A field strain of minute virus of mice (MVMm) exhibits age- and strain-specific pathogenesis

Abstract

The influence of mouse strain, immune competence and age on the pathogenesis of a field strain of minute virus of mice (MVMm) was examined in BALB/c, C3H, C57BL/6 and SCID mice experimentally infected as neonates, weanlings and adults. Sera, bodily excretions and tissues were harvested at 7, 14, 28 and 56 days after inoculation and evaluated by serology, quantitative PCR and histopathology. Seroconversion to recombinant viral capsid protein 2 was consistently observed in all immunocompetent strains of mice, regardless of the age at which they were inoculated, while seroconversion to the viral nonstructural protein 1 was only consistently detected in neonate inoculates. Viral DNA was detected by quantitative PCR in multiple tissues of immunocompetent mice at each time point after inoculation, with the highest levels being observed in neonate inoculates at 7 days after inoculation. In contrast, viral DNA levels in tissues and bodily excretions increased consistently over time in immunodeficient SCID mice, regardless of the age at which they were inoculated, with mortality being observed in neonatal inoculates between 28 and 56 days after inoculation. Overall, productive infection was observed more frequently in immunocompetent mice inoculated as neonates as compared to those inoculated as weanlings or adults, and immunodeficient SCID mice developed persistent, progressive infection, with mortality being observed in mice inoculated as neonates. Importantly, the clinical syndrome observed in experimentally infected SCID neonatal mice recapitulates the clinical presentation reported for the naturally infected immunodeficient NOD µ-chain knockout mice from which MVMm was initially isolated.

Introduction

Despite its discovery more than a half century ago, minute virus of mice (MVM) is still detected in contemporary laboratory mouse colonies [1–4]. MVM remains a concern in laboratory mice due to its potential to impact research by inducing clinical disease [5–11], haematopoietic suppression [8–11], in vitro and in vivo immunomodulatory effects [6, 12–14], tumour suppression [6, 15, 16] and contamination of cell cultures and tissues originating from mice [12, 17–22]. MVM can potentially be transmitted among research facilities due to its environmental stability [23, 24], its potential to induce persistent infection in mice and cell lines [10, 25], and the difficulties associated with eradicating the virus from infected laboratory mouse colonies [3, 26].

During an epidemiological investigation of mice naturally infected with parvoviruses, our laboratory identified a strain of MVM that is distinct from the previously reported prototype (MVMp), immunosuppressive (MVMi) and Cutter (MVMc) strains of MVM. This novel MVM strain was predominant among the MVM isolates surveyed, with 91.3 % prevalence, and was named MVMm [27]. Sequence analysis of the viral genome indicated that MVMm is 95.5, 96.0 and 96.1 % homologous to MVMp, MVMi and MVMc, respectively, with similar organization of the genome. MVMm was subsequently isolated from immunodeficient NOD µ-chain knockout mice exhibiting a clinical disease syndrome characterized by growth retardation, reduced fecundity and premature death [28]. This was the first report of MVM inducing clinical disease in naturally infected mice.

Natural infection with MVMp and MVMi with associated clinical disease or pathology in mice has not been reported, and neither of these strains were detected in a prior epidemiological survey of parvovirus-positive samples obtained from random sources [27]. This lack of detection is not surprising, since these two MVM strains were isolated from contaminated cell culture systems over four decades ago [12, 29]. The more common murine parvovirus, mouse parvovirus (MPV), similarly has not been reported to cause clinical disease in naturally or experimentally infected mice [3]. However, differences in susceptibility to infection have been demonstrated between various strains and ages of mice experimentally inoculated with MPV [30–32]. Pathogenesis studies indicated that experimental infection with MVMi induces clinical disease and pathology characterized by haematopoietic suppression and cerebellar hypoplasia in BALB/c mice inoculated as neonates, and premature death in severe combined immunodeficient (SCID) mice inoculated as neonates, with widespread tissue distribution of the virus [5–11]. Recent studies indicated that MVMi infectivity differs by sex [33], and can persist in experimentally infected immunocompetent mice [34, 35]. By contrast, mice experimentally infected with MVMp do not display clinical disease or pathology, and virus distribution is limited to the upper respiratory tract [6]. The pathogenesis of the Cutter strain of MVM (MVMc), a cell culture contaminant, has not been assessed.

