Persistent Expression of Shrimp-virus Antigens in Two Insect Cell Lines Challenged with Two Shrimp Viruses

Abstract

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.

Successful propagation of shrimp yellow head virus in immortal mosquito cells

“…In contrast to whole cells, culture supernatant solutions were negative for YHV using the same RT-PCR protocol, suggesting that YHV target material was not released into the culture medium, at least not in sufficient quantity to be detected by the methods used. This result corresponded to previous results showing a lack of recognizable YHV viral particles or nucleocapsids in YHV-imunopositive C6/36 cells by transmission electron microscopy (Sriton et al 2009) and suggested that transmission of YHV in the insect cells most likely occurred during daughter cell production or by endocytosis.…”

“…Although it is not proposed that these cultures should or would replace YHV extracts or purified YHV as inoculum for challenge tests with shrimp, the cultures might constitute a convenient, additional tool for laboratory studies on some topics, such as aspects of hostviral interaction and efficacy of double-stranded RNA constructs for knockdown of viral antigens. It remains to be determined whether homogenates of early passages of Sf9 cells persistently immunopositive for YHV antigens (Sriton et al 2009) would also cause YHD in shrimp. …”

“…C6/36 mosquito cells (a single cell-type clone obtained from the American Type Culture Collection under catalogue number CRL-1660) were maintained in supplemented Leibovitz's (L-15) medium (Gibco Invitrogen) and persistently infected with YHV as previously described (Sriton et al 2009). Briefly, this consisted of exposing the cells to YHV inoculum for 2 h, followed by washing before addition of fresh culture medium.…”

“…Using serial split-passage of whole C6/36 cells challenged with YHV, we successfully produced cultures with 100% YHV-immunopositive cells as previously described (Sriton et al 2009). They were immunopositive with monoclonal antibodies against capsid protein p20 and envelope proteins gp64 and gp116.…”

“…Our subsequent work with insect viruses revealed that persistent infections with 1 or 2 insect viruses could be simply achieved by serial split-passage of whole cells (Burivong et al 2004, Kanthong et al 2008. When this technique was used with shrimp yellow head virus (YHV) and white spot syndrome virus (WSSV), both mosquito (C6/36) and lepidopteran (Sf9) cell lines were obtained that persistently expressed the appropriate viral antigens as those detected by confocal microscopy (Sriton et al 2009). However, transmission electron microscopy did not show any recognizable viral particles of YHV or WSSV in the immunopositive insect cells, and no tests were carried out to establish whether the intact viral genomes of the 2 viruses were being replicated in the insect cells.…”