Vesicular Stomatitis Virus Genomic RNA PersistsIn Vivoin the Absence of Viral Replication

Abstract

Our previous studies using intranasal inoculation of mice with vesicular stomatitis virus (VSV) vaccine vectors showed persistence of vector genomic RNA (gRNA) for at least 60 days in lymph nodes in the absence of detectable infectious virus. Here we show high-level concentration of virus and gRNA in lymph nodes after intramuscular inoculation of mice with attenuated or single-cycle VSV vectors as well as long-term persistence of gRNA in the lymph nodes. To determine if the persistence of gRNA was due to ongoing viral replication, we developed a tagged-primer approach that was critical for detection of VSV mRNA specifically. Our results show that VSV gRNA persists long-term in the lymph nodes while VSV mRNA is present only transiently. Because VSV transcription is required for replication, our results indicate that persistence of gRNA does not result from continuing viral replication. We also performed macrophage depletion studies that are consistent with initial trapping of VSV gRNA largely in lymph node macrophages and subsequent persistence elsewhere in the lymph node.Vesicular stomatitis virus (VSV) is a nonsegmented, negative-strand RNA virus and the prototype of the Rhabdoviridae family. VSV encodes 5 structural proteins: nucleocapsid protein (N), phosphoprotein (P), matrix protein (M), the surface glycoprotein (G), and the RNA-dependent RNA polymerase (L) (19). Live-attenuated vaccine vectors based on VSV have been developed and approved for clinical trials. Attenuated VSV-based vaccine vectors expressing foreign proteins induce potent immune responses and protect against viral and bacterial disease in several animal models, including nonhuman primates (9, 13, 15, 16, 17, 21, 25-27, 29, 30). A live-attenuated VSV-based Ebola virus vaccine vector has also been used in a person following a possible Ebola virus exposure (http://blogs .sciencemag.org/scienceinsider/2009/03/researchers-aro.html).Highly attenuated and single-cycle VSV vectors have been extensively characterized previously in our laboratory and elsewhere (4,17,23,24,26). The highly attenuated live VSV vector VSV-CT1 has a truncation of the VSV G cytoplasmic domain from 29 amino acids to 1 amino acid (32). Compared to recombinant wild-type (rwt) vector (rwtVSV), the CT1 vector grows to approximately 20-fold-lower titers in tissue culture. The single-cycle VSV vector (VSV⌬G) has a deletion of the VSV G gene but can be grown in complementing cells expressing the VSV G protein (33). This virus can infect cells and replicate for a single cycle but does not produce infectious progeny in the absence of complementing VSV G protein.In previous studies, we found that VSV vaccine vector genomic RNA (gRNA) persists in the cervical draining lymph nodes for at least 60 days after intranasal (i.n.) inoculation with rwtVSV and VSV-CT1 vectors, although infectious virus could be recovered for only the first 4 days after inoculation (34).VSV-encoded antigen is also known to persist for at least 6 weeks after acute infection (35). Long-term persistence of live vir...

Re-engineering Vesicular Stomatitis Virus to Abrogate Neurotoxicity, Circumvent Humoral Immunity, and Enhance Oncolytic Potency

“…For all the other tissues tested, both rVSV and rVSV(GP) were rapidly cleared with the exception of the spleen where viral gRNA persisted for at least 40 dpi (Supplementary Fig. S3), consistent with earlier reports (32).…”

A Viable Recombinant Rhabdovirus Lacking Its Glycoprotein Gene and Expressing Influenza Virus Hemagglutinin and Neuraminidase Is a Potent Influenza Vaccine

“…with rVSV, virus was recovered only from muscle and draining lymph nodes. This finding is consistent with results from previous studies using rVSVs (48). These experiments indicate that VSV⌬G-H5N1 does not replicate extensively in vivo.…”

“…with 2 ϫ 10 7 PFU of VSV⌬G-H5N1 or 2 ϫ 10 5 PFU of a control rVSV expressing VSV G and a hybrid influenza virus hemagglutinin (VSV-cH5/ 1). Tissues (leg muscle, lung, spleen, and draining lymph nodes) were isolated and homogenized at either 24 h or 96 h, as described previously, and assayed for infectious virus on BHK-21 cells (48). We detected no infectious VSV⌬G-H5N1 in any tissue at either time point.…”

“…Mice were euthanized either 24 h or 96 h after vaccination, and tissues (lung, spleen, draining lymph nodes, and leg muscle) were isolated and frozen. Homogenates were prepared in 2.5% DMEM, as previously described (48), and frozen at Ϫ80°C prior to inoculation of cell monolayers. BHK-21 cells that were plated onto 24-well plates the day prior were then exposed to 20 l of the supernatant and observed for viral CPE for 72 h. Any well demonstrating CPE within this time period was subjected to a plaque assay to determine the titer of recovered virus.…”

Measles immunometrics

“…Any or all of these cells could be the source of MV RNA, as MV RNA persists not just in blood, but also urine, lymphoid tissue, and lung secretions. MV can persistently infect neurons, spreading between cells directly without budding (4 (14) and be accompanied by viral immunogens capable of activating CD8 + T cells (15,16). It would certainly make sense for the immune system to retain the genetic information of previous viral infections to maintain memory by occasionally translating retained viral mRNA, although it is clear that antigen persistence is not an absolute prerequisite for maintaining memory T cells (17).…”