Given the genetic disparity of MVMm as compared to other MVM strains, its high prevalence in naturally infected mice, its isolation from naturally infected mice afflicted with clinical disease and the variability in pathogenesis displayed by MVMp and MVMi in experimentally infected mice, the current studies aimed to demonstrate the influence of strain, immunocompetence and age at which mice were inoculated on MVMm infectivity and pathogenesis. We hypothesized that immunodeficient animals would exhibit increased susceptibility to experimental MVMm infection compared to immunocompetent strains, that immunocompetent strains would differ in their susceptibility and that the age at which mice were inoculated would influence tissue and excreted viral DNA loads, clinical disease and pathogenesis.

Results

MVMm infection in immunocompetent neonatal mice

Neonatal mice representing three immunocompetent strains (BALB/c, C3H, C57BL/6) were oronasally inoculated with MVMm. Serum samples collected at 7, 14, 28 and 56 days post-inoculation (p.i.) were monitored for antibody response to MVMp recombinant nonstructural protein 1 (NS1) and viral capsid protein 2 (VP2) antigens by multiplex fluorescent immunoassay (MFI). Seroconversion to the VP2 antigen was consistently observed in all strains, with BALB/c mice seroconverting by 7 days p.i., and C3H and C57BL/6 mice seroconverting by 14 days p.i. Seroconversion to NS1 antigen was also consistently observed, with BALB/c and C3H mice seroconverting by 14 days p.i., and C57BL/6 mice by 28 days p.i. (Fig. 1). DNA was extracted from tissue and bodily excretion samples collected from MVMm-infected neonatal mice at 7, 14, 28 and 56 days p.i. and evaluated by quantitative PCR (qPCR). Viral DNA was consistently detected in lymphoid tissues in all three immunocompetent mouse strains at all four time points evaluated, with the highest viral DNA levels being detected at 7 and 14 days p.i., while viral DNA was only consistently detected in the intestinal tissues of all mouse strains at 7 and 14 days p.i. (Fig. 2). Viral DNA was consistently detected in other (extra-intestinal, non-lymphoid) tissues at 7, 14 and 28 days p.i. in all mouse strains. Viral DNA was inconsistently detected in bodily excretions (faeces, nasopharyngeal lavage fluid, urine), with most positive samples being detected at 7 days p.i. and no positive samples being detected at 56 days p.i. Overall, seroconversion during the early course of infection (as observed in neonatally infected BALB/c mice) correlated with less viral DNA detection in tissues at 28 and 56 days p.i., while seroconversion later in the course of infection (as observed in neonatally infected C57BL/6 mice) correlated with more consistent detection of viral DNA at 28 and 56 days p.i.

Fig. 1.

Heat map demonstrating seroconversion to MVM recombinant nonstructural protein 1 (NS1) and viral capsid protein 2 (VP2) at 7, 14, 28 and 56 days p.i. by immunocompetent mice (BALB/c, C3H, C57BL/6) infected as neonates (a), weanlings (b), or adults (c). Darker colours correlate with higher serum antibody levels, and lighter colours correlate with lower serum antibody levels, measured in median fluorescent intensity (mfi).

Fig. 2.

Heat map demonstrating viral DNA loads (reciprocally transformed Ct values) of lymphoid tissues (mesenteric and peripheral lymph nodes, spleen, thymus), gastrointestinal tissues (jejunum, colon), other tissues (bone marrow, pancreas, liver, kidney, gonad, salivary gland, heart, lung, brain) and bodily excretions (faeces, nasopharyngeal lavage fluid, urine) measured at 7, 14, 28 and 56 days p.i. in BALB/c, C3H, C57BL/6 and SCID mice infected as neonates (a), weanlings (b), or adults (c). SCID mice inoculated as neonates did not survive to 56 days p.i. Darker colours correlate with higher viral DNA loads, and lighter colours correlate with lower viral DNA loads.

MVMm infection in immunocompetent weanling mice

Serological and qPCR analysis of weanling immunocompetent mouse inoculates was performed as for neonate inoculates (Figs 1 and 2). Seroconversion to recombinant VP2 antigen was observed at 14, 28, and 56 days p.i. in all immunocompetent mouse strains. Seroconversion to recombinant NS1 antigen was observed, though less consistently, in C3H and C57BL/6 mice at these same time points. Seroconversion to recombinant NS1 antigen was not observed in any BALB/c mice at any time point after inoculation. The majority of C57BL/6 animals had detectable viral DNA in lymphoid and other (extra-intestinal, non-lymphoid) tissues collected at all four time points after inoculation, while detection was less reproducible in the intestine and bodily excretions in this mouse strain. Viral DNA was inconsistently detected in tissues and bodily excretions collected from BALB/c and C3H mice. The viral DNA levels detected in immunocompetent weanling mice were comparable to the levels observed at 28 and 56 days p.i. for neonatal mice.

