Aedes Albopictus Cells Research Articles

Pan-flavivirus analysis reveals sfRNA-independent, 3' UTR-biased siRNA production from an insect-specific flavivirus.

RNA interference (RNAi) plays an essential role in mosquito antiviral immunity, but it is not known whether viral small interfering RNA (siRNA) profiles differ between mosquito-borne and mosquito-specific viruses. A pan-Orthoflavivirus analysis in Aedes albopictus cells revealed that viral siRNAs were evenly distributed across the viral genome of most representatives of the Flavivirus genus. In contrast, siRNA production was biased toward the 3' untranslated region (UTR) of the genomes of classical insect-specific flaviviruses (cISF), which was most pronounced for Kamiti River virus (KRV), a virus with a unique, 1.2 kb long 3' UTR. KRV-derived siRNAs were produced in high quantities and almost exclusively mapped to the 3' UTR. We mapped the 5' end of KRV subgenomic flavivirus RNAs (sfRNAs), products of the 5'-3' exoribonuclease XRN1/Pacman stalling on secondary RNA structures in the 3' UTR of the viral genome. We found that KRV produces high copy numbers of a long, 1,017 nt sfRNA1 and a short, 421 nt sfRNA2, corresponding to two predicted XRN1-resistant elements. Expression of both sfRNA1 and sfRNA2 was reduced in Pacman-deficient Aedes albopictus cells; however, this did not correlate with a shift in viral siRNA profiles. We suggest that cISFs, particularly KRV, developed a unique mechanism to produce high amounts of siRNAs as a decoy for the antiviral RNAi response in an sfRNA-independent manner.IMPORTANCEThe Flavivirus genus contains diverse mosquito viruses ranging from insect-specific viruses circulating exclusively in mosquito populations to mosquito-borne viruses that cause disease in humans and animals. Studying the mechanisms of virus replication and antiviral immunity in mosquitoes is important to understand arbovirus transmission and may inform the development of disease control strategies. In insects, RNA interference (RNAi) provides broad antiviral activity and constitutes a major immune response against viruses. Comparing diverse members of the Flavivirus genus, we found that all flaviviruses are targeted by RNAi. However, the insect-specific Kamiti River virus was unique in that small interfering RNAs are highly skewed toward its uniquely long 3' untranslated region. These results suggest that mosquito-specific viruses have evolved unique mechanisms for genome replication and immune evasion.

Negevirus Piura Suppresses Zika Virus Replication in Mosquito Cells.

We investigated the interaction between the insect-specific virus, Piura virus (PIUV), and the arbovirus Zika virus (ZIKV) in Aedes albopictus cells. We performed coinfection experiments in C6/36 cells. Piura virus (Cor 33 strain, Colombia) and ZIKV (PRVABC58 strain, Puerto Rico) were co-inoculated into C6/36 cells using two multiplicity of infection (MOI) combinations: 0.1 for both viruses and 1.0 for ZIKV, 0.1 for PIUV. Wells were infected in triplicate with either PIUV and ZIKV coinfection, ZIKV-only, or PIUV-only. Mock infected cells served as control wells. The cell suspension was collected daily 7 days post-infection. Zika virus load was titrated by TCID50 on Vero 76 cells. The ZIKV-only infection and PIUV and ZIKV coinfection experiments were also quantified by RT-qPCR. We also investigated whether ZIKV interfered in the PIUV replication. PIUV suppressed the replication of ZIKV, resulting in a 10,000-fold reduction in ZIKV titers within 3 days post-infection. PIUV viral loads were not reduced in the presence of ZIKV. We conclude that, when concurrently infected, PIUV suppresses ZIKV in C6/36 cells while ZIKV does not interfere in PIUV replication.

Evidence of Differences in Cellular Regulation of Wolbachia-Mediated Viral Inhibition between Alphaviruses and Flaviviruses.

