Detection and clearance of a mosquito densovirus contaminant from laboratory stocks of Zika virus

BACKGROUND The Zika virus (ZIKV) epidemics that affected South America in 2016 raised several research questions and prompted an increase in studies in the field. The transient and low viraemia observed in the course of ZIKV infection is a challenge for viral isolation from patient serum, which leads to many laboratories around the world sharing viral strains for their studies. C6/36 cells derived from Aedes albopictus larvae are commonly used for arbovirus isolation from clinical samples and for the preparation of viral stocks. OBJECTIVES Here, we report the contamination of two widely used ZIKV strains by Brevidensovirus, here designated as mosquito densovirus (MDV). METHODS Molecular and immunological techniques were used to analyse the MDV contamination of ZIKV stocks. Also, virus passages in mammalian cell line and infecting susceptible mice were used to MDV clearance from ZIKV stocks. FINDINGS MDV contamination was confirmed by molecular and immunological techniques and likely originated from C6/36 cultures commonly used to grow viral stocks. We applied two protocols that successfully eliminated MDV contamination from ZIKV stocks, and these protocols can be widely applied in the field. As MDV does not infect vertebrate cells, we performed serial passages of contaminated stocks using a mammalian cell line and infecting susceptible mice prior to re-isolating ZIKV from the animals' blood serum. MDV elimination was confirmed with immunostaining, polymerase chain reaction (PCR), and analysis of the mosquitoes that were allowed to feed on the infected mice. MAIN CONCLUSIONS Since the putative impact of viral contaminants in ZIKV strains generally used for research purposes is unknown, researchers working in the field must be aware of potential contaminants and test viral stocks to certify sample purity.

Pathogenicity and infectivity of the Thai-strain densovirus (AThDNV) in Anopheles minimus S.L.

We report the first experimental infection of an anopheline, An. minimus s.l., with the Thai-strain densovirus, AThDNV. Two hundred first-instar larvae were raised in 2 virus concentrations; mortality in the low virus concentration was no different than that in controls (9.5% vs 7.5%, respectively). Mortality in the high virus concentration was 17.5%. Among surviving adults, infection rates were 33.3% (low concentration) and 15.5% (high concentration), as judged by PCR-screening. Infection rates did not differ between males and females. All orally-infected females transmitted densovirus to at least some of their offspring. Vertical transmission rates ranged from 25.0-53.8%. Densovirus infection did not appear to affect fecundity, either in the number of eggs laid or the number of eggs hatched.