Vesicular stomatitis New Jersey virus (VSNJV) infects keratinocytes and is restricted to lesion sites and local lymph nodes in the bovine, a natural host Charles Scherer, Vivian O’Donnell, William Golde, Douglas Gregg, D. Mark Estes, Luis Rodriguez To cite this version: Charles Scherer, Vivian O’Donnell, William Golde, Douglas Gregg, D. Mark Estes, et al.. Vesicular stomatitis New Jersey virus (VSNJV) infects keratinocytes and is restricted to lesion sites and local lymph nodes in the bovine, a natural host. Veterinary Research, BioMed Central, 2007, 38 (3), pp.375-390. 10.1051/vetres:2007001. hal-00902859 HAL Id: hal-00902859 https://hal.archives-ouvertes.fr/hal-00902859 Submitted on 1 Jan 2007 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Vet. Res. 38 (2007) 375–390 375 c INRA, EDP Sciences, 2007 DOI: 10.1051/vetres:2007001 Original article Vesicular stomatitis New Jersey virus (VSNJV) infects keratinocytes and is restricted to lesion sites and local lymph nodes in the bovine, a natural host Charles F.C. Sa,b,c,VivianO’Da,d, William T. Ga, Douglas Ga,D.MarkEb,c,LuisL.Ra* a Plum Island Animal Disease Center, Agricultural Research Service, United States Department of Agriculture, PO Box 848, Greenport, NY 11944, USA b University of Missouri, College of Veterinary Medicine, Department of Veterinary Pathobiology, Columbia, MO 65251, USA c Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX 77555, USA d Department of Pathobiology, University of Connecticut, Storrs CT 06269, USA (Received 16 August 2006; accepted 19 October 2006) Abstract – Inoculation of vesicular stomatitis New Jersey virus (VSNJV) by skin scarification of the coronary-band in cattle, a natural host of VSNJV, resulted in vesicular lesions and 6−8log10 TCID50 increase in skin virus titers over a 72 h period. Virus infection was restricted to the lesion sites and lymph nodes draining those areas but no virus or viral RNA was found in the blood or in 20 other organs and tissues sampled at necropsy. Scarification of flank skin did not result in lesions or a significant increase in viral titer indicating that viral clinical infection is restricted to skin inoculation at sites where lesions naturally occur. Viral antigens co-localized primarily with keratinocytes in the coronary band, suggesting these cells are the primary site of viral replication. Viral antigen also co-localized with few MHC-II positive cells, but no co-localization was observed in cells positive for macrophage markers. Although granulocyte infiltration was observed in lesions, little viral antigen co-localized with these cells. This is the first detailed description of VSNJV tissue distribution and infected cell characterization in a natural host. The pathogenesis model shown herein could be useful for in-vivo tracking of virus infection and local immune responses. vesicular stomatitis / bovine / pathogenesis / confocal microscopy / keratinocytes 1. INTRODUCTION application as a vector for experimental vaccine delivery and for anti-tumor ther- Vesicular stomatitis virus (VSV) is apy [1,18]. In the nature VSV is an impor- widely used as a laboratory research tool tant livestock pathogen causing vesicular for RNA virus evolution and to evaluate stomatitis (VS) a disease characterized by immune function. More recently it has had the appearance of vesicular lesions in the mouth, feet and udders, of cattle, pigs * Corresponding author: and horses. In cattle and swine these [email protected] lesions are clinically undistinguishable Article available at http://www.edpsciences.org/vetres or http://dx.doi.org/10.1051/vetres:2007001 376 C.F.C. Scherer et al. from foot-and-mouth disease, a devastat- to the description of gross pathology and ing disease of livestock [20]. VSV has histopathology of the lesions without spe- been shown to be transmitted by insect cific identification of cell types involved in bites [6,14,27] but transmission by di- early viral infection. rect contact between animals has also been This study describes early events of demonstrated [15, 23]. The mechanisms of vesicular stomatitis New Jersey virus (VS- disease remain unclear, although field ob- NJV) infection in cattle utilizing a novel servations indicate that many infections coronary band scarification inoculation do not result in overt clinical disease and model combined with analyses by im- many susceptible species living in endemic munohistochemistry, confocal microscopy areas possess neutralizing antibody titers to and real-time RT-PCR. The tissue distribu- VSV [16,19]. tion of virus and the identity of the cell Most knowledge pertaining to VSV types infected during early phases of dis- pathogenesis is derived from studies in- ease are described. volving laboratory rodents where clini- cal signs are not