The intracellular bacterium Wolbachia is increasingly being utilised in control programs to limit the spread of arboviruses by Aedes mosquitoes. Achieving a better understanding of how Wolbachia strains can reduce viral replication/spread could be important for the long-term success of such programs. Previous studies have indicated that for some strains of Wolbachia, perturbations in lipid metabolism and cholesterol storage are vital in Wolbachia-mediated antiviral activity against the flaviviruses dengue and Zika; however, it has not yet been examined whether arboviruses in the alphavirus group are affected in the same way. Here, using the reporters for the alphavirus Semliki Forest virus (SFV) in Aedes albopictus cells, we found that Wolbachia strains wMel, wAu and wAlbB blocked viral replication/translation early in infection and that storage of cholesterol in lipid droplets is not key to this inhibition. Another alphavirus, o'nyong nyong virus (ONNV), was tested in both Aedes albopictus cells and in vivo in stable, transinfected Aedes aegypti mosquito lines. The strains wMel, wAu and wAlbB show strong antiviral activity against ONNV both in vitro and in vivo. Again, 2-hydroxypropyl-β-cyclodextrin (2HPCD) was not able to rescue ONNV replication in cell lines, suggesting that the release of stored cholesterol caused by wMel is not able to rescue blockage of ONNV. Taken together, this study shows that alphaviruses appear to be inhibited early in replication/translation and that there may be differences in how alphaviruses are inhibited by Wolbachia in comparison to flaviviruses.

Full genome characterization and evolutionary analysis of Banna virus isolated from Culicoides, mosquitoes and ticks in Yunnan, China.

Banna virus (BAV), a potential pathogen that may cause human encephalitis, is the prototype species of genus Seadornaviru within the family Reoviridae, and has been isolated from a variety of blood-sucking insects and mammals in Asia. Culicoides, Mosquitoes, and Ticks were collected overnight in Yunnan, China, during 2016-2023 using light traps. Virus was isolated from these collected blood-sucking insects and grown using Aedes albopictus (C6/36) cells. Preliminary identification of the virus was performed by agarose gel electrophoresis (AGE). The full genome sequences of the BAVs were determined by full-length amplification of cDNAs (FLAC) and sequenced using next-generation sequencing. In this study, 13 strains BAV were isolated from Culicoides, Mosquitoes and Ticks. Their viral genome consisted of 12 segments of double-stranded RNA (dsRNA), and with three distinct distribution patterns. Sequence analysis showed that Seg-5 of four strains (SJ_M46, SJ_M49, JC_M19-13 and JC_C24-13) has 435 bases nucleotide sequence insertions in their ORF compared to other BAVs, resulting in the length of Seg-5 up to 2128 nt. There are 34 bases sequence deletion in Seg-9 of 3 strains (WS_T06, MS_M166 and MS_M140). Comparison of the coding sequences of VP1, VP2, VP5, VP9 and VP12 of the 13 BAV strains, the results show that VP1, VP2 and VP12 are characterised by high levels of sequence conservation, while VP9 is highly variable, under great pressure to adapt and may be correlated with serotype. While also variable, VP5 appears to be under less adaptive pressure than VP9. Additionally, phylogenetic analysis indicates that the 13 BAV strains locate in the same evolutionary cluster as BAVs isolated from various blood-sucking insects, and are clustered according to geographical distribution. The data obtained herein would be beneficial for the surveillance of evolutionary characteristics of BAV in China and neighboring countries as well as extend the knowledge about its genomic diversity and geographic distribution.

A Chimeric Classical Insect-Specific Flavivirus Provides Complete Protection Against West Nile Virus Lethal Challenge in Mice.

West Nile virus (WNV), an arthropod-borne flavivirus, can cause severe symptoms, including encephalitis, and death, posing a threat to public health and the economy. However, there is still no approved treatment or vaccine available for humans. Here, we developed a novel vaccine platform based on a classical insect-specific flavivirus (cISF) YN15-283-02, which was derived from Culicoides. The cISF-WNV chimera was constructed by replacing prME structural genes of the infectious YN15-283-02 cDNA clone with those of WNV and successfully rescued in Aedes albopictus cells. cISF-WNV was nonreplicable in vertebrate cells and nonpathogenic in type I interferon receptor (IFNAR)-deficient mice. A single-dose immunization of cISF-WNV elicited considerable Th1-biased antibody responses in C57BL/6 mice, which was sufficient to offer complete protection against lethal WNV challenge with no symptoms. Our studies demonstrated the potential of the insect-specific cISF-WNV as a prophylactic vaccine candidate to prevent infection with WNV.

Isolation and Genetic Evolution of Dengue Virus from the 2019 Outbreak in Xishuangbanna, Yunnan Province, China.

