Tick-Borne Transmission of Murine Gammaherpesvirus 68

Abstract

Herpesviruses are a large group of DNA viruses infecting mainly vertebrates. Murine gammaherpesvirus 68 (MHV68) is often used as a model in studies of the pathogenesis of clinically important human gammaherpesviruses such as Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus. This rodent virus appears to be geographically widespread; however, its natural transmission cycle is unknown. Following detection of MHV68 in field-collected ticks, including isolation of the virus from tick salivary glands and ovaries, we investigated whether MHV68 is a tick-borne virus. Uninfected Ixodes ricinus ticks were shown to acquire the virus by feeding on experimentally infected laboratory mice. The virus survived tick molting, and the molted ticks transmitted the virus to uninfected laboratory mice on which they subsequently fed. MHV68 was isolated from the tick salivary glands, consistent with transmission via tick saliva. The virus survived in ticks without loss of infectivity for at least 120 days, and subsequently was transmitted vertically from one tick generation to the next, surviving more than 500 days. Furthermore, the F1 generation (derived from F0 infected females) transmitted MHV68 to uninfected mice on which they fed, with MHV68 M3 gene transcripts detected in blood, lung, and spleen tissue of mice on which F1 nymphs and F1 adults engorged. These experimental data fulfill the transmission criteria that define an arthropod-borne virus (arbovirus), the largest biological group of viruses. Currently, African swine fever virus (ASFV) is the only DNA virus recognized as an arbovirus. Like ASFV, MHV68 showed evidence of pathogenesis in ticks. Previous studies have reported MHV68 in free-living ticks and in mammals commonly infested with I. ricinus, and neutralizing antibodies to MHV68 have been detected in large mammals (e.g., deer) including humans. Further studies are needed to determine if these reports are the result of tick-borne transmission of MHV68 in nature, and whether humans are at risk of infection.

Keywords: Ixodes ricinus; MHV68; arbovirus; gammaherpesvirus; tick; transmission.

Figure 1

Tick-borne virus transmission and tick infection following injection of ticks with MHV68. (A) Virus detected by nested PCR. Lanes S1-S7, salivary glands of virus-injected ticks fed on uninfected mice for 5 days; lanes B1-B5, blood samples of five mice 15 days after tick infestation; L1, HyperLadder; L2, GeneRuler DNA ladder; lane C1, MHV68 DNA (positive control); lane C2, nested PCR without template (negative control); lane C3, MHV68 DNA nested PCR 1st round product with outer primers (positive control); lane C4, 1st PCR round with outer primers without template (negative control). ^** Indicates MHV68 ORF50 gene PCR product of 382 base pairs. (B) Infectivity as determined by plaque formation in BHK-21 cells. Cells inoculated with homogenate of virus infected tick and observed by (a) light microscopy (magnification x50) and (b–d) specific immunofluorescence staining (magnification x200). (b) Single plaque shown of a maximum 5 plaques observed per tick; (c) control, uninfected cells; and (d) cells inoculated with homogenate of uninfected tick.

Figure 2

MHV68 detection in blood samples from mice infested with naturally infected nymphs or adult ticks. (A) Lanes N1-N7, blood samples of mouse 1-7 exposed to nymphs molted from larvae that had engorged on infected mice; lane N8, blood of mouse infested with uninfected nymphs. (B) Lane A1, blood of mouse infested with uninfected adult ticks; lanes A2-A14, blood samples of 13 mice exposed to adults molted from nymphs that had engorged on infected mice. All blood samples were collected 15 days after tick infestation. Lanes L1, L2, C1–C4 as for Figure 1A. ^**Indicates MHV68 ORF50 gene PCR product of 382 base pairs.

Figure 3

MHV68 detection in blood samples from mice infested with F0 females or F1 nymphs. (A) Lanes A15-A34, blood samples of 20 mice infested with infected F0 females; blood collected 15 days after tick infestation. ^**Indicates MHV68 ORF50 gene nested PCR product of 382 base pairs. (B) Lanes 1n²⁶, 2n¹⁷, 3n¹⁸, 4n²⁸, 5n¹⁹, 6n²⁰, 7n³², 8n³³, and 9n²⁴, blood samples of mice infested with F1_i nymphs; N9, N10 blood samples of control mice infested with F1_c nymphs. ⁺Indicates MHV68 M3 gene one step RT-PCR product of 520 base pairs. Lanes L2, C1–C4 as for Figure 1A.

Figure 4

MHV68 genome load in organs of mice exposed to F1 infected ticks. Box and whisker plots of genome equivalent copies per ml blood or mg lung or spleen from mice 15 days after infestation with either F1_i nymphs or F1_i adult ticks. All controls were negative (see Table 2).

Figure 5

MHV68 detection in lung and spleen samples from mice infested with F1_i adults and in F1_i female ticks. (A) Lung (a,c) and spleen (b,d) samples of mice infested with F1_i adults examined by nested PCR (a,b) and RT-PCR (c,d). Lanes 1a²⁶, 2a¹⁷, 3a¹⁸, 4a²⁸, 5a¹⁹, 6a²⁰, 7a³², 8a³³, and 9a²⁴ samples of mice infested with F1_i adult ticks; A35, A36, samples of control mice infested with F1_c adults. Lanes L2, C1–C4 as for Figure 1A. ^** Indicates MHV68 ORF50 gene nested PCR product of 382 base pairs; ⁺⁺ indicates MHV68 M3 gene nested RT-PCR product of 241 base pairs. (B) Semi-thin sections of frozen whole body of F1_i females fed for 4 days. (a,b) F1_i tick from mouse 3a¹⁸ and 5a¹⁹ stained with anti-MHV68 rabbit polyclonal serum; (c) uninfected tick (F6 generation of breeding) stained with anti-MHV68 rabbit polyclonal serum; (d) F1_i tick from mouse 3a¹⁸ stained with rabbit polyclonal serum against PB1-F2 protein of influenza virus A (H1N1) (negative control). MD, cells of midgut diverticula; L, lumen of midgut diverculum. Scale bar, 200 μm.