Abstract
Abstract
Murine gammaherpesvirus-68 (MHV-68), a natural pathogen of mice, is being evaluated as a model of Epstein Barr Virus (EBV) infection for use in investigation of the effects of immunomodulatory therapy on herpesvirus pathogenesis in humans. Immunosuppressive agents are used for treatment of a variety of autoimmune diseases as well as for prevention of tissue rejection after organ transplantation and can result in recrudescence of latent herpesvirus infections. Prior to examination of MHV-68 as a suitable model for EBV, better characterization of the MHV-68 model was desirable. Characterization of the MHV-68 model involved development of assays for detecting virus and for demonstration of safety when present in murine colonies. Limited information is available in the literature regarding MHV-68 transmission, although recent reports indicate the virus is not horizontally spread in research facilities. To further determine transmission potential, immunocompetent and immunodeficient mice were infected with MHV-68 and co-habitated with naïve animals. Molecular pathology assays were developed to characterize the MHV-68 model and to determine viral transmission. Horizontal transmission of virus was not observed from infected animals to naïve cagemates after fluorescence microscopy assays and quantitative PCR (qPCR). Serologic analysis complemented these studies and was used as a method of monitoring infection amongst murine colonies. Overall, these findings demonstrate that MHV-68 infection can be controlled and monitored in murine research facilities, and the potential for unintentional infection is low.
Introduction
The gammaherpesvirus family includes Epstein Barr Virus (EBV) and Kaposi Sarcoma-associated Herpesvirus (KSHV), both of which are known to contribute to human disease. Murine gammaherpesvirus-68 (MHV-68) is being evaluated as a murine model of EBV infection. Gammaherpesviruses are large double-stranded DNA viruses characterized by a life cycle of lytic and latent infections (Kieff & Rickinson, 2001; Roizman & Knipe, 2001). During the lytic phase of infection, infectious virions are produced. During latency, most viral genes are silenced and there is an absence of infectious virus production (Barton et al., 2011; Sunil-Chandra et al., 1992a, b, 1993). Latent infections permit lifelong viral persistence through evasion of the host immune response. Re-activation of gammaherpesvirus lytic gene expression and virus production may be triggered by insult to the immune system (Hwang et al., 2008; Sunil-Chandra et al., 1994; Yarilin et al., 2004).
Immunosuppressive therapy is used for treatment of immune disorders and for prevention of tissue rejection after organ transplant. However, immunosuppressive treatments may be associated with an increased risk for the development of neoplasia or post-transplant lymphoproliferative disorder (PTLD). In some cases, viral recrudescence of latent gammaherpesvirus infections that occur from EBV and KSHV may be linked with these disorders (Mesri et al., 2010; Rezk & Weiss, 2007; Schwartz, 2001). Direct small animal models to examine these human viruses do not exist, indicating a need for better understanding of gammaherpesvirus pathogenesis. Murine gammaherpesvirus-68 (MHV-68) may be a relevant model for studying gammaherpesvirus pathogenesis as it relates to human disease (Aligo et al., 2014). However, in vivo models to examine these areas have yet to be fully characterized and an understanding of the mechanism of viral transmission in animal populations is lacking. Further characterization of the MHV-68 model is essential for understanding viral pathogenesis. Expanded knowledge of MHV-68 transmission for use in vivarium populations is also required to ensure the well-being of animal colonies.
MHV-68 was originally isolated from wood voles in Slovakia (Blaskovic et al., 1980; Nash et al., 2001). Infected mice may develop lymphoma over a long period of time, although this process may be accelerated after drug-induced immunodeficiency (Kulkarni et al., 1997; Sunil-Chandra et al., 1994; Tarakanova et al., 2005, 2008). MHV-68 has been studied in the laboratory for at least three decades. To date, there is very little published literature about MHV-68 epidemiology, and horizontal transmission has not been observed in the laboratory (Barton et al., 2011). The reasons for this are unknown, but some hypotheses include a lack of understanding of the natural viral host as well as an inadequate understanding of unexplored routes of infection. In a recent report, the authors hypothesized the laboratory setting for animal caging prevents normal physiological interactions between mice that would occur in the wild (Francois et al., 2013). This same study found transmission of virus from female mice to naïve males after sexual contact, but horizontal transmission from infected female mice to naïve females was not observed (Francois et al., 2013).
The focus of the present study was to first establish assays to detect and monitor the course of MHV-68 infection in vitro and in vivo. The first experiments utilized a latently infected B-cell lymphoma line to develop tools to examine MHV-68 gene expression and protein production. These cells were next used to examine horizontal transmission in vivo and were also used to generate viral stocks. Viral stocks were next used to infect immunocompromised mice and determine if the virus was horizontally transmitted to naïve cagemates. Immunocompetent animals were not used in the infection studies as viral infection enters latency due to action of the immune system. Latent infections may be difficult to detect and the experiments described in this manuscript were specifically designed to maximize the probability that horizontal transmission would be detected if it occurred. The data from these experiments were used to further characterize the MHV-68 model and to determine if infection is horizontally transmitted amongst laboratory cagemates.
Materials and methods
Mice
Balb/c, SCID, and SCID/Beige mice (female, 5–8-weeks-of-age) were obtained from, respectively, The Jackson Laboratory (Bar Harbor, ME), SAGE Labs (Boyertown, PA), and Charles River Laboratories (Wilmington, MA). All mice were maintained in the vivarium at Janssen Research and Development, LLC (Radnor, PA) under Specific Pathogen Free (SPF) conditions. Mice were provided ad libitum access to filtered water and autoclaved LabDiet® 5010 (LabDiet, St. Louis, MO). Mice were group housed on ALPHA-dri® bedding in filter-topped static rodent housing with a 12-h light cycle in a facility maintained at 21–22 °C with a 40–60% humidity.
Janssen Research and Development, LLC complies with recommendations of the Guide for Care and Use of Laboratory Animals with respect to restraint, husbandry, procedures, feed and fluid regulation, and veterinary care. The animal care and use program at Janssen Research and Development, LLC is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC), which assures compliance with accepted standards for the care and use of laboratory animals. The Institutional Animal Care and Use Committee (IACUC) at Janssen Research and Development, LLC approved all studies.
Animals were individually identified with ear tags placed ∼1 week prior to the start of the study. Animals were evaluated daily throughout the study for clinical signs of infection. Qualitative parameters including the appearance of skin color, fur condition, body condition, respiration, presence of feces or appetite, hydration, posture, alertness, responsiveness, and conspecific interaction were evaluated. Any variance from normal clinical observations (smooth and clean hair coat, normal respiration, animal is bright, alert, responsive, social, eating/drinking, etc.) was noted in the study file. Quantitative parameters included recording the body weight of each animal twice weekly.