Background: Dengue virus (DENV) can be divided into four serotypes-DENV-1, DENV-2, DENV-3, and DENV-4. In humans, infection leads to dengue fever (DF), dengue hemorrhagic fever, and dengue shock syndrome, both widely prevalent in tropical and subtropical regions. In 2019, a severe outbreak of DF occurred in Xishuangbanna, Yunnan province. Objective: To investigate the etiology and genotype of the causative agents of this severe dengue outbreak in Xishuangbanna. Methods: Between October and November 2019, the sera of patients clinically diagnosed with DF were collected in the first People's Hospital of Xishuangbanna. RNA was extracted from the sera and amplified by RT-PCR with flavivirus primers. Flavivirus-positive sera were then used to inoculate Aedes albopictus cells (C6/36); viral RNA was extracted from these cells, amplified, and sequenced with DENV E gene-specific primers. Sequence splicing and nucleotide homology genetic evolution analysis were carried out by biological software (DNAStar). Unique mutations in the E genes of isolated DENV were analyzed by SWISS-MODEL and PyMOL. Results: Of the 60 samples collected from DF patients, 39 tested positively with flavivirus primers. The DENV was isolated from 25 of the 39 positive seras, of which 20 showed cytopathic effects (CPE) and 5 were no CPE. In these 25 isolated nucleic acids, 21 strains of DENV-1, 3 strains of DENV-2, and 1 strain of DENV-3 were identified according to the sequence of E protein. In the four unique mutations (D52, Y149, L312, T386), D52 and Y149 in the E protein of DENV-1 were predicted to be exposed on the surface of the prefusion conformation. Conclusion: The 2019 outbreak of DF in Xishuangbanna area of Yunnan Province consists of at least three serotypes of DENV-1, DENV-2, and DENV-3, and the sources of these virus strains are of mixed and complicated origin.

Co-infection of dengue and Zika viruses mutually enhances viral replication in the mosquito Aedes aegypti

BackgroundThe mosquito Aedes aegypti transmits two of the most serious mosquito-borne viruses, dengue virus (DENV) and Zika virus (ZIKV), which results in significant human morbidity and mortality worldwide. The quickly shifting landscapes of DENV and ZIKV endemicity worldwide raise concerns that their co-circulation through the Ae. aegypti mosquito vector could greatly exacerbate the disease burden in humans. Recent reports have indicated an increase in the number of co-infection cases in expanding co-endemic regions; however, the impact of co-infection on viral infection and the detailed molecular mechanisms remain to be defined.MethodsC6/36 (Aedes albopictus) cells were cultured in Dulbecco's modified Eagle medium/Mitsuhashi and Maramorosch Insect Medium (DMEM/MM) (1:1) containing 2% heat-inactivated fetal bovine serum and 1× penicillin/streptomycin solution. For virus propagation, the cells were infected with either DENV serotype 2 (DENV2) strain 16681 or ZIKV isolate Thailand/1610acTw (MF692778.1). Mosquitoes (Ae. aegypti UGAL [University of Georgia Laboratory]/Rockefeller strain) were orally infected with DENV2 and ZIKV through infectious blood-feeding.ResultsWe first examined viral replication activity in cells infected simultaneously, or sequentially, with DENV and ZIKV, and found interspecies binding of viral genomic transcripts to the non-structural protein 5 (NS5). When we challenged Ae. aegypti mosquitos with both DENV2 and ZIKV sequentially to probe similar interactions, virus production and vector susceptibility to infection were significantly enhanced.ConclusionsOur results suggest that DENV2 and ZIKV simultaneously establishing infection in the Ae. aegypti mosquito vector may augment one another during replication. The data also implicate the homologous NS5 protein as a key intersection between the flaviviruses in co-infection, highlighting it as a potential target for vector control.

Differential Gene Expression Pattern of Importin β3 and NS5 in C6/36 Cells Acutely and Persistently Infected with Dengue Virus 2.

The establishment of persistent dengue virus infection within the cells of the mosquito vector is an essential requirement for viral transmission to a new human host. The mechanisms involved in the establishment and maintenance of persistent infection are not well understood, but it has been suggested that both viral and cellular factors might play an important role. In the present work, we evaluated differential gene expression in Aedes albopictus cells acutely (C6/36-HT) and persistently infected (C6-L) with Dengue virus 2 by cDNA-AFLP. We observed that importin β3 was upregulated in noninfected cells compared with C6-L cells. Using RT-qPCR and plaque assays, we observed that Dengue virus levels in C6-L cells essentially do not vary over time, and peak viral titers in acutely infected cells are observed at 72 and 120 h postinfection. The expression level of importin β3 was higher in acutely infected cells than in persistently infected cells; this correlates with higher levels of NS5 in the nucleus of the cell. The differential pattern of importin β3 expression between acute and persistent infection with Dengue virus 2 could be a mechanism to maintain viral infection over time, reducing the antiviral response of the cell and the viral replicative rate.

