Mosquito cell-derived West Nile virus replicon particles mimic arbovirus inoculum and have reduced spread in mice

Abstract

Half of the human population is at risk of infection by an arthropod-borne virus. Many of these arboviruses, such as West Nile, dengue, and Zika viruses, infect humans by way of a bite from an infected mosquito. This infectious inoculum is insect cell-derived giving the virus particles distinct qualities not present in secondary infectious virus particles produced by infected vertebrate host cells. The insect cell-derived particles differ in the glycosylation of virus structural proteins and the lipid content of the envelope, as well as their induction of cytokines. Thus, in order to accurately mimic the inoculum delivered by arthropods, arboviruses should be derived from arthropod cells. Previous studies have packaged replicon genome in mammalian cells to produce replicon particles, which undergo only one round of infection, but no studies exist packaging replicon particles in mosquito cells. Here we optimized the packaging of West Nile virus replicon genome in mosquito cells and produced replicon particles at high concentration, allowing us to mimic mosquito cell-derived viral inoculum. These particles were mature with similar genome equivalents-to-infectious units as full-length West Nile virus. We then compared the mosquito cell-derived particles to mammalian cell-derived particles in mice. Both replicon particles infected skin at the inoculation site and the draining lymph node by 3 hours post-inoculation. The mammalian cell-derived replicon particles spread from the site of inoculation to the spleen and contralateral lymph nodes significantly more than the particles derived from mosquito cells. This in vivo difference in spread of West Nile replicons in the inoculum demonstrates the importance of using arthropod cell-derived particles to model early events in arboviral infection and highlights the value of these novel arthropod cell-derived replicon particles for studying the earliest virus-host interactions for arboviruses.

Growth and adaptation of Zika virus in mammalian and mosquito cells

“…This is the first description of how ZIKV RNA levels correspond to infectious virus particles. The ZIKV ratio is similar to reported ratios for the flaviviruses DENV and yellow fever virus, which have GE:PFU ratios of 1-5x10 3 [ 52 , 53 ], but is approximately 10-fold higher than WNV [ 54 – 56 ]. The GE:PFU ratio of ZIKV mam was 2–7 fold higher than ZIKV mos , similar to the difference observed between mammalian cell- and mosquito cell-produced WNV [ 55 ].…”

“…C6/36 cells do not demonstrate CPE, and may produce virus longer than Vero cells, which succumb to infection. Similarly, C6/36 cells demonstrate sustained production of subgenomic replicon particles for up to 10 days after transfection [ 54 ]. The disparity in maintaining high levels of virus may reflect host physiological differences with important implications on flavivirus transmission.…”

New reverse genetics and transfection methods to rescue arboviruses in mosquito cells

“…Transfection methods using regular lipid-based transfection reagent are supposed to have mainly been optimized to be used on mammalian cell lines that are cultured at 37 °C. Accordingly, when applying the same transfection protocol to other vertebrates or invertebrate cells like fish or mosquito cells, which are cultured at lower temperature (5–15 °C), the efficiency of transfection decreases and is often below 10% 14 , 25 . While a low percentage of transfection may be sufficient for some studies, the rescue of recombinant viruses from overlapped DNA amplicons often requires higher transfection efficiency.…”

“…However, whether the ISA method could be adapted to Aedes mosquito cells remained to be demonstrated. Second, previous studies using electroporation and/or transfection with regular lipids-based reagents of RNA in Aedes mosquito cells demonstrated a very low efficiency of RNA delivery (ranging from 0.2 to 10%) 14 , 15 . Therefore, new powerful method of nucleic acids delivery in Aedes mosquito cells is needed and especially to be applied to the ISA reverse genetics system.…”

Biological and phylogenetic characteristics of West African lineages of West Nile virus

“…The ratio of particles per infectious unit in the initial viral stocks ranged from 8 to 600 [ 98 ]. Our viral stocks had a similar ratio of particles per infectious unit as that seen produced by fully infectious extracellular WN virus particles [ 100 ] and mosquito-derived replicon WN virus particles [ 101 ]. Variances in replication efficiency between studies observed during in vitro infection could be explained to differences in the viral strain and to the infection conditions i .…”