Cells
A20 cells are a lymphoma cell line isolated from a spontaneous reticulum cell neoplasm found in an aged BALB/cAnN mouse (Kim et al., 1979). A20-HE-2 cells are A20 cells latently infected with MHV-68-EGFP (enhanced green fluorescent protein) virus. These cells were obtained from Dr Craig Forrest and Dr Samuel Speck (Emory University, Atlanta, GA) who had previously characterized them (see Forrest & Speck, 2008). All cells used in these studies were either provided directly from Forrest and Speck or were re-derived from these cells. Both A20 and A20-HE-2 cells were maintained in RPMI 1640 medium containing 1X Glutamax, 10% fetal bovine serium (FBS), 1% penicillin-streptomycin solution, and 55 μM 2-mercaptoethanol (all from Life Technologies, Carlsbad, CA). A20-HE-2 cells were supplemented with 1 mg Hygromycin B/ml (EMD, Millipore, Billerica, MA). A 24-h treatment of A20-HE-2 cells with 40 ng PMA/ml (phorbol-12-myristate-13-acetate; Sigma, St. Louis, MO) was sufficient for viral re-activation according to the method of Forrest & Speck (2008).
Baby Hamster Kidney (BHK-21) cells, an adherent cell line derived from 1-day-old hamsters (Macpherson & Stoker, 1961), are traditionally used for viral growth/titration experiments as they are highly permissive for growth of several viral species. Vero cells are kidney epithelial cells originally isolated from an African green monkey (Yasumura & Kawakita, 1963). Vero cells are also highly permissive for several viral species and commonly used in viral growth and/or titration experiments. Here, BHK-21 and Vero cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) containing 1X Glutamax, 10% FBS, 1% penicillin-streptomycin, and 1 mM sodium pyruvate (all Life Technologies). The BHK-21 cell cultures were also supplemented with 5% Tryptose Phosphate Broth (Sigma).
Virus stocks
The MHV-68-Hygromycin B/EGFP virus used in these studies was the WUMS strain (ATCC VR1465) and was obtained from A20-HE-2 cells after a 24-h PMA treatment (Forrest & Speck, 2008). The virus was first collected from the supernatant of re-activated cells after three rounds of freeze–thawing. Virus stocks were then amplified after 1 week post-infection in BHK-21 cells permissive to MHV-68 lytic growth. Once BHK-21 monolayers showed visible signs of cytopathic effects (CPE), cells and supernatant were harvested and subjected to three rounds of freeze–thawing. The cellular debris was removed by centrifugation and the supernatant aliquoted and stored at −80 °C. Viral titers from these stocks were determined by plaque assay on Vero cells and calculated as plaque forming units (PFU)/ml (Dulbecco, 1952; Dulbecco & Vogt, 1953). Viral titers determined from these experiments were used to calculate the amount of virus (as PFU) required for in vivo infection experiments.
Immunoblotting
Prior to immunoblotting, cells were lysed in ice cold lysis buffer containing 150 mM NaCl, 50 mM Tris [pH 8.0], 1 mM EDTA, 1% NP-40, 0.1% SDS, and complete protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN). The protein concentration of each lysate was determined by the Bradford method (Pierce, Rockford, IL). An aliquot (100 μg) of total protein was then resolved via SDS-PAGE and the proteins transferred to a PVDF membrane (Life Technologies). Western blotting was then performed using either a rabbit anti-MHV-68 antibody (provided by Dr Craig McLahan and colleagues at Janssen R&D; 1:2000 dilution) or a chicken anti-ORF59 antibody (Gallus Immunotech, Cary, NC; 1:2000 dilution) incubated with the membrane at room temperature for 1 h. Rabbit anti-GFP (1:5000 dilution) and mouse anti-β-tubulin (1:5000 dilution) antibodies were obtained from Abcam (Cambridge, MA). Horseradish peroxidase (HRP)-conjugated secondary antibodies were used to detect the rabbit (Vector Labs, Burlingame, CA; 1:5000 dilution), mouse (Vector Labs; 1:5000 dilution), and chicken (Abcam; 1:5000 dilution) primary antibodies. All antibodies were diluted in phosphate-buffered saline, pH 7.5 (PBS, Life Technologies) containing 0.15% Tween-20 (Sigma) and 5% non-fat dry milk (Rockland, Gilbertsville, PA). The membrane was washed between antibody binding steps by rinsing three times with PBS containing 0.15% Tween-20 (PBS-T) for 15 min at room temperature. After a final rinsing with PBS-T to remove unbound antibody, enhanced chemi-luminescence substrate (ECL, GE Healthcare, Piscataway, NJ) was used to detect the bound proteins and the signals were visualized with an ImageQuant LAS 4000 digital imaging system (GE Healthcare).
Flow cytometry
Cells were fixed and processed for flow cytometry using a FoxP3 staining kit (BD Biosciences, San Jose, CA) according to manufacturer protocols and then stained with rabbit anti-MHV-68 antibody (1:6000 dilution). This kit had been optimized by the manufacturer for FoxP3 staining, and has previously been used with good results for analysis of some intracellular markers in our laboratory (unpublished observations). The cells were then stained with a 1:200 dilution of anti-rabbit DyLight 650 antibody (Abcam). Flow cytometry was then immediately performed on a Guava 8HT flow cytometer (EMD Millipore) using Guava Express Pro software. A minimum of 5000 events/sample was acquired.
qPCR
Total RNA was isolated from cells or tissue using a RNeasy Kit according to the manufacturer protocols (Qiagen, Valencia, CA). cDNA was synthesized using 8–20 ng total RNA using a High Capacity cDNA-RNA Synthesis Kit (Life Technologies) and random primers; qPCR was performed using Taqman probes (Life Technologies) for latent (mLANA) or lytic (RTA) MHV-68-specific genes (probe/primer sequences available upon request). Viral gene expression in tissues and cells was compared to RNA transcript levels of latently infected A20-HE-2 cells. All qPCR reactions were run on a Viia7™ real-time PCR instrument (Life Technologies).