Syncytial and Congregative Effects of Dengue and Zika Viruses on the Aedes Albopictus Cell Line Differ among the Viral Strains.

Dengue viruses (DENV) and Zika viruses (ZIKV) are transmitted from human to human or from non-human primates to humans by mosquito biting, so the viral interaction with mosquito cells is one key step within the viral life cycle. Therefore, our objective is to know how DENV or ZIKV interacts with mosquito cells. Immunofluorescence assay and a direct visualization system are combined to monitor the syncytial or congregative effects of DENVs and ZIKVs on C6/36 cells. we studied the cytopathic effects of DENVs and ZIKVs on the mosquito cells, C6/36 which are widely used in the laboratory for the infections of DENV and ZIKV. Our results show that all strains of DENV-1 and DENV-2, most DENV-4 and some DENV-3 strains caused syncytial effects on C6/36 cells, while some DENV-3 and DENV-4 strains, and all the tested ZIKV strains caused cell congregation after infection but no cell fusion. In addition, we detected a range of pH environments from 6.0 to 8.0 that support the virus-caused cell fusion and figured out that the optimal pH condition is 7.5 at which the viral production is also the best. Furthermore, viral replication may be required for DENV's syncytial effects on C6/36 cells because the UV-inactivated virus failed to cause cell fusion. Syncytial and congregative effects of DENV and ZIKV on the Aedes albopictus cells differ among the viral strains. Syncytial effects of DENV on C6/36 are important for viral replication.

Genetic Characterization of DH13M98, Umatilla Virus, Isolated from Culex tritaeniorhynchus Giles in Yunnan Province, China

Background: In August 2013, a virus strain (DH13M98) was isolated from Culex tritaeniorhynchus Giles collected in Mangshi, the southwestern border area of Yunnan Province, China. The virus replicated and caused cytopathic effects (CPE) in Aedes albopictus (C6/36) cells, but not in baby hamster Syrian kidney (BHK-21) cells. Materials and Methods: Agarose gel electrophoresis (AGE) analysis revealed that the DH13M98 virus was a 10-segment double-stranded RNA (dsRNA) virus, with a "1-1-1-2-1-1-2-1" pattern. The full genome of the DH13M98 virus was sequenced by full-length amplification of complementary DNAs (FLAC). Results: Phylogenetic analysis of the viral RNA-dependent RNA polymerase (Pol), major subcore-shell (T2), and major core-surface (T13) protein showed that DH13M98 clustered with Umatilla virus (UMAV), and the amino acid (aa) sequences of DH13M98 shared more than 89.5% (Pol), 95% (T2), and 91.1% (T13) identity with UMAV. However, the aa identity of outer capsid protein one (OC1) of DH13M98 with other UMAV was 57.1-79.2%, suggesting that DH13M98 was UMAV, but distinct from other strains of UMAV from the United States, Japan, and Germany at OC1, and it may be a high variant strain of UMAV, even a new serotype. Conclusion: This is the first isolation of UMAV in China, which enriches the resources of virus species in China and provides new insights into the genetic diversity and geographical distribution of the virus.

Circulation of Ngari Virus in Livestock, Kenya.