MVMm infection in immunocompetent adult mice

Serological and qPCR analysis of adult immunocompetent mouse inoculates was performed as for neonate and weanling inoculates (Figs 1 and 2). Seroconversion to recombinant VP2 antigen was consistently observed in all strains, with BALB/c mice and C3H mice seroconverting by 14 days p.i., and C57BL/6 mice by 28 days p.i. Seroconversion to recombinant NS1 was less consistently observed in all three mouse strains, and only at 28 and 56 days p.i. An adaptive lasso model revealed that the age at which mice were inoculated significantly (P<0.0001) affected seroconversion to NS1, while the number of days p.i. significantly (P<0.0001) predicted seroconversion to VP2 in all immunocompetent strains. The majority of C57BL/6 animals had detectable viral DNA in lymphoid and other (extra-intestinal, non-lymphoid) tissues, while detection was less reproducible in the intestine and bodily excretions in this mouse strain. Viral DNA was inconsistently detected in tissues and bodily excretions collected from BALB/c and C3H mice. The viral DNA levels detected in adult mice were comparable to the levels observed at 28 and 56 days p.i. for neonatal mice.

MVMm infection in SCID mice

Neonatal, weanling and adult SCID mice were inoculated oronasally with MVMm. Consistently increasing levels of viral DNA were detected in all tissues and excretions of infected mice in each age group at 7, 14, 28, and 56 days p.i., except for infected neonates that were found dead (seven mice) or became moribund and were euthanized (one mouse) prior to 56 days p.i. Principal component analysis (PCA), which transforms the data to highlight any variance, demonstrated complete separation between neonatally infected BALB/c, C3H, C57BL/6 and SCID mice at 7 days p.i. when plotted along the axes of greatest variation (Fig. 3). PCA determined that serology and viral DNA load in excretions separated data the most significantly. PCA did not find consistent separation of data between BALB/c, C3H, C57BL/6 and SCID weanling and adult mice at any time point (Figs 1, 2). The CBC results from blood collected at 28 days p.i. revealed neonates inoculated with MVMm developed statistically significant anaemia, leukopenia and thrombocytosis (P<0.05) as compared to mock inoculates (Fig. 4). Histopathology was performed on SCID mice inoculated as neonates. Rare intranuclear inclusion bodies were detected in the spleen and mesenteric lymph node of SCID mice inoculated with MVMm as neonates, while these inclusions were not observed in mock-inoculated SCID neonates. No histological lesions were observed in the peripheral lymph node, thymus, heart, lung, salivary gland, jejunum, colon, liver, pancreas, kidney, gonad, bone marrow or brain.

Fig. 3.

Principal component analysis (PCA) of viral DNA loads and serum antibody levels to NS1 and VP2 explains 52.1 % (PC 1) and 36 % (PC 2) of the variation between neonatally infected BALB/c, C3H, C57BL/6 and SCID mice at 7 days p.i., respectively, along the axes of greatest data variation (a). A loading plot showing the respective variables that determined the principal components, with those that cluster within the same quadrant exhibiting co-variability (b). PCA contribution scores revealed that serology (NS1 and VP2), and viral DNA loads in excretions provided the most significant contributions to the principal components (c).

Fig. 4.

White blood cell (WBC), neutrophil (NEU), lymphocyte (LYM), red blood cell (RBC) and platelet (PLT) counts from SCID neonates inoculated with MVMm or a mock inoculate and evaluated at 28 days p.i. Bars are standard error bars and P-values were calculated from a t-test (two tailed).

Discussion

MVMm is the most recently isolated strain of minute virus of mice and the only strain to be isolated directly from naturally infected mice displaying clinical disease. Previous reports have determined that MVMm is the most prevalent strain in contemporary laboratory mouse colonies [27] and can cause death in immunodeficient mice [28]. The present studies aimed to differentiate strain susceptibility, tissue tropism and pathogenesis amongst three immunocompetent mouse strains and one immunodeficient mouse strain infected with MVMm at multiple ages to better understand the progression and detection of infections in contemporary laboratory mouse colonies.

