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.

A rapid polymerase chain reaction protocol to detect adenovirus in eye swabs

Purpose: Viruses of the Adenoviridae family are associated with many clinical syndromes, processing 50 serotypes. These agents and viruses of the Herpesviridae family are the two major agents responsible for viral conjunctivitis, and a rapid diagnosis is important due to the epidemic character of adenoviral infection. Methods: We developed a PCR without DNA extraction for adenovirus using primers thath amplify a 300 tp gragament of the hexon capsid protein gene from many serotypes. Results: Swab samples from cornea of seven patients with keratoconjunctivitis were analyzed, and one of them was PCR positive for adenovirus. The sequence of this fragment shows a 100 percent homology with the sequence of adenovirus type 8. Conclusion: Sequencing of 300 bp from the hexon gene allows to identity almost all Ad serotypes, including all serotypes related to epidemic keratokonjuntivitis (8, 19,37) and almost all serotypes involved with Ad-associated conjunctivitis.