Ngari virus (NRIV) is a mosquito-borne reassortant orthobunyavirus that causes severe febrile illness and hemorrhagic fever in humans and small ruminants. Due to limited diagnostics and surveillance, NRIV has only been detected sporadically during Rift Valley fever virus outbreaks. Little is known on its interepidemic maintenance and geographic distribution. In this study, sera from cattle, goats, and sheep were collected through a cross-sectional survey after the rainy seasons between 2020 and 2021 in two pastoralist-dominated semiarid ecosystems, Baringo and Kajiado counties in Kenya. NRIV was detected in 11 apparently healthy animals (11/2,039, 0.54%) by RT-PCR and isolated in cell culture from seven individuals. Growth analyses displayed efficient replication in cells from sheep and humans in contrast to weak replication in goat cells. NRIV infection of a wide variety of different vector cells showed only rapid replication in Aedes albopictus cells but not in cells derived from other mosquito species or sandflies. Phylogenetic analyses of complete-coding sequences of L, M, and S segments of four viruses showed that the Kenyan sequences established a monophyletic clade most closely related to a NRIV sequence from a small ruminant from Mauritania. NRIV neutralizing reactivity in cattle, goats, and sheep were 41.6% (95% CI = 30 to 54.3), 52.4% (95% CI = 37.7 to 66.6), and 19% (95% CI = 9.7 to 33.6), respectively. This is the first detection of NRIV in livestock in Kenya. Our results demonstrate active and undetected circulation of NRIV in the three most common livestock species highlighting the need for an active one-health surveillance of host networks, including humans, livestock, and vectors. IMPORTANCE Surveillance of vectors and hosts for infection with zoonotic arthropod-borne viruses is important for early detection and intervention measures to prevent outbreaks. Here, we report the undetected circulation of Ngari virus (NRIV) in apparently healthy cattle, sheep, and goats in Kenya. NRIV is associated with outbreaks of hemorrhagic fever in humans and small ruminants. We demonstrate the isolation of infectious virus from several animals as well as presence of neutralizing antibodies in 38% of the tested animals. Our data indicate active virus circulation and endemicity likely having important implications for human and animal health.

Full-genome characterisation of a putative novel serotype of Yonaguni orbivirus isolated from cattle in Yunnan province, China.

In July 2019, a novel viral strain (JH2019C603) was isolated from sentinel cattle in Jinghong City, in the subtropical region of Yunnan Province, China. The virus replicated and caused cytopathological effects in both Aedes albopictus (C6/36) and Baby Hamster Syrian Kidney (BHK-21) cells. Agarose gel electrophoresis analysis revealed a viral genome comprised of 10 segments of double-stranded RNA, with a 1-2-2-1-1-1-1-1 migration pattern. Complete genome sequences of the JH2019C603 virus were determined through full-length cDNA amplification. Phylogenetic analysis based on the amino acid (aa) sequences of RNA-dependent RNA Polymerase (Pol), Major subcore (T2) and Major core-surface (T13) showed that JH2019C603 clustered with Yonaguni orbivirus (YONOV) from Japan, with aa identities relative to YONOV of 97.7% (Pol), 99.0% (T2) and 98.5% (T13). However, phylogenetic analysis based on the aa sequences of the outer capsid protein one and two (OC1 and OC2) showed that JH2019C603 formed an independent branch in the phylogenetic tree, and its aa identity with YONOV was only 55.4% (OC1) and 80.8% (OC2), respectively. Compared with the prototype of YONOV, a notable sequence deletion was observed in the 3' non-coding region of NS1, with the NS1 of JH2019C603 encoded within segment 7 (Seg-7), in contrast to YONOV, which contains NS1 in Seg-6. These results indicate that JH2019C603 belongs to the YONOV lineage and might be a novel serotype or a highly variant strain of YONOV. These findings will facilitate the identification of new isolates and clarify their geographical distribution, epidemiology, genetic diversity and possible disease associations.

Impact of CHIKV Replication on the Global Proteome of Aedes albopictus Cells.

Arboviruses are some of the important causative agents of mosquito-mediated viral diseases. These viruses are transmitted between vector and host during the blood meal. Upon viral entry, host replication machinery is hijacked, supporting new virus particle production and thereby allowing viral survival in the host. In this process, host proteins interact with viral proteins to either facilitate viral replication, or they may provide antiviral defense mechanisms. In this study, we analyzed the impact of chikungunya virus (CHIKV) infection on the global proteome of Dicer active Aedes albopictus cells during the early and late time points of infection. We utilized a bottom-up approach of global proteomics analysis, and we used label-free quantitative mass spectrometry to identify the global protein signatures of Ae. albopictus at two different time points upon CHIKV infection. The mass spectrometry data analysis of the early time point revealed that proteins belonging to pathways such as translation, RNA processing, and cellular metabolic processes were less in abundance, whereas those belonging to pathways such as cellular catabolic process and organic substance transport were significantly abundant. At later time points, proteins belonging to pathways such as cellular metabolic processes, primary metabolic process, organonitrogen compound metabolic process, and organic substance metabolic process were found to be decreased in their presence, whereas those belonging to pathways such as RNA processing, gene expression, macromolecule metabolic processing, and nitrogen compound metabolic processing were found to be abundant during CHIKV infection, indicating that modulation in gene expression favoring cell survival occurs at a later time point, suggesting a survival strategy of Aedes cells to counter prolonged CHIKV infection.