vesicular in nature, but rather the infection manifests as encephali- 2. MATERIALS AND METHODS tis and death depending on host factors such as age, route of inoculation and vi- 2.1. Animals and virus ral strain [7, 9]. Neurological symptoms have not been reported in natural VSV Adult (18 to 24 months) Holstein hosts (cattle, swine, horses) [20]. The basic steers weighing 500−700 lb were obtained mechanisms of VSV infection remain un- from an experimental-livestock provider clear, but experiments in swine suggest that (Thomas-Morris Inc., PA, USA) and kept the inoculation site determines clinical out- in the biosafety level 3 facility at Plum come. Vesicular lesions are observed only Island Animal Disease Center for at least when the virus is intradermally inoculated one week prior to initiation of the exper- at specific sites where lesions are observed iments. All animal inoculations were per- during natural infections (snout, lip, feet) formed with a VSNJV field strain obtained and instead subclinical infection occurs from tongue epithelium of a bovine nat- when virus is inoculated intradermally at urally infected during the 1995 epidemic other sites (i.e. ear, abdomen) or by the in Colorado (95COB). This virus was intranasal or intravenous routes [11, 15]. identified as VSNJV by virus neutraliza- Recently, it was further shown that vesic- tion and sequencing of the complete viral ular lesions only developed when exper- genome [21]. The virus was propagated by imentally infected black flies (Simulium passing once in baby hamster kidney cells vittatum) were allowed to feed on the snout (BHK-21) infected at 0.01 multiplicity of but not when flies fed on the abdomen of infection. Viral stock was titrated in BHK- swine [14]. 21 cells, and kept in aliquots at −70 ◦C. Little is known about the basic cellu- lar and molecular mechanisms mediating VSV pathogenesis in its natural hosts. In- 2.2. Inoculation procedure formation regarding the primary sites of virus replication and the cell types involved Animals were sedated with xylazine and in supporting viral growth and those in- the coronary band areas were shaved, prior volved in controlling the infection remain to the epidermis being pricked 20 times us- scarce. Previous studies on VSV inocu- ing a dual tip skin test applicator (Duotip- lation in natural hosts have been limited Test, Lincoln Diagnostics, Decatur, IL, Vesicular stomatitis: pathogenesis in cattle 377 USA). Virus inoculum was placed on the trogen for RNA extraction, virus isolation scarified area in 100 µL of Dulbecco Mod- and confocal microscopy. Oropharyngeal ified Eagle Medium containing 2% fe- fluid (OPF), plasma and blood samples tal bovine serum (FBS) (DMEM2). The were also obtained and kept at −70 ◦C un- area under the inoculum was then scar- til being processed. In four of the animals ified 20 additional times with the ani- inoculated in the coronary band, biopsies mals restrained in a stationary position for were also taken 20 min after inoculation. 1−2 min until the inoculum was adsorbed. Eight animals were euthanized at 72 hpi A total of thirteen animals were used in this and the following tissues were collected for study. Six animals, housed in three sep- RT-PCR and virus isolation: prescapular, 7 arate rooms, received 10 TCID50/foot in popliteal, axillary, mediastinal, mesenteric, each of all four feet; one animal (#102) was iliac, prefemoral, retropharyngeal, parotid, inoculated only on the right feet and mock- and submandibular lymph nodes; coronary inoculated on the left feet. Animals 12 band, tongue, tonsils, lung, heart, liver, 7 and 148 were inoculated with 10 TCID50 spleen, kidney, small intestine (duode- of VSNJV by intradermal injection or scar- num), large intestine (cecum), snout skin, ification on the skin of the flank and kept mandibular salivary glands, nasal epithe- in separate rooms. One animal (#699) was lium and brain (olfactory area). Animals 12 7.0 inoculated by injection of 10 TCID50 and 148, inoculated on the flank, were eu- distributed in four sites on the dorsal ep- thanized at 48 hpi and only skin samples ithelium of the tongue and house alone. All and major lymph nodes were collected. inoculations were performed with the same viral stock. Three non-inoculated animals kept in separate rooms were used as negative con- 2.4. Virus isolation trols and sampled similarly. Clinical signs, temperature, appetite, and attitude were Tissues were macerated using a mor- evaluated daily for all animals. Clinical tar and pestle in 2 mL of Minimal Es- disease was scored by determining the size sential Medium containing 400 U/mL of and number of lesions, a value of 1 was penicillin, 400 U/mL of streptomycin, and giventosmalllesionsandavalueof2for 10 µg/mL of amphotericin B (MEM).