In vivo infection of SCID and SCID/Bg mice
Infection of SCID mice was done using virus from a master stock containing 9 × 106 PFU/ml DMEM. Virus from this master stock was diluted in DMEM to a final concentration of 2 × 106 PFU/ml. On Day 0, all animals received individual study numbers (via tattoo or ear tag) and were weighed. Five animals were removed from the communal cage, anesthetized with CO2/O2 and administered 50 µl of the virus inoculum (100 000 PFU) via the intra-nasal (IN) route. In this study 100 000 PFU of virus/mouse was delivered, as previous experiments in our laboratory at 1000, 10 000, and 100 000 PFU showed the highest infectivity was achieved at 100 000 PFU (unpublished observations). This amount of virus also ensured high-level infection so there would be a high probability of detecting any horizontally-transmitted virus. The mice were placed into a fresh cage after infection and allowed to recover from anesthesia to allow them time to expel any excess infectious fluid from their noses and prevent contamination of the cage that housed naive animals. After 5 h, the infected animals were placed back into the original cage with the untreated animals.
Infection of SCID/Bg mice was done as above with the exception that the infected mice were administered 50 µl of virus inoculum from the undiluted master stock containing 9 × 106 PFU/ml DMEM. All infected SCID/Bg mice were administered with 450 000 PFU of virus. This amount of virus was the most concentrated amount of virus our laboratory could deliver based on the viral stock titer. This dose was chosen to further ensure MHV-68 was not horizontally transmitted.
Implantation of A20-HE-2 cells
Methods for tumor implantation were similar to a published study from our group with a different B-cell lymphoma cell line (see Rafferty et al., 2012). Briefly, A20-HE-2 cells (in a volume of 200 μl of PBS/mouse) were implanted subcutaneously (SC) into the right flank of six Balb/c mice and six SCID/Bg mice using a 26-gauge 1/2” needle attached to a 1 ml syringe. Cells were provided for implantation at a concentration of 1.5 × 106 cells/ml such that 3 × 105 cells were delivered to each mouse. This dose of cells was chosen based on previous findings with the A20 and A20-HE-2 cell lines in our laboratory in studies that examined a range of implantation doses (from 1 × 105–1 × 106 cells/mouse; unpublished observations). Implantation of 3 × 105 cells/mouse generally gave rise to measureable tumors within 1-month post-implantation. In the present study, tumors were measured by caliper (width and length) twice weekly and tumor volume estimated using: tumor volume = width2 × length/2. Mice were observed until tumor volume reached 2000 mm3, at which point the mouse was euthanized by CO2 asphyxiation.
Spleen and tumor single cell suspensions
Animals that were implanted with A20-HE-2 cells were terminated and necropsied at 28 days post-implant for analysis of horizontal transmission of MHV-68. All spleens and any tumors were harvested in PBS and placed on ice. Single cell suspensions were prepared in PBS after homogenizing each tissue through a 40-μM cell strainer (BD Biosciences) with the rubber stopper of a 3 ml syringe (BD Biosciences). Cell viabilities were assessed by trypan blue exclusion assays on an automated cell counter (Countess®, Life Technologies). An aliquot (5 × 105) of cells was re-suspended in 0.5 ml PBS and freeze–thawed 3-times on dry ice to release potential intracellular virus into the supernatant. The supernatants were then aliquoted onto BHK-21 cell monolayers and incubated for 1 week. Any virus present in the samples would infect the BHK-21 cells and spread in the cultures which, in turn, were analysed by fluorescence microscopy for EGFP. As controls, BHK-21 monolayers were also either mock-infected or infected with MHV-68-EGFP virus.
Serology
For serologic analysis, serum was isolated from blood collected from 12 uninfected Balb/c control mice and 12 mice infected with 100 000 PFU MHV-68-EGFP/mouse at Days 1, 4, and 10 post-infection. At each timepoint, four mice/group were euthanized via CO2 asphyxiation. After termination, a thoracotomy was performed on each mouse and 0.5 ml blood obtained from the heart using a 25-gauge, ½” needle attached to a 1 ml syringe.
Anti-MHV68 antibodies in serum were analysed via an indirect immunofluorescence protocol that employed MHV-68-EGFP-infected Vero cells as antigen for detection of the presence of anti-MHV-68 antibodies. Briefly, cells were seeded in 96-well glass bottom plates (Sensoplate from Greiner Bio-One; Monroe, NC) designed for fluorescence applications and allowed to attach overnight. The next day, the cells were infected with MHV-68-EGFP at a multiplicity of infection (MOI) of five per cell for ∼24 h. Infection of Vero cells was confirmed by fluorescence microscopy for EGFP. The cell monolayers were then washed twice with PBS and fixed in freshly made 4% paraformaldehyde (Alfa Aesar, Ward Hill, MA) in PBS. The cells were subsequently permeabilized with 0.1% Triton X-100 (Sigma) in PBS. The monolayers were then treated with 3% BSA (bovine serum albumin; Life Technologies) diluted in PBS as a blocking agent. Sera from naïve and MHV-68-infected mice were then diluted into blocking buffer and used as primary antibody. Detection of anti-MHV-68 antibodies was performed using an anti-mouse IgG Dylight 649/650 secondary antibody (1:200 dilution; Abcam). As controls for the assay, rabbit anti-MHV-68 antiserum was tested on both mock-infected and MHV-68-EGFP-infected Vero cells and detected with an anti-rabbit IgG Dylight 649/650 secondary (1:200 dilution; Abcam). Other negative controls included staining mock and MHV-68-EGFP-infected Vero cells treated with secondary antibodies only. In all cases, 4′,6-diamidino-2-phenylindole (DAPI) was used as a nuclear stain (Life Technologies).
Fluorescence intensities for EGFP, Dylight 649/650, and DAPI were obtained using a Synergy HT plate reader (BioTek, Winooski, VT). EGFP intensities were plotted to ensure all antigen cells were infected uniformly (data not shown). Ratios for Dylight 649/50:DAPI were calculated to normalize each well for similar cell number. For each group of stained cells, the average ratio was taken and plotted. Error bars were plotted using the standard error of the mean (SEM) for each group. Staining data were averaged from four animals/group run in triplicate. The samples were then examined by fluorescence microscopy using an Eclipse Ti inverted fluorescence microscope (Nikon, Melville, NY) with a 40× oil immersion lens. Digital images were taken with CoolSNAP EZ CCD camera and processed in Nikon Elements Advanced Research (AR) imaging software.
Statistics and data analysis
All graphical data were plotted using the group mean for each data point. All error bars were plotted using ±SEM with the exception of the qPCR data. For qPCR analyses, relative expression levels were calculated according to the ΔΔCt method using murine GAPDH (mGAPD) as the housekeeping gene. All ΔΔCt calculations were done using Viia7™ (Life Technologies) software. Error bars were plotted using the software settings for relative quantitation and the 95% confidence interval (α = 0.05). Calculations for statistical significance were conducted with a Student’s t-test (unpaired). A p-value ≤ 0.05 was accepted as significant.