The Dengue Virus Nonstructural Protein 1 (NS1) Interacts with the Putative Epigenetic Regulator DIDO1 to Promote Flavivirus Replication in Mosquito Cells.

Dengue virus (DENV) NS1 is a multifunctional protein essential for viral replication. To gain insights into NS1 functions in mosquito cells, the protein interactome of DENV NS1 in C6/36 cells was investigated using a proximity biotinylation system and mass spectrometry. A total of 817 mosquito targets were identified as protein-protein interacting with DENV NS1. Approximately 14% of them coincide with interactomes previously obtained in vertebrate cells, including the oligosaccharide transferase complex, the chaperonin containing TCP-1, vesicle localization, and ribosomal proteins. Notably, other protein pathways not previously reported in vertebrate cells, such as epigenetic regulation and RNA silencing, were also found in the NS1 interactome in mosquito cells. Due to the novel and strong interactions observed for NS1 and the epigenetic regulator DIDO1 (Death-Inducer Obliterator 1), the role of DIDO1 in viral replication was further explored. Interactions between NS1 and DIDO1 were corroborated in infected mosquito cells, by colocalization and proximity ligation assays. Silencing DIDO1 expression results in a significant reduction in DENV and ZIKV replication and progeny production. Comparison of transcription analysis of mock or DENV infected cells silenced for DIDO1 revealed variations in multiple gene expression pathways, including pathways associated with DENV infection such as RNA surveillance, IMD, and Toll. These results suggest that DIDO1 is a host factor involved in the negative modulation of the antiviral response necessary for flavivirus replication in mosquito cells. Our findings uncover novel mechanisms of NS1 to promote DENV and ZIKV replication, and add to the understanding of NS1 as a multifunctional protein. IMPORTANCE Dengue is the most important mosquito-borne viral disease to humans. Dengue virus NS1 is a multifunctional protein essential for replication and modulation of innate immunity. To gain insights into NS1 functions, the protein interactome of dengue virus NS1 in Aedes albopictus cells was investigated using a proximity biotinylation system and mass spectrometry. Several protein pathways, not previously observed in vertebrate cells, such as transcription and epigenetic regulation, were found as part of the NS1 interactome in mosquito cells. Among those, DIDO1 was found to be a necessary host factor for dengue and Zika virus replication in mosquito cells. Transcription analysis of infected mosquito cells silenced for DIDO1 revealed alterations of the IMD and Toll pathways, part of the antiviral response in mosquitoes. The results suggest that DIDO1 is a host factor involved in modulation of the antiviral response and necessary for flavivirus replication.

Genomic Analyses of the Fungus Paraconiothyrium sp. Isolated from the Chinese White Wax Scale Insect Reveals Its Symbiotic Character.

The Chinese white wax scale, Ericerus pela, is an insect native to China. It harbors a variety of microbes. The Paraconiothyrium fungus was isolated from E. pela and genome sequenced in this study. A fungal cytotoxicity assay was performed on the Aedes albopictus cell line C6/36. The assembled Paraconiothyrium sp. genome was 39.55 Mb and consisted of 14,174 genes. The coding sequences accounted for 50.75% of the entire genome. Functional pathway analyses showed that Paraconiothyrium sp. possesses complete pathways for the biosynthesis of 20 amino acids, 10 of which E. pela lacks. It also had complementary genes in the vitamin B groups synthesis pathways. Secondary metabolism prediction showed many gene clusters that produce polyketide. Additionally, a large number of genes associated with ‘reduced virulence’ in the genome were annotated with the Pathogen–Host Interaction database. A total of 651 genes encoding carbohydrate-active enzymes were predicted to be mostly involved in plant polysaccharide degradation. Pan-specific genomic analyses showed that genes unique to Paraconiothyrium sp. were enriched in the pathways related to amino acid metabolism and secondary metabolism. GO annotation analysis yielded similar results. The top COG categories were ‘carbohydrate transport and metabolism’, ‘lipid transport and metabolism’, and ‘secondary metabolite biosynthesis, transport and catabolism’. Phylogenetic analyses based on gene family and pan genes showed that Paraconiothyrium sp is clustered together with species from the Didymosphaeriaceae family. A multi-locus sequence analysis showed that it converged with the same branch as P. brasiliense and they formed one group with fungi from the Paraconiothyrium genus. To validate the in vitro toxicity of Paraconiothyrium sp., a cytotoxicity assay was performed. The results showed that medium-cultured Paraconiothyrium sp. had no harmful effect on cell viability. No toxins were secreted by the fungus during growth. Our results imply that Paraconiothyrium sp. may establish a symbiotic relationship with the host to supply complementary nutrition to E. pela.

