Adventitious viruses persistently infect three commonly used mosquito cell lines

Weger-Lucarelli, James; Rückert, Claudia; Grubaugh, Nathan D.; Misencik, Michael J.; Armstrong, Philip M.; Stenglein, Mark D.; Ebel, Gregory D.; Brackney, Doug E.

doi:10.1016/j.virol.2018.06.007

Cited by 34 publications

(26 citation statements)

References 55 publications

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“…Importantly, Rps7 was amplified when genomic DNA was treated with RNase, but not DNase, confirming the purity of the DNA samples (Fig 2A). Although we cannot exclude the possibility that the primers we designed missed smaller fragments of integrated PCLV sequence, our data suggest that PCLV fragments are not integrated into the genome of parental Aag2 cells, which is in agreement with data from other research groups [56].…”

Section: Resultssupporting

confidence: 90%

“…Cell fusing agent virus (CFAV; family Flaviviridae , genus Flavivirus ) was the first insect-specific virus discovered and has long been known to persistently infect Aag2 cells and other Ae. aegypti cell lines [3,55,56]. In addition, we previously discovered Aag2 cells to be persistently infected with the insect-specific virus Phasi Charoen-like virus (PCLV; order Bunyavirales , family Phenuiviridae , genus Phasivirus ) [57].…”

Section: Introductionmentioning

confidence: 99%

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Aedes aegypti (Aag2)-derived clonal mosquito cell lines reveal the impact of pre-existing persistent infection with the insect-specific bunyavirus Phasi Charoen-like virus on arbovirus replication

Fredericks

Wallace

Russell

et al. 2019

Preprint

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BackgroundAedes aegypti is a vector mosquito of major public health importance, transmitting arthropod-borne viruses (arboviruses) such as chikungunya, dengue, yellow fever and Zika viruses. Wild mosquito populations are persistently infected at high prevalence with insect-specific viruses that do not replicate in vertebrate hosts. In experimental settings, acute infections with insect-specific viruses have been shown to modulate arbovirus infection and transmission in Ae. aegypti and other vector mosquitoes. However, the impact of persistent insect-specific virus infections that more closely mimic the situation in nature has not been investigated extensively. Cell lines are useful models for studying virus-host interactions, however the available Ae. aegypti cell lines are poorly defined and heterogenous cultures.Methodology/Principle FindingsWe generated single cell-derived clonal cell lines from the commonly used Ae. aegypti cell line Aag2. Two of the fourteen Aag2-derived clonal cell lines generated harboured markedly and consistently reduced levels of the insect-specific bunyavirus Phasi Charoen-like virus (PCLV) known to persistently infect Aag2 cells. In contrast to studies with acute insect-specific virus infections in cell culture and in vivo, we found that pre-existing persistent PCLV infection had no major impact on the replication of the flaviviruses dengue virus and Zika virus, the alphavirus Sindbis virus, or the rhabdovirus vesicular stomatitis virus. We also performed a detailed characterisation of the morphology, transfection efficiency and immune status of our Aag2-derived clonal cell lines, and have made a clone that we term Aag2-AF5 available to the research community as a well-defined cell culture model for arbovirus-vector interaction studies.Conclusions/SignificanceOur findings highlight the need for further in vivo studies that more closely recapitulate natural arbovirus transmission settings in which arboviruses encounter mosquitoes harbouring persistent rather than acute insect-specific virus infections. Furthermore, we provide the well-characterised Aag2-derived clonal cell line as a valuable resource to the arbovirus research community.AUTHOR SUMMARYMosquito-borne viruses usually only infect humans through the bite of a mosquito that carries the virus. Viruses transmitted by the ‘yellow fever mosquito’ Aedes aegypti, including dengue virus, Zika virus, yellow fever virus and chikungunya virus, are causing an ever-increasing number of human disease cases globally. Mosquito-borne viruses have to infect and replicate inside the mosquito before they are transmitted to humans, and the presence of other infectious agents can change the efficiency of virus transmission. Mosquitoes are known to be infected with ‘insect-specific viruses’ that only infect mosquitoes and cannot cause human disease. We have shown here that in laboratory cell cultures derived from the Aedes aegypti mosquito, pre-existing infection with an insect-specific virus called Phasi Charoen-like virus does not affect the infection and growth of the mosquito-borne viruses dengue virus, Zika virus, Sindbis virus or vesicular stomatitis virus. Compared to previous research, our research is more reflective of conditions that mosquito-borne viruses encounter in nature, and our results provide important new insights into whether and how insect-specific viruses affect mosquito-borne virus transmission. Ultimately, this information could inform ongoing research into whether insect-specific viruses could be used to prevent the transmission of mosquito-borne viruses to reduce global disease burdens.

