Kanthong, N. | Search bioRxiv

bioRxiv2022

Host reverse transcriptase helps establish and maintain persistent dengue virus infections in C6/36 insect cells

C6/36 cells challenged with Dengue virus serotype 2 (DENV-2 strain NGC) show initial cytopathic effects (CPE) they overcome within 3 split passages and resume normal growth despite persistent infection with DENV-2. We hypothesized that tolerated persistent infections also required persistent host reverse-transcriptase (HRT) activity, and this was subsequently proven using the RT inhibitor AZT (azidothymidine, a nucleoside analogue antiretroviral drug) to treat C6/36 cells challenged with DENV-1. We confirmed this using the RT inhibitor tenofovir disoproxil fumarate (TDF) with C6/36, U4.4cells challenged with DENV-1. However, it has never been determined whether the persistently tolerated infections are also dependent on persistent HRT activity. This brief report reveals that persistent HRT activity is required to maintain persistent DENV-2 infections. Specifically, TDF treatment (0.1 mM) revealed minor toxicity to naive C6/36 cells but led to cell death instead of accommodation upon concomitant challenge with DENV-2, as previously reported for DENV-1. However, TDF treatment of stable, grossly normal C636 cell cultures persistently infected with DENV-2 for up to 30 split passages reverted to CPE, supporting our earlier hypothesis that persistent host RT activity is also required to maintain persistent infections. This is a practical tool for research applications in virology of crustaceans and insects for screening living, grossly healthy, imported animals for listed viral pathogens.

immunology↗

bioRxiv2022

Shrimp vaccination with insect-adapted yellow head virus (YHV) extends survival upon YHV challenge

This short paper on yellow head virus Type1 (YHV-1) describes preliminary research worthy of further study. YHV-1 disease outbreaks can cause severe mortality in the cultivated shrimp Penaeus (Penaeus) monodon and Penaeus (Litopenaeus) vannamei. No practical preventative treatment such as vaccination has been reported. However, it has been shown that C6/36 mosquito cell cultures can adapt to YHV-1 and become persistently immunopositive for the virus after 30 split-cell passages or more. Shrimp injection with homogenates from low passages caused yellow head disease (YHD) but from high passages did not, even though injection resulted in immunopositive hemocytes in the injected shrimp. This suggested YHV-1 attenuation during insect cell passaging and the possibility of using cell homogenates as a vaccine to protect shrimp against virulent YHV-1. To test this hypothesis, we injected shrimp with 30th passage homogenates to test for protection against YHD upon subsequent challenge with virulent YHV-1. Results confirmed earlier work that hemocytes of the infected shrimp became both reverse transcriptase PCR positive and immunopositive for YHV-1 but exhibited no mortality. Similarly, there was no mortality in the control group injected with homogenate from YHV-1 negative insect cells. When subsequently challenged with YHV-1, shrimp in the positive control group injected with homogenate from naive insect cells gave 100 percent mortality within 7 days post challenge while total mortality in the group injected with YHV-1 homogenate did not occur until day 9 post challenge. Kaplan-Meier log-rank survival analysis revealed that survival curves for the two groups were significantly different (p<0.001) and that the mean survival time for the test group (6.5 days) was significantly longer than that in the positive control group (5.4 days). The results confirmed that shrimp injection with YHV-1 immunopositive insect-cell homogenate gave transient resistance to YHV infection and that further research into the possible use of insect cell lines to produce shrimp antiviral vaccines is warranted.

microbiology↗