Transcriptome Analysis of an Aedes albopictus Cell Line Single- and Dual-Infected with Lammi Virus and WNV.

Understanding the flavivirus infection process in mosquito hosts is important and fundamental in the search for novel control strategies that target the mosquitoes’ ability to carry and transmit pathogenic arboviruses. A group of viruses known as insect-specific viruses (ISVs) has been shown to interfere with the infection and replication of a secondary arbovirus infection in mosquitoes and mosquito-derived cell lines. However, the molecular mechanisms behind this interference are unknown. Therefore, in the present study, we infected the Aedes albopictus cell line U4.4 with either the West Nile virus (WNV), the insect-specific Lammi virus (LamV) or an infection scheme whereby cells were pre-infected with LamV 24 h prior to WNV challenge. The qPCR analysis showed that the dual-infected U4.4 cells had a reduced number of WNV RNA copies compared to WNV-only infected cells. The transcriptome profiles of the different infection groups showed a variety of genes with altered expression. WNV-infected cells had an up-regulation of a broad range of immune-related genes, while in LamV-infected cells, many genes related to stress, such as different heat-shock proteins, were up-regulated. The transcriptome profile of the dual-infected cells was a mix of up- and down-regulated genes triggered by both viruses. Furthermore, we observed an up-regulation of signal peptidase complex (SPC) proteins in all infection groups. These SPC proteins have shown importance for flavivirus assembly and secretion and could be potential targets for gene modification in strategies for the interruption of flavivirus transmission by mosquitoes.

Small RNA Response to Infection of the Insect-Specific Lammi Virus and Hanko Virus in an Aedes albopictus Cell Line

RNA interference (RNAi)-mediated antiviral immunity is believed to be the primary defense against viral infection in mosquitoes. The production of virus-specific small RNA has been demonstrated in mosquitoes and mosquito-derived cell lines for viruses in all of the major arbovirus families. However, many if not all mosquitoes are infected with a group of viruses known as insect-specific viruses (ISVs), and little is known about the mosquito immune response to this group of viruses. Therefore, in this study, we sequenced small RNA from an Aedes albopictus-derived cell line infected with either Lammi virus (LamV) or Hanko virus (HakV). These viruses belong to two distinct phylogenetic groups of insect-specific flaviviruses (ISFVs). The results revealed that both viruses elicited a strong virus-derived small interfering RNA (vsiRNA) response that increased over time and that targeted the whole viral genome, with a few predominant hotspots observed. Furthermore, only the LamV-infected cells produced virus-derived Piwi-like RNAs (vpiRNAs); however, they were mainly derived from the antisense genome and did not show the typical ping-pong signatures. HakV, which is more distantly related to the dual-host flaviviruses than LamV, may lack certain unknown sequence elements or structures required for vpiRNA production. Our findings increase the understanding of mosquito innate immunity and ISFVs’ effects on their host.

Induction of cell migration by transient Wolbachia pipientis infection in Aedes albopictus cell line

