Vesicular stomatitis virus genomic RNA persists in vivo in 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.

FIG. 1.

Replication and spread of VSV vaccine vectors following i.m. inoculation. Eight-week-old BALB/c mice were inoculated i.m. with 5 × 10⁵ PFU of rwtVSV (solid squares) or VSV-CT1 (open triangles). Viral titers were determined at the times indicated from the hind leg muscle (A and C) and popliteal lymph node (B and D) as described in Materials and Methods. Each point represents an individual animal. Each bar represents the mean value for all animals at that time point. The number of points indicates the number of animals (typically four) tested at each time.

FIG. 2.

Persistence of VSV gRNA following i.m. inoculation. Eight-week-old BALB/c mice were inoculated with 5 × 10⁵ PFU of rwtVSV (solid squares) (A and B), VSV-CT1 (open triangles) (C and D), and VSVΔG (solid circles) (E and F). Leg muscle and popliteal lymph nodes (PLN) were harvested at the indicated times postinoculation. RNA was isolated from the tissue, and a reverse transcription, real-time PCR assay was performed to quantify gRNA. Each point represents the result from an individual animal. Copy numbers below 1 × 10⁴ copies (background of the assay) are all indicated arbitrarily as 10⁰. Each bar represents the mean value for all animals at that time point.

FIG. 3.

Diagram of tagged-primer assay specific for positive-strand VSV N mRNA. The first 5 nucleotides at the 3′ end of the tagged primer (boxed) are complementary to the VSV N mRNA just preceding the poly(A) tail and not to other VSV mRNAs. The lowercase sequence following the T₁₉ tract is the unrelated sequence tag. cDNA generated with this primer is then amplified with a VSV N internal primer, a primer corresponding only to the tag sequence, and a labeled probe, as described in Materials and Methods.

FIG. 4.

Lack of persistence of VSV N mRNA following i.m. inoculation. Eight-week-old BALB/c mice were inoculated with 5 × 10⁵ PFU of rwtVSV (solid squares) (A and B), VSV-CT1 (open triangles) (C and D), or VSVΔG (solid circles) (E and F). Leg muscle and popliteal lymph nodes (PLN) were harvested at the indicated times postinoculation, RNA was prepared from the tissues, and N mRNA was quantified using the tagged-primer method. The graph shows the number of mRNA copies per 10 mg of tissue, with each point representing an individual animal. Copy numbers below 1 × 10⁴ copies (background of the assay) are all indicated arbitrarily as 10⁰. Bars represent the mean values.

FIG. 5.

Lack of persistence of VSV N mRNA following i.n. inoculation. Eight-week-old BALB/c mice were inoculated with 5 × 10⁵ PFU of rwtVSV. Lungs (A) and cervical lymph nodes (LN) (B) were harvested at various times postinoculation, RNA was prepared from the tissues, and N mRNA was quantified using the tagged-primer method. The graph shows the number of mRNA copies per 10 mg of tissue, with each point representing values determined from an individual animal. Copy numbers below 1 × 10⁴ copies (background of the assay) are all indicated arbitrarily as 10⁰. Bars represents the mean values.

FIG. 6.

Effects of lymph node macrophage depletion on trapping of gRNA. (A) Representative flow cytometry plots of PLN cells stained with anti-CD11b and anti-CD169 at 5 days following injection of control PBS liposomes or clodronate liposomes into the footpad. The numbers circled are the percentages of cells that were CD11b⁺ CD169⁺ in the PLN. (B) VSV gRNA levels in the PLN of control or macrophage-depleted mice at 6 h after i.m. injection with 3.5 × 10⁷ PFU of rwtVSV. Depletions were performed 5 days prior to injection. (C and D) VSV gRNA levels in the PLN of mice that were inoculated i.m. with 3.5 × 10⁷ PFU of rwtVSV (C) or VSVΔG (D). Five days after inoculation, PLN macrophages were depleted with clodronate liposomes, and the levels of gRNA in the PLN of control or depleted mice were measured 5 days later (10 d.p.i.). (E) VSV gRNA levels in the PLN of mice that were inoculated i.m. with 3.5 × 10⁷ PFU of VSVΔG. Fifty days after inoculation, PLN macrophages were depleted with clodronate liposomes, and the levels of gRNA in the PLN of control or depleted mice were measured 10 days later (60 d.p.i.). Control experiments showed that macrophage depletion was still complete at 10 days after clodronate treatment. Each point represents the gRNA level determined from an individual animal. Bars represent the mean values.