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ABSTRACT-Experiments with crustacean viruses are hampered by lack of susceptible continuous cell lines. To overcome this problem, immortal mosquito and lepidopteran cell lines were both separately challenged with a shrimp DNA virus (white spot syndrome virus: WSSV, = PRDV) and RNA virus (yellow head virus: YHV) followed by serial, split-passage with immunohistochemical monitoring by confocal laser microscopy using labeled monoclonal antibodies to shrimp viral antigens. Stable, immortal cultures with 100% of the cells expressing shrimp-virus antigens were obtained, although the infected cells appeared grossly normal by phase contrast microscopy. Nor did they show any ultrastructural modifications characteristic of the challenge viruses. These persistently-expressing insect cell cultures were stable and could be continuously passaged, stored and revived as required. Since disparate viruses and insect cells were used, this appears to be a generic process that may be applicable to other shrimp viruses as well.

BackgroundIt is known that insects and crustaceans can carry simultaneous, active infections of two or more viruses without showing signs of disease, but it was not clear whether co-infecting viruses occupied the same cells or different cells in common target tissues. Our previous work showed that successive challenge of mosquito cell cultures followed by serial, split-passage resulted in stabilized cultures with 100% of the cells co-infected with Dengue virus (DEN) and an insect parvovirus (densovirus) (DNV). By addition of Japanese encephalitis virus (JE), we tested our hypothesis that stable, persistent, triple-virus co-infections could be obtained by the same process.ResultsUsing immunocytochemistry by confocal microscopy, we found that JE super-challenge of cells dually infected with DEN and DNV resulted in stable cultures without signs of cytopathology, and with 99% of the cells producing antigens of the 3 viruses. Location of antigens for all 3 viruses in the triple co-infections was dominant in the cell nuclei. Except for DNV, this differed from the distribution in cells persistently infected with the individual viruses or co-infected with DNV and DEN. The dependence of viral antigen distribution on single infection or co-infection status suggested that host cells underwent an adaptive process to accommodate 2 or more viruses.ConclusionsIndividual mosquito cells can accommodate at least 3 viruses simultaneously in an adaptive manner. The phenomenon provides an opportunity for genetic exchange between diverse viruses and it may have important medical and veterinary implications for arboviruses.

Research on crustacean viruses is hampered by the lack of continuous cell lines susceptible to them. To overcome this problem, we previously challenged immortal mosquito and lepidopteran cell lines with shrimp yellow head virus (YHV), followed by serial, split-passage of whole cells, and showed that this produced cells that persistently expressed YHV antigens. To determine whether such insect cultures positive for YHV antigens could be used to infect shrimp Penaeus monodon with YHV, culture supernatants and whole-cell homogenates were used to challenge shrimp by injection. Shrimp injected with culture supernatants could not be infected. However, shrimp injection-challenged with whole-cell homogenates from Passage 5 (early-passage) of such cultures died with histological and clinical signs typical for yellow head disease (YHD), while homogenates of mock-passaged, YHV-challenged cells did not. By contrast, shrimp challenged with cell homogenates of late-passage cultures became infected with YHV, but survived, suggesting that YHV attenuation had occurred during its long-term serial passage in insect cells. Thus, YHV could be propagated successfully in C6/36 mosquito cells and used at low passage numbers as a source of inoculum to initiate lethal infections in shrimp. This partially solves the problem of lack of continuous shrimp cell lines for cultivation of YHV.

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