDF proof of your manuscript carefully, as this is the last chance to correct any scientific or type-setting errors. Please note that major changes, or those which affect the scientific understanding of the work, will likely cause delays to the publication date of your manuscript. Note: Proofs for Front Matter articles (Editorial, Viewpoint, Symposium, Review, etc...) are generated on a different schedule and may not be made available as quickly.

Soon after your final files are uploaded, the early version of your manuscript will be published online unless you opted out of this process. The date of the early version will be your article's publication date. The final article will be published to the same URL, and all versions of the paper will be accessible to readers.

Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Shaden Kamhawi

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

Paul Brindley

co-Editor-in-Chief

PLOS Neglected Tropical Diseases