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Section: Resultssupporting

confidence: 90%

Section: Introductionmentioning

confidence: 99%

Aedes aegypti (Aag2)-derived clonal mosquito cell lines reveal the impact of pre-existing persistent infection with the insect-specific bunyavirus Phasi Charoen-like virus on arbovirus replication

Fredericks

Wallace

Russell

et al. 2019

Preprint

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“…Schultz et al screened their mosquito cell lines and discovered a persistent infection of CFAV and PCLV [ 104 ]. Further, Weger-Lucarelli et al screened the four widely used mosquito cell lines C6/36, U4.4, Aag2 and HSU and confirmed that all four cell lines were persistently infected with several viruses [ 109 ]. This should to be considered when designing an experiment or analysing data generated from these cell lines.…”

Section: Vector Competence Studiesmentioning

confidence: 99%

Insect-specific virus evolution and potential effects on vector competence

2019

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The advancement in high-throughput sequencing technology and bioinformatics tools has spurred a new age of viral discovery. Arthropods is the largest group of animals and has shown to be a major reservoir of different viruses, including a group known as insect-specific viruses (ISVs). The majority of known ISVs have been isolated from mosquitoes and shown to belong to viral families associated with animal arbovirus pathogens, such as Flaviviridae, Togaviridae and Phenuiviridae . These insect-specific viruses have a strict tropism and are unable to replicate in vertebrate cells, these properties are interesting for many reasons. One is that these viruses could potentially be utilised as biocontrol agents using a similar strategy as for Wolbachia . Mosquitoes infected with the viral agent could have inferior vectorial capacity of arboviruses resulting in a decrease of circulating arboviruses of public health importance. Moreover, insect-specific viruses are thought to be ancestral to arboviruses and could be used to study the evolution of the switch from single-host to dual-host. In this review, we discuss new discoveries and hypothesis in the field of arboviruses and insect-specific viruses.

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“…Of note, different strains of a particular cell line do not necessarily contain the same persistent infections. For example, Weger-Lucarelli et al identified only CYV in U4.4 cells [218], while our U4.4 cells are additionally infected with the ISVs PCLV and CFAV. Cross-contamination between insect cell lines is a risk, as in most laboratories cell lines are passaged in parallel.…”

Section: Discussionmentioning

confidence: 53%

“…With the introduction of next-generation sequencing (NGS) technologies and metagenomics into the field of virology, it becomes increasingly clear that many insect cell lines and insects, including mosquitoes, carry persistently infecting ISVs [217][218][219][220]. To investigate the presence of ISVs in our Cx.t and U4.4 cells, the libraries of small RNA reads of these two cell lines were subjected to de novo assembly.…”

Section: Discovery Of Insect-specific Viruses By De Novo Assembly Of mentioning

confidence: 99%

Molecular determinants of arbovirus transmission by mosquitoes

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Adventitious viruses persistently infect three commonly used mosquito cell lines

Cited by 34 publications

References 55 publications

Aedes aegypti (Aag2)-derived clonal mosquito cell lines reveal the impact of pre-existing persistent infection with the insect-specific bunyavirus Phasi Charoen-like virus on arbovirus replication

Aedes aegypti (Aag2)-derived clonal mosquito cell lines reveal the impact of pre-existing persistent infection with the insect-specific bunyavirus Phasi Charoen-like virus on arbovirus replication

Insect-specific virus evolution and potential effects on vector competence

Molecular determinants of arbovirus transmission by mosquitoes

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