Fluorescence quantitation for serology assays was collected using Gen5™ Data Analysis Software (Biotek). All fluorescence data were exported into Excel (Microsoft Corporation, Redmond, VA) or Prism 5 (GraphPad Software, La Jolla, CA) for statistical analyses. Calculations for statistical significance were conducted with a Student’s t-test (unpaired). A p-value ≤ 0.05 was accepted as significant.
Results
MHV-68 infection and reactivation in vitro can be monitored by complementary assays
Prior to testing for the horizontal transmission of MHV-68, it was important to establish assays for detecting MHV-68 infection. A20-HE-2 cells were obtained from Dr Craig Forrest and Dr Samuel Speck (Emory University). These cells are an A20 B-cell lymphoma line latently infected with an MHV-68 virus containing a hygromycin resistance protein fused to enhanced green fluorescent protein (EGFP) (Forrest & Speck, 2008). Latency has been shown to be the default pathway of the virus in these cells, where the viral genome can be selected for and maintained in the presence of hygromycin B in the culture medium. Viral re-activation can be induced in these cells after treatment with PMA.
To induce MHV-68 recrudescence from a latent state, A20-HE-2 cells were treated with PMA (40 ng/ml) for 24 h and analyzed for viral re-activation (Figure 1) similar to a previous report using these cells (Forrest & Speck, 2008). As shown in Figure 1A, PMA induced robust viral re-activation from A20-HE-2, as shown by an increase in EGFP fluorescence in PMA-treated cells compared with untreated A20-HE-2 cells. Parental A20 cells were completely negative for EGFP fluorescence (Figure 1c and data not shown). Further characterization of A20, untreated A20-HE-2, and PMA-treated A20-HE-2 cell lines was performed by Western blotting for MHV-68-EGFP proteins, i.e. ORF-59 lytic antigen (anti-ORF-59), MHV-68 (MHV-68 antisera), and EGFP (anti-GFP). As shown in Figure 1B, PMA treatment of A20-HE-2 cells resulted in an increase in MHV-68 and EGFP protein production. Lysates from MHV-68-infected BHK-21 cells were included as positive controls as they are permissive for MHV-68 lytic infection and are commonly used to expand MHV-68 virus stocks (Cipkova-Jarcuskova et al., 2013).
Published online:
07 August 2015Figure 1. MHV-68 infection and re-activation in vitro can be monitored by complementary assays. A20 cells were infected with a MHV-68 variant containing an EGFP/Hygromycin B fusion cassette (Forrest and Speck, 2008); Hygromycin B selection gives rise to cells latently infected with MHV-68 EGFP/Hygromycin B (A20-HE-2 cells). Re-activation of MHV-68 was achieved via 24-hr stimulation of cells with PMA and detected through increased (A) EGFP fluorescence or (B) after immunoblotting against GFP or MHV-68-specific antibody (note arrows to MHV-68-specific bands). As controls, lysates from mock or MHV-68-EGFP-infected BHK-21 cells, which are permissive for MHV-68 infection, were included. Immunoblotting vs. β-tubulin was included as a protein loading control. (C) Viral re-activation in A20 parental cells and A20-HE-2 cells ± PMA was also assayed by flow cytometry. EGFP or MHV-68 protein levels were compared amongst these cell lines. (D) qPCR was employed to assay levels of lytic (RTA) and latent (mLANA) RNA transcripts in A20 parental cells and A20-HE-2 cells ± PMA treatment. Expression levels were plotted relative to A20-HE-2 cells without PMA. Asterisks indicate statistically different values in MHV-68 mRNA levels in A20-HE-2 cells treated with PMA as compared to untreated A20-HE-2 cells (Student’s t-test, p < 0.05).
![]({"type":"image","src":"/na101/home/literatum/publisher/tandf/journals/content/iimt20/2015/iimt20.v012.i04/1547691x.2014.980020/20150807/images/medium/iimt_a_980020_f0001_c.jpg"})
Figure 1. MHV-68 infection and re-activation in vitro can be monitored by complementary assays. A20 cells were infected with a MHV-68 variant containing an EGFP/Hygromycin B fusion cassette (Forrest and Speck, 2008); Hygromycin B selection gives rise to cells latently infected with MHV-68 EGFP/Hygromycin B (A20-HE-2 cells). Re-activation of MHV-68 was achieved via 24-hr stimulation of cells with PMA and detected through increased (A) EGFP fluorescence or (B) after immunoblotting against GFP or MHV-68-specific antibody (note arrows to MHV-68-specific bands). As controls, lysates from mock or MHV-68-EGFP-infected BHK-21 cells, which are permissive for MHV-68 infection, were included. Immunoblotting vs. β-tubulin was included as a protein loading control. (C) Viral re-activation in A20 parental cells and A20-HE-2 cells ± PMA was also assayed by flow cytometry. EGFP or MHV-68 protein levels were compared amongst these cell lines. (D) qPCR was employed to assay levels of lytic (RTA) and latent (mLANA) RNA transcripts in A20 parental cells and A20-HE-2 cells ± PMA treatment. Expression levels were plotted relative to A20-HE-2 cells without PMA. Asterisks indicate statistically different values in MHV-68 mRNA levels in A20-HE-2 cells treated with PMA as compared to untreated A20-HE-2 cells (Student’s t-test, p < 0.05).
A20 and A20-HE-2 cells were also examined by flow cytometry for EGFP fluorescence and MHV-68 protein expression using anti-MHV-68 antisera. As shown in Figure 1C, ∼9% of A20-HE-2 cells were positive for EGFP fluorescence; this percentage increased to ∼30% after PMA treatment. Staining with anti-MHV-68 antiserum can also be used to further differentiate lytic expression in re-activated cells as compared to latently infected cells; PMA-treated A20-HE-2 cells were ∼24% double-positive for EGFP and MHV-68 protein expression as compared to ∼2% double-positive background levels in A20 and untreated A20-HE-2 cells (Figure 1C).
Finally, quantitative PCR (qPCR) was employed to assay latent and lytic MHV-68 gene expression in A20, untreated A20-HE-2 and PMA-treated A20-HE-2 cells. In Figure 1D, gene expression of a lytic transcript (RTA; Replication Transactivator) and a latent transcript (mLANA; murine Latency Associated Nuclear Antigen) was examined. Gene expression of both of these transcripts was normalized to the untreated A20-HE-2 cell line. In both cases, expression of these genes was increased in PMA-treated A20-HE-2 cells, as was previously reported in Forrest & Speck (2008). However, gene expression of RTA was ∼100–150-fold higher in PMA-treated A20-HE-2 cells relative to that in the untreated A20-HE-2 cells. In contrast, mLANA expression was only ∼3-fold higher in PMA-treated A20-HE-2 cells relative to in the untreated cells. Neither transcript was detected in A20 parental cells.