• The effect of transient Wolbachia infection was investigated in vitro. • Transient Wolbachia infection induced the migration of Aedes albopictus cells. • Wolbachia infection induced production of nitrite and reactive oxygen species. • Wolbachia infection elevated the expression of Toll pathway-related genes. • Early adaptation of Wolbachia in original host insect affects insect cell features. Wolbachia is a genus of maternally transmitted bacteria having an endosymbiotic relationship with arthropod and nematode species. These bacteria manipulate host development, sex-determination, and reproduction. In addition, they are known to potentially suppress vector-borne diseases by interfering with pathogen transmission. Although the occurrence of Wolbachia infection in insects has been known, the underlying mechanisms that mediate their interactions remain unclear. To examine the influence of Wolbachia adaptation on the host, we infected w AlbA and w AlbB strain from Aedes albopictus into the C6/36 cell line derived from Ae. albopictus . The transient Wolbachia infection was characterized by induction of cell migration without cell proliferation. The production of nitrite and reactive oxygen species (ROS) was induced by transient Wolbachia infection. Cells with transient Wolbachia infection exhibited elevated expression of Toll-like receptor 6 (TLR6) and myeloid differentiation primary response 88 (Myd88). Conversely, the expression of TLR2, TLR4, TLR7, Cactus, Ankyrin, and Argonaute2 (AGO2) was inhibited upon Wolbachia infection. These results suggest that Wolbachia has an influence on the cell migration ability as well as host innate immune response in vitro . Considering these results, transient Wolbachia strain transfer in C6/36 cells might be an important approach for studying Wolbachia- host interactions and might help gain a deeper understanding of the early adaptation of Wolbachia in the original host insect.

Infection, dissemination, and transmission efficiencies of Zika virus in Aedes aegypti after serial passage in mosquito or mammalian cell lines or alternating passage in both cell types

BackgroundZika virus (ZIKV) is an arthropod-borne virus (arbovirus) with an urban transmission cycle that primarily involves humans and Aedes aegypti. Evidence suggests that the evolution of some arboviruses is constrained by their dependency on alternating between disparate (vertebrate and invertebrate) hosts. The goals of this study are to compare the genetic changes that occur in ZIKV after serial passaging in mosquito or vertebrate cell lines or alternate passaging in both cell types and to compare the replication, dissemination, and transmission efficiencies of the cell culture-derived viruses in Ae. aegypti.MethodsAn isolate of ZIKV originally acquired from a febrile patient in Yucatan, Mexico, was serially passaged six times in African green monkey kidney (Vero) cells or Aedes albopictus (C6/36) cells or both cell types by alternating passage. A colony of Ae. aegypti from Yucatan was established, and mosquitoes were challenged with the cell-adapted viruses. Midguts, Malpighian tubules, ovaries, salivary glands, wings/legs and saliva were collected at various times after challenge and tested for evidence of virus infection.ResultsGenome sequencing revealed the presence of two non-synonymous substitutions in the premembrane and NS1 regions of the mosquito cell-adapted virus and two non-synonymous substitutions in the capsid and NS2A regions of both the vertebrate cell-adapted and alternate-passaged viruses. Additional genetic changes were identified by intrahost variant frequency analysis. Virus maintained by continuous C6/36 cell passage was significantly more infectious in Ae. aegypti than viruses maintained by alternating passage and consecutive Vero cell passage.ConclusionsMosquito cell-adapted ZIKV displayed greater in vivo fitness in Ae. aegypti compared to the other viruses, indicating that obligate cycling between disparate hosts carries a fitness cost. These data increase our understanding of the factors that drive ZIKV adaptation and evolution and underscore the important need to consider the in vivo passage histories of flaviviruses to be evaluated in vector competence studies.Graphical abstract

Expression profile of the PIWI mRNAs protein family in human cells experimentally infected with Dengue Virus 4

Objective: Evaluate the messenger RNAs (mRNA) PIWI proteins expression during infection with VDEN 4 in human hepatocyte cells. Materials and Methods: VDEN4 strain H778494 (JQ513335) was used, which was stored in Aedes albopictus cell culture (Clone C6 / 36) at the Arbovirology and Hemorrhagic Fevers section of the Evandro Chagas Institute. The techniques of cell cultures, stock (C6/36), inoculation, extraction, viral load quantification, RTqPCR and statistical analyzes were performed at the Viral Biogenesis Laboratory. Results: According to the results obtained, two cells (HepG2 and Huh7.5) demonstrated a higher level of viral replication at 72 hours post infection (hpi). The PIWI 2 target alter its mRNA expression during VDEN4 infection. The PIWI 4 target expression, was observed an altered expression in the infected cells. Thus, it was found that they are in different poles, while the viral load in the first three days showed high expression. The expression of mRNA was low in relation to the normal rate given by the uninfected cells. Conclusion: In our findings, it was observed that PIWI2 and 4 proteins have an inverse relationship to the viral infection process by VDEN4, when there is an increase in viral replication, these two proteins end up having a significant reduction in their expression. Probably, this reduction of expression is involved with the biogenesis process of apoptosis-regulating microRNAs.