Multiplex serology was used to evaluate antibody induction following MVMm inoculation, and utilized purified MVMp VP2 and NS1 antigens, which share high amino acid homology with their MVMm counterparts [27]. Seroconversion to VP2 was consistently observed by 28 days p.i. in immunocompetent mice inoculated as neonates, weanlings and adults. The majority of BALB/c and C3H mice became seropositive at 7 and 14 days p.i., with C57BL/6 mice seroconverting at 14 and 28 days p.i. The antibody response to NS1 was less consistent, but was observed from 14 to 56 days p.i., with C57BL/6 mice again seroconverting at a delayed rate. These data indicate that mouse strain-specific host factors play a role in antibody response to MVMm. The pattern observed is similar to those in previous studies that suggested mouse strain differences in seroconversion to another murine parvovirus (MPV-1) may be due to differences in the predominant T-helper cell phenotype (Th1 vs Th2) exhibited by that mouse strain [30, 32]. Interestingly, principal component analysis indicated that mouse strain-related differences in seroconversion to MVMm are present at an extremely early age, with clear separation being observed among the four strains of neonate inoculates at 7 days p.i. While it is possible that maternal antibodies to VP2 were detected, the amount of MVMm inoculum from inoculated neonates to which naïve dams were exposed would have been far less than that received by the adult mice inoculated directly with MVMm, and the latter cohort did not seroconvert to VP2 by 7 days p.i. The NS1 protein is not present in infectious parvovirus virions, so antibody response to this antigen implies viral entry and transcription, thereby indicating productive infection of mice by MVMm [32]. NS1 seroconversion was more prevalent among neonatal inoculates than in mice inoculated as weanlings or adults, regardless of animal strain. Collectively, these results are similar to the findings from prior studies that documented high rates of NS1 seroconversion among neonatal inoculates following inoculation with MPV1 or hamster parvovirus [36, 37]. The adaptive lasso statistical model was able to predict decreasing seroconversion to NS1 with increasing inoculation age across all strains, and supports the hypothesis that age at infection plays a pivotal role in the ability of MVMm to reproduce actively within host cells.

MVM DNA was detected in immunocompetent mice by qPCR, albeit inconsistently. Viral DNA was primarily detected in intestinal tissue during the early stages of infection, while detection in lymphoid tissues was observed through all time points. This temporal pattern of tissue tropism for MVMm-infected mice is similar to that observed in experimentally induced MVMi infections [6, 34]. Interestingly, PCA highlighted that the variation seen between mouse strains inoculated as neonates was influenced most by viral loads in excretions, while the data collected from weanlings and adults did not show distinct separation between strains (Figs 1 and 2, respectively). These data support the hypothesis that there is strain-related variation in response to MVMm infection. Overall, viral DNA was more likely to be detected in mice inoculated as neonates as compared to weanling and adults. There was also an increased rate of detection in weanling C3H and C57BL/6 mice as compared to weanling BALB/c. These findings are similar to those in previous reports that focused on MVM and MPV pathogenesis, which demonstrated mouse age- and strain-related susceptibilities [6, 30–32, 34]. Notably, the viral DNA levels detected in some tissue samples were significantly higher than those present in inocula, which definitively indicates viral genome replication consistent with productive infection.

Increasing levels of viral DNA in the lymphoid and non-intestinal tissues of SCID mice inoculated as neonates, weanlings, or adults were detected throughout the 56-day course of infection. These findings suggest persistent, productive infection by MVMm. Histology was performed on tissues from SCID mice inoculated as neonates, as this cohort displayed the most consistently positive viral DNA results, and because immunodeficient mice have been shown to be more susceptible to MVMi infection [3, 34, 35]. Amphophilic intranuclear inclusion bodies were detected in the spleen and mesenteric lymph node of multiple neonatal SCID mice, providing morphological evidence of viral replication. In addition, statistically significant anaemia, leukopenia, neutropenia, lymphopenia and thrombocytosis were observed at 28 days p.i., the final time point at which tissues and blood could be sampled from neonatal inoculates. These data indicate that MVMm persistently and productively infects immunodeficient SCID mice, with eventual mortality, likely due to haematopoietic suppression of both red and white blood cell lineages.