MHV-68 is not horizontally transmitted to immunocompetent or immunodeficient mice from A20-HE-2 cells
Once the assays for MHV-68 detection were established, this study sought to determine if MHV-68 from A20-HE-2 cells would re-activate in vivo. These studies were designed to also evaluate if virus could be transmitted to naïve cagemates. As contamination of murine colonies with infectious agents can be devastating for vivariums, these experiments were performed not only to validate in vitro assays but also to establish the conditions required to safely handle MHV-68-infected mice in large murine colonies. These experiments were conducted to fully characterize if horizontal transmission of MHV-68 could occur amongst mice and warn of any potential problems associated with viral transmission in the laboratory.
To determine if MHV-68 was re-activated from A20-HE-2 cells in vivo, immunocompetent Balb/c mice (syngeneic strain for A20 and A20-HE-2 cells) and immunodeficient SCID/Bg mice (lacking B-, T-, and NK cell immunity) were implanted subcutaneously with 3 × 105 A20-HE-2 cells/mouse and co-habitated with naïve Balb/c and SCID/Bg mice for 28 days or until clinical signs of infection/distress were observed (see environmental conditions listed in Table 1). The cell dose for implant was selected due to historical evidence suggesting this dose of A20-HE-2 cells would induce measurable subcutaneous tumors within 28 days after implantation, as described in the Materials and methods (unpublished observations). These cells were utilized for virus transmission studies because it was reasoned that any tumors that developed might be a site of viral recrudescence that could be investigated for the presence of virus by the methods described in Figure 1. One other hypothesis was that, if lytic virus was re-activated in these tumors, then the likelihood of viral spread to other tissues/animals was increased. Re-activated virus in these samples might also serve as an internal control for the experiment.
Table 1. Animal housing for A20-HE-2 cell implants.
The results of the A20-HE-2 MHV-68 horizontal transmission experiment are displayed in Figure 2 and summarized in Table 2. Once the study was terminated after 28 days, any tumors that developed—as well as spleens from all of the animals—were harvested and processed to single cell suspensions. The spleen was investigated because splenic B-cells represent a major organ of MHV-68 tropism (Barton et al., 2011; Usherwood et al., 1996; Weck et al., 1996, 1997). Figure 2A presents data from control BHK-21 cultures that were either mock-infected or infected with MHV-68-EGFP virus. Figures 2B and c include representative images from the spleen and tumor cell suspensions. Interestingly, every tumor derived from a SCID/Bg implanted mouse was positive for MHV-68, while only one of the six SCIB/Bg spleens contained detectable virus by the assay methods used (Figure 2C). In contrast, MHV-68 was only detectable in two of the four tumors that developed in Balb/c hosts and was not found in any of the Balb/c spleens (Figure 2C). No virus was detected in any of the tissues tested from naïve mice (Figure 2B). Further, three of the six SCID/Bg mice implanted with A20-HE-2 cells were unexpectedly found dead toward the end of the study. MHV-68 infection is lethal in immunocompromised mice as latency can never be established (Kulkarni et al., 1997); it is likely that these hosts died as a result of the infection. However, none of the naïve mice exhibited any clinical signs of infection or had any detectable virus (see summary in Table 2). These results suggest to us that MHV-68 is not horizontally transmitted to cagemates in the laboratory. These data also indicate that B-, T-, and/or NK cell immunity were important for maintenance of MHV-68 infection.
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07 August 2015Figure 2. MHV-68 from A20-HE-2 cells was not horizontally transmitted to immunocompetent or immunodeficient mice. To determine if latently-infected A20-HE-2 cells were re-activated in the absence of in vivo immunosurveillance, 3 × 105 cells were injected into Balb/c and SCID/Beige mice. Naïve mice without A20-HE-2 cells were co-housed with these mice to test for viral transmission. At 28 days post-implant, all mice were euthanized and spleens and tumors were processed into single cell suspensions for analysis of the presence of MHV-68-EGFP (via co-culture with permissive BHK-21 cells). Presence of EGFP in cells during assessments with a fluorescence microscope was indicative of a positive MHV-68-EGFP-infected host. As controls, mock- and MHV-68-EGFP-infected BHK-21 cells (A) were imaged alongside co-cultures containing freeze–thawed supernatants from spleen or tumor cells from naïve mice (B) or mice implanted with A20-HE-2 cells (C). No virus was detected from any mouse not dosed with A20-HE-2 cells (indicating MHV-68 was not transmitted among cagemates).
![]({"type":"image","src":"/na101/home/literatum/publisher/tandf/journals/content/iimt20/2015/iimt20.v012.i04/1547691x.2014.980020/20150807/images/medium/iimt_a_980020_f0002_c.jpg"})
Figure 2. MHV-68 from A20-HE-2 cells was not horizontally transmitted to immunocompetent or immunodeficient mice. To determine if latently-infected A20-HE-2 cells were re-activated in the absence of in vivo immunosurveillance, 3 × 105 cells were injected into Balb/c and SCID/Beige mice. Naïve mice without A20-HE-2 cells were co-housed with these mice to test for viral transmission. At 28 days post-implant, all mice were euthanized and spleens and tumors were processed into single cell suspensions for analysis of the presence of MHV-68-EGFP (via co-culture with permissive BHK-21 cells). Presence of EGFP in cells during assessments with a fluorescence microscope was indicative of a positive MHV-68-EGFP-infected host. As controls, mock- and MHV-68-EGFP-infected BHK-21 cells (A) were imaged alongside co-cultures containing freeze–thawed supernatants from spleen or tumor cells from naïve mice (B) or mice implanted with A20-HE-2 cells (C). No virus was detected from any mouse not dosed with A20-HE-2 cells (indicating MHV-68 was not transmitted among cagemates).
Table 2. Summary: A20-HE-2 cells do not horizontally transmit MHV-68 to Balb/c or SCID/Bg mice.