In summary, several strains and ages of mice were infected with a clinically relevant field strain of MVM. High viral DNA loads in tissues and excretions, with consistent NS1 antibody response in animals infected as neonates, demonstrated an age-related difference in susceptibility to MVMm. The variation in the amount of viral DNA found in tissues and excretions over the time course of infection, along with the differing seroconversion rates among immunocompetent strains, indicated strain-related variability in MVMm pathogenesis. SCID mice uniquely exhibited persistent and productive infection at all ages, with morbidity and mortality being seen in mice infected as neonates. Haematopoietic suppression, as evidenced by the anaemia and leukopenia noted in neonates at 28 days p.i., likely contributed to lethality. The increasing tissue viral DNA loads among SCID mice inoculated as weanlings and adults suggested that mortality may have been observed in those animals if the time course after infection had been prolonged beyond the 56 days p.i. end point for the study. These data and conclusions support the hypotheses that age, strain and immunocompetence factors convey differences in the susceptibility, clinical disease and pathogenesis of MVMm infection in mice. Importantly, the clinical syndrome observed in experimentally infected SCID neonatal mice recapitulates the clinical presentation reported for the naturally infected immunodeficient NOD µ-chain knockout mice from which MVMm was isolated.

Methods

Mice

Four- and 9-week-old male and female BALB/cAnNHsd (BALB/c), C3H/HeNHsd (C3H) and C57BL/6NHsd (C57BL/6) mice were obtained from Envigo (Indianapolis, IN, USA). Four- and 9-week-old male and female C.B-17/IcrHsd-Prkdc^scid (SCID) mice were obtained from an intramural breeding colony. Mice of each age were placed on experiment, with additional breeding-age mice being used to produce the neonatal mice needed. The mouse strains were chosen based on previous reports of susceptibility to mouse parvovirus infections [5, 6, 10, 32, 36]. All mice were specified to be free of murine viruses (mouse hepatitis virus, minute virus of mice, mouse parvovirus, mouse rotavirus, encephalomyelitis virus, pneumonia virus of mice, Sendai virus, lymphocytic choriomeningitis virus, murine norovirus, ectromelia virus, Hantaan virus, mouse adenovirus, mouse cytomegalovirus, respiratory enteric virus III, K virus, lactic dehydrogenase-elevating virus, polyoma virus and mouse thymic virus), pathogenic bacteria and endo- and ectoparasites by intramural and vendor-supplied health surveillance reports.

Each experimental group was housed separately in microisolater caging. All manipulations of mice were performed in a class II biological safety cabinet using standard microisolation techniques. Animals were housed in sterilized static microisolation caging on aspen chip bedding (changed weekly) in a biocontainment facility at a temperature of 22–24 °C and a humidity of 30–70 %, with 12–15 air exchanges per hour, and a 14/10 h light/dark cycle. Irradiated Teklad Global 19 % Protein diet (breeders) or Teklad NIH-31 diet (weanlings and adults) (Envigo, Madison, WI, USA) and hyperchlorinated water were provided ad libitum. The University of Arizona Institutional Animal Care and Use Committee approved all animal procedures, which were performed in accordance with the Guide for the Care and Use of Laboratory Animals [38]. The animal care and use programme of the University of Arizona is fully accredited by AAALAC International.

Viral infections

MVMm was isolated and propagated as described previously [27]. Multiple strains of mice (BALB/c, C3H, C57BL/6, SCID) were inoculated with MVMm as neonates (1 day old), weanlings (4 weeks of age), or adults (9 weeks of age). Neonates were inoculated oronasally with 10 µl of viral inoculum, a dosage of log₁₀ TCID₅₀ 5.6. Weanling and adult mice were inoculated orally with a dosage of log₁₀ TCID₅₀ 5.9 of viral inoculum. Mock-infected mice [Tris-EDTA buffer (pH 8.7)] were included for studies in SCID mice. Clinical observations of MVMm-infected mice were performed daily throughout the course of the study. Mice were euthanized at 7, 14, 28 or 56 days p.i., or when observed to be moribund, by carbon dioxide inhalation. Blood was collected by cardiocentesis from each mouse. Serum obtained from immunocompetent mice was diluted 1 : 5 (vol/vol) in phosphate-buffered saline and stored at −80 °C until evaluation by MFI. Whole blood from SCID mice was collected in an EDTA microtainer and a complete blood count (CBC) was performed with a Hemavet 850 Multispecies Hematology Analyzer (Drew Scientific, Miami Lakes, FL, USA). Various tissues and bodily excretions were collected from each mouse and included mesenteric lymph node, spleen, peripheral lymph node, thymus, jejunum, colon, heart, lung, salivary gland, liver, pancreas, kidney, gonad, bone marrow, brain, faeces, urine and nasopharyngeal lavage fluid. Representative samples of each tissue from SCID mice were fixed in 4 % paraformaledehyde for histopathology or stored frozen at −80 °C for DNA extraction. Routine histotechnology was performed on fixed tissues with paraffin-embedding, 5 µM sections, and haematoxylin and eosin staining. DNA was extracted from fresh frozen specimens (approximately 20 mg tissue or 50 µl of fluid) using a MagneSil KF Genomic DNA extraction kit (Promega Corp., Madison, WI, USA) and a KingFisher robotic extraction station (Thermo Fisher Scientific, Waltham, MA, USA) per the manufacturer’s recommendations. Extracted DNA was stored at −20 °C until evaluation by quantitative PCR.