SCID mice infected with MHV-68 do not horizontally transmit virus to naïve cagemates
MHV-68 transmission was not detected from A20-HE-2 cells to immunocompromised mice. However, that experiment did not address if transmission of virus to naïve mice occurred at levels below the limit of detection. Also, at the time of the experiment, serological assays were not in place to examine potential low levels of infection in naïve animals. Further, viral shedding may or may not have occurred due to the method used to infect these mice. Therefore, immunodeficient SCID mice were infected with 100 000 PFU (Table 3) of MHV-68 via the intra-nasal route as described in the Materials and methods. As MHV-68 cannot establish latency in immunodeficient animals, SCID mice were chosen to eliminate complications in detecting virus due to latency. This dose of virus was chosen based on historical experiments in our laboratory that suggest mice are infected at high levels (data not shown). A high level of infection was desirable to ensure a strong probability of detecting any horizontal transmission.
Table 3. Treatment regimen for viral infection of SCID mice.
The infected mice were then co-housed with naïve mice and monitored over the course of ∼75 days. MHV-68 cannot establish latency in immunodeficient animals and all infected SCID mice exhibited clinical signs of infection by ∼21 days post-infection, including weight loss starting at around Day 16, although statistically significant weight differences between these groups were not observed (Figure 3A). At necropsy, all spleens and lungs were harvested for MHV-68 gene expression by qPCR against a lytic protein (RTA, see Figure 3B); all were highly positive for MHV-68 infection. RTA levels were at least as high as that found in A20-HE-2 + PMA-treated control cells. Conversely, the naïve mice housed until Day 75 never showed clinical signs of infection or any sustained weight loss (Figure 3A). qPCR confirmed the lungs and spleens of these hosts did not express any detectable level of MHV-68 gene expression. These results were consistent with our previous observations (Figure 2) that suggested the virus was not horizontally transmitted.
Published online:
07 August 2015Figure 3. MHV-68 infection is not horizontally transmitted amongst SCID cagemates. Immunocompromised mice lacking functional T- and B-cells (SCID) were infected intra-nasally with MHV-68-EGFP and then co-habitated with naïve SCID hosts. All mice were monitored for clinical signs of infection and body weights were monitored twice weekly (A). When clinical signs appeared, the mice were euthanized and MHV-68 gene expression in spleen and lung analyzed by qPCR (B). Naïve animals were housed an additional 50 days post-infection and then analyzed. Gene expression was relative to that noted in latently-infected A20-HE-2 cells and compared with A20-HE-2 cells re-activated by PMA treatment. Asterisks indicate statistically different values in MHV-68 mRNA levels of naïve SCID spleens compared to infected SCID spleens and naïve SCID lungs compared to infected SCID lungs (Student’s t-test, p < 0.05).
![]({"type":"image","src":"/na101/home/literatum/publisher/tandf/journals/content/iimt20/2015/iimt20.v012.i04/1547691x.2014.980020/20150807/images/medium/iimt_a_980020_f0003_c.jpg"})
Figure 3. MHV-68 infection is not horizontally transmitted amongst SCID cagemates. Immunocompromised mice lacking functional T- and B-cells (SCID) were infected intra-nasally with MHV-68-EGFP and then co-habitated with naïve SCID hosts. All mice were monitored for clinical signs of infection and body weights were monitored twice weekly (A). When clinical signs appeared, the mice were euthanized and MHV-68 gene expression in spleen and lung analyzed by qPCR (B). Naïve animals were housed an additional 50 days post-infection and then analyzed. Gene expression was relative to that noted in latently-infected A20-HE-2 cells and compared with A20-HE-2 cells re-activated by PMA treatment. Asterisks indicate statistically different values in MHV-68 mRNA levels of naïve SCID spleens compared to infected SCID spleens and naïve SCID lungs compared to infected SCID lungs (Student’s t-test, p < 0.05).
SCID/Bg mice infected with a concentrated dose of MHV-68 did not horizontally transmit virus to naïve cagemates
Finally, to further ensure that MHV-68 infection was not horizontally transmitted, SCID/Bg mice were employed to examine the combined role of T-, B-, and NK cell immunity on viral transmission. As shown in Table 4, a concentrated dose of virus, equivalent to the highest dose deliverable based on the viral stock (∼450 000 PFU/mouse), was provided via the intra-nasal route. Naïve SCID/Bg mice were then co-habitated with the infected mice. All mice were then monitored for clinical signs of infection. Infected mice began to lose weight at ∼Day 12, although statistically significant weight differences were not observed (Figure 4A). The infected animals succumbed to infection between days 17–21 post-infection. High levels of RTA expression were identified in the spleens and lungs of all the infected hosts (Figure 4B). The naïve mice necropsied and examined for presence of RTA after an additional 3 weeks of housing again showed no clinical signs of infection. All naïve mice were negative for any detectable MHV-68 lytic transcripts. These data strongly indicate that MHV-68 was not transmitted horizontally among co-housed mice.
Published online:
07 August 2015Figure 4. MHV-68 infection with a concentrated viral dose is not horizontally transmitted amongst SCID/Bg cagemates. Immunocompromised mice lacking functional T-, B-, or NK cells (SCID/Bg) were infected intra-nasally with a concentrated dose of MHV-68-EGFP and then co-habitated with naïve SCID/Bg mice. (A) All mice were monitored for clinical signs of infection and body weights were monitored twice weekly. When clinical signs appeared, the mice were euthanized and MHV-68 gene expression in spleen and lung analyzed by qPCR (B). Naïve animals were housed an additional ∼40 days post-infection and then analyzed. Gene expression was relative to that noted in latently-infected A20-HE-2 cells and compared with A20-HE-2 cells re-activated by PMA treatment. Asterisks indicate statistically different values in MHV-68 mRNA levels of naïve SCID/Bg spleens compared to infected SCID/Bg spleens and naïve SCID/Bg lungs compared to infected SCID/Bg lungs (Student’s t-test, p < 0.05).
![]({"type":"image","src":"/na101/home/literatum/publisher/tandf/journals/content/iimt20/2015/iimt20.v012.i04/1547691x.2014.980020/20150807/images/medium/iimt_a_980020_f0004_c.jpg"})
Figure 4. MHV-68 infection with a concentrated viral dose is not horizontally transmitted amongst SCID/Bg cagemates. Immunocompromised mice lacking functional T-, B-, or NK cells (SCID/Bg) were infected intra-nasally with a concentrated dose of MHV-68-EGFP and then co-habitated with naïve SCID/Bg mice. (A) All mice were monitored for clinical signs of infection and body weights were monitored twice weekly. When clinical signs appeared, the mice were euthanized and MHV-68 gene expression in spleen and lung analyzed by qPCR (B). Naïve animals were housed an additional ∼40 days post-infection and then analyzed. Gene expression was relative to that noted in latently-infected A20-HE-2 cells and compared with A20-HE-2 cells re-activated by PMA treatment. Asterisks indicate statistically different values in MHV-68 mRNA levels of naïve SCID/Bg spleens compared to infected SCID/Bg spleens and naïve SCID/Bg lungs compared to infected SCID/Bg lungs (Student’s t-test, p < 0.05).
Table 4. Treatment regimen for viral infection of SCID/Bg mice.
MHV-68 infection can be monitored in vivo via serologic analysis.
To further ensure the safety of animal colonies that harbor MHV-68-infected animals, a sensitive surveillance method to track a sub-clinical in vivo infection that did not rely on the termination of animals for necropsy was needed. A plate-based fluorescence assay was next developed to monitor seroconversion of infected animals. As ‘antigen’ for detection of anti-MHV-68 antibody, Vero cells were seeded into 96-well plates and infected with MHV-68-EGFP; infection was confirmed by fluorescence microscopy for the EGFP. Sera from naïve or infected mice were used undiluted as a primary antibody to determine the timeline of anti-MHV-68 antibody production. Specifically, here, serum from uninfected Balb/c controls and mice infected with 100 000 PFU MHV-68/mouse was harvested at Days 1, 4, and 10 post-infection. As shown in Figure 5a, uninfected Vero cells were completely negative for EGFP fluorescence and for red fluorescence (using rabbit-derived anti-MHV-68 polyclonal antibody; Figure 1). Conversely, MHV-68-EGFP-infected Vero cells exhibited robust EGFP fluorescence and strong staining with anti-MHV-68 antibody (Figures 5B and 5C). While naïve animal sera were negative for anti-MHV-68 antibodies at every timepoint assayed, infected mice began to develop detectable anti-MHV-68 antibody production by day 10 (Figures 5B and C). The relative level of antibody produced was expressed in terms of the ratio of red fluorescence (e.g. anti-MHV-68; Dylight 650 fluorescence) to the amount of ‘antigen’ cells seeded/well (e.g. nuclei from viable cells present per well; DAPI fluorescence). Based on the results here, this assay could be used in a facility harboring MHV-68-infected animals to monitor infection of hosts over time and could serve as a safety measure to monitor for viral spread among other animals in the colony.
Published online:
07 August 2015Figure 5. Monitoring anti-MHV-68 antibody production in infected mice. Examination of MHV-68 antibody production was performed via immunofluorescence staining on permissive Vero cells infected with MHV-68-GFP according to the Materials and methods. (A) Control sera used to validate immunofluorescence method. Serum from naïve and MHV-68-EGFP-infected mice was assayed for anti-MHV-68 antibody. (B) Representative fluorescence images are shown for sera from naïve and infected animals at days 4 and 10 post-infection. (C) Fluorescence intensities of sera collected from all animals was also quantified in terms of the ratio of Dylight 650 (anti-Mouse IgG) intensity vs an internal nuclear standard (DAPI). Asterisk indicates statistically different anti-MHV-68 antibody levels in naive Balb/c mice at day 10 post-infection as compared to infected animals at the same time-point (Student’s t-test, p < 0.05).
![]({"type":"image","src":"/na101/home/literatum/publisher/tandf/journals/content/iimt20/2015/iimt20.v012.i04/1547691x.2014.980020/20150807/images/medium/iimt_a_980020_f0005_c.jpg"})
Figure 5. Monitoring anti-MHV-68 antibody production in infected mice. Examination of MHV-68 antibody production was performed via immunofluorescence staining on permissive Vero cells infected with MHV-68-GFP according to the Materials and methods. (A) Control sera used to validate immunofluorescence method. Serum from naïve and MHV-68-EGFP-infected mice was assayed for anti-MHV-68 antibody. (B) Representative fluorescence images are shown for sera from naïve and infected animals at days 4 and 10 post-infection. (C) Fluorescence intensities of sera collected from all animals was also quantified in terms of the ratio of Dylight 650 (anti-Mouse IgG) intensity vs an internal nuclear standard (DAPI). Asterisk indicates statistically different anti-MHV-68 antibody levels in naive Balb/c mice at day 10 post-infection as compared to infected animals at the same time-point (Student’s t-test, p < 0.05).
Discussion
MHV-68 infection in mice is being evaluated as a model of human EBV infection and may also be a suitable model for examination of gammaherpesvirus recrudescence. We have previously discussed how the MHV-68 model may be of further use for testing the effects of immunomodulatory compounds on viral recrudescence (Aligo et al., 2014). The focus of this report was to first establish assays for examination of MHV-68 infection. Once assays were in place, the potential for horizontal transmission amongst cohoused mice was investigated. Horizontal transmission of virus to naïve animals could impact concurrent studies within a vivarium. Fact-based containment and surveillance strategies for housing MHV-68-infected mice are necessary.
MHV-68 assay development was performed using a MHV-68-EGFP latently infected A20 B-cell lymphoma line (Forrest & Speck, 2008). Treatment of these cells with PMA induced MHV-68 re-activation as indicated by increases in EGFP fluorescence and protein levels, as well as an increase in MHV-68-specific gene expression and protein production. Viral re-activation and increases in EGFP fluorescence and protein expression were linked in these studies, which indicate that EGFP might be a suitable marker in the absence of robust tools (e.g. specific antibodies) for tracking MHV-68-EGFP re-activation. Measurement of MHV-68 lytic gene expression is preferable, however, as low levels of EGFP fluorescence were present in the latently infected cell line, as reported previously (Forrest & Speck, 2008). Therefore, qPCR is likely the method of choice for examination of MHV-68 infection/re-activation.
The next experiments focused on determining if latent virus from A20-HE-2 cells was capable of recrudescence in immunocompromised/immunocompetent animals, and, if so, if the infection could be transmitted to naïve cagemates. The first experiment used these cells because they had already been characterized as latently infected (Forrest & Speck, 2008) and, so, any lytic virus present would likely indicate viral recrudescence. Use of these cells for in vitro work was also considered extremely valuable. Finally, the ability to implant cells that were already latently infected without having to directly infect animals and wait for latency establishment was an attractive component of using A20-HE-2 cells.
It was hypothesized that Balb/c mice implanted with A20-HE-2 cells might never show signs of detectable infection due to innate immune mechanisms that maintained latency or levels of infection that were below the limit of detection. In contrast, it was reasoned that immunodeficient mice would eventually develop clinical signs of infection as there was no immune system in place to halt the spread of infection to other tissues. These mice also were the most likely to transmit virus if horizontal transmission occurred. Interestingly, none of the spleens from immunocompetent hosts assayed in the present study were positive for virus (Figure 2 and Table 2). These observations suggested to us that either re-activated virus was not detectable by the methods used or that immune mechanisms prevented viral spread. Alternatively, a latent infection might have been established that would have been undetectable by the assay. In contrast with these data, all immunodeficient mice implanted with A20-HE-2 cells developed tumors that were positive for virus infection. Only one animal showed systemic dissemination of virus and had detectable virus in splenic tissue. However, it is plausible that virus load in spleens may have been below the limit of detection for the assay. It is also possible we did not detect virus in immunocompromised spleens because the virus was poorly disseminated systemically from the latently-infected tumor cells. These tumor cells were delivered subcutaneously (as described in Materials and methods), and so the kinetics of viral distribution/dissemination throughout the animal from any infected tumor cells were unknown. As the study end-points solely related to animal health/tumor burden it was possible there was simply inadequate time for complete infection of the animal. Most importantly, none of the naïve immunocompetent or immunodeficient animals tested positive for viral infection or developed tumors. Further, no clinical signs of infection were ever observed in these animals. Horizontal transmission of virus amongst these animals was, therefore, unlikely.
To further determine if horizontal transmission of MHV-68 occurred in the laboratory, SCID mice were infected with a standard viral dose as well as SCID/Bg mice with a concentrated viral dose. This experiment was done in part due to questions about the robustness of using latently-infected cells for horizontal transmission studies. Infection of animals with well-defined doses of virus was also desirable to help ensure reproducibility of the assay. The use of immunodeficient animals also eliminated confounding factors associated with latency as MHV-68 cannot establish latency without functional B- and T-lymphocytes. Infected SCID animals all succumbed to infection, while naïve animals never developed clinical signs or exhibited any viral gene expression by qPCR. This experiment strongly indicated that horizontal transmission of MHV-68 did not occur among cagemates. As SCID animals lack functional B-and T-lymphocytes, we hypothesized these cell types may not play a major role in transmission of MHV-68 among murine populations. Likewise, SCID/Bg mice infected with a concentrated dose of virus also succumbed to infection, while naïve animals were completely negative for virus. SCID/Bg animals lack functional B-, T-, and NK lymphocytes, and we, therefore, hypothesized that B-, T-, and NK lymphocytes may not play a key role in MHV-68 transmission.
How then is MHV-68 infection transmitted in the wild? A recent report examined this question and determined that the most likely route of transmission of MHV-68 is via the sexual route from infected females to naïve males (Francois et al., 2013). This same report indicated that horizontal transmission of virus was not seen amongst female cagemates. This report was consistent with the current findings here, but all of those experiments used immunocompetent (Balb/c) mice and a single dose of virus (10 000 PFU/mouse). All of the animals used in the current experiments were female; it appeared unlikely that horizontal transmission of MHV-68 would be observed in future studies, provided one gender was used for infection.
The results of the current studies allow us to suggest it is possible to better ensure the safety of animals used in laboratories via serologic examination (as opposed to euthanization) to assess for any possible MHV-68 seroconversion. The data here indicated a positive serology result could be detected fairly early (e.g. within 10 days) of any initial infection. This assay will likely be quite useful in animal monitoring as it can be performed with small volumes of sera. Analyses of MHV68+ sera collected from ‘wild’ mice have also been reported previously in the literature, indicating that this assay may be useful for animal health monitoring, even when low levels of virus are present (Blasdell et al., 2003; Hughes et al., 2010).
Conclusion
In conclusion, this study identified assays that can be used for the determination of MHV-68 infections. The study also examined the possibility of horizontal transmission of virus amongst laboratory cagemates and determined the unlikelihood of viral transmission among animals of the same gender. Taken together, these results establish a platform through which future studies may examine insults to the immune system and viral recrudescence in MHV-68-infected hosts.
Declaration of interest
This work was supported by Janssen Research and Development, LLC, a Division of Johnson and Johnson Pharmaceutical Research and Development, LLC. All Janssen authors are stockholders with Johnson and Johnson. The authors report no conflicts of interest.
| Cage | Total mice per cage | Caging arrangement (3 × 105 cells/mouse; subcutaneous implant) |
|---|---|---|
| 1 | 8 | 2 Balb/c mice (no A20-HE-2) |
| 2 SCID/Bg mice (no A20-HE-2) | ||
| 2 Balb/c mice (+A20-HE-2) | ||
| 2 SCID/Bg mice (+A20-HE-2) | ||
| 2 | 8 | 2 Balb/c mice (no A20-HE-2) |
| 2 SCID/Bg mice (no A20-HE-2) | ||
| 2 Balb/c mice (+A20-HE-2) | ||
| 2 SCID/Bg mice (+A20-HE-2) | ||
| 3 | 8 | 2 Balb/c mice (no A20-HE-2) |
| 2 SCID/Bg mice (no A20-HE-2) | ||
| 2 Balb/c mice (+A20-HE-2) | ||
| 2 SCID/Bg mice (+A20-HE-2) |
| Group | Animals positive for virus |
|---|---|
| SCID/Bg, No A20-HE-2 | 0/6 |
| Balb/c, No A20-HE-2 | 0/6 |
| SCID/Beige + A20-HE-2 (spleen) | 1/6 |
| SCID/Beige + A20-HE-2 (tumor) | 6/6 |
| Balb/c + A20-HE-2 (spleen) | 0/6 |
| Balb/c + A20-HE-2 (tumor) | 2/4* |
*Sample size in this group is four because only four of the six animals developed measureable tumors.
| Group | Route of infection | Infectious dose (PFU) | Inoculation volume | Number of mice/timepoint | Key end-points assessed |
|---|---|---|---|---|---|
| I | None | 0 (Naïve) | 0 | 5 | Body, spleen, and lung weights |
| II | Intra-nasal | 100 000 | 50 µl | 5 | Viral titer in spleen and lungs |
| Viral genome number in spleen and lungs by qRT-PCR |
Environmental conditions: Housing Arrangement = five MHV-68-infected SCID mice and five non-infected (10 mice/cage).
| Group | Route of infection | Infectious dose (PFU) | Inoculation volume | Number of mice/timepoint | Key end-points assessed |
|---|---|---|---|---|---|
| I | None | 0 (Naïve) | 0 | 5 | Body, spleen, and lung weights |
| II | Intra-nasal | 450 000 | 50 µl | 5 | Viral titer in spleen and lungs |
| Viral genome number in spleen and lungs by qRT-PCR |
Environmental conditions: Housing Arrangement = five MHV-68-infected SCID/Bg mice and five non-infected (10 mice/cage).
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