Quantitative polymerase chain reaction (qPCR)

Extracted DNA was screened by an MVM-specific qPCR assay as previously described [27, 36, 39]. Reactions were performed with a Stratagene Mx3000P qPCR system (Agilent Technologies, Santa Clara, CA, USA) and the products were analysed by the accompanying software. Each 20 µl reaction consisted of 1× TaqMan buffer [50 mM KCl, 10 µM EDTA, 10 mM Tris-HCl (pH 8.3) and 60 nM passive reference], 5.5 mM MgCl2, 200 µM (each) dATP, dCTP and dGTP, 400 µM dUTP, 300 nM primers, 100 nM probe, 0.2 U of AmpErase uracil-N-glycosylase (UNG), 0.5 U AmpliTaq Gold Polymerase (Thermo Fisher Scientific, Carlsbad, CA, USA) and 2 µl template DNA. The thermal cycling conditions consisted of 50 °C for 2 min for UNG incubation, polymerase activation at 95 °C for 10 min and then 45 cycles of 95 °C for 15 s followed by 60 °C for 1 min. Samples were considered positive if they exhibited a cycle threshold (Ct) <35. Ct values were reciprocally transformed using an assay-derived logarithmic scale based on the standard curve.

Serology

The MFI format was used to evaluate sera for the presence of virus-specific antibodies. Baculovirus-expressed recombinant MVMp NS1 and recombinant MVMp VP2 were prepared as previously described [40, 41]. Purified recombinant NS1 and VP2 antigens were covalently coupled to carboxylated polystyrene microspheres (Luminex Corporation, Austin, TX, USA) at a coupling concentration of 25 µg of protein per 5×106 microspheres according to the manufacturer’s recommended protocols. Ovalbumin, A92L mouse fibroblast cell lysate and Hi-Five insect cell lysate were similarly coated to microspheres to serve as control antigens, with the cell lysates being prepared by three freeze-thaw cycles. Microspheres were stored at 4 °C in the dark until use. Evaluation of mouse sera for recombinant NS1- and VP2-specific antibodies was performed automatically (LiquiChip Workstation, Qiagen, Valencia, CA, USA) as described previously [27]. Briefly, antigen-coated microspheres were incubated for 60 min with dilute sera at a final dilution of 1 : 500 in 100 µl diluent, washed twice, incubated with phycoerythrin-conjugated F(ab’)2 fragment goat antimouse IgG (H+L) secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA, USA), washed twice and resuspended in stop solution containing formalin. The microplate was then shaken for 5 min and analysed. Baseline values of 100 (VP2) and 250 (NS1) were used to discriminate negative and positive samples. The baselines had been determined previously as the mean plus 5 standard deviations of results obtained for 50 serum samples from mice known to be negative for murine parvovirus infections.

Statistical analyses

Statistical analyses were performed using R statistical software (R Foundation for Statistical Computing, Vienna, Austria) or JMP, version 13 (SAS Institute Inc., Cary, NC, USA). Specifically, principal component analysis (PCA) was used to identify those principal components that accounted for the variation within data [42]. Generalized regression (adaptive lasso) was used with validation (leave-one-out) to predict NS1 and VP2 using the predictors [43]. Log₁₀ or Johnson Sb distributional transformation were used to transform covariates and dependent variables. Complete blood count parameters obtained from mock and MVMm-infected SCID mice were compared with a Student’s t-test (95 % confidence intervals, two tails) to determine statistical significance. A P-value of 0.05 was considered statistically significant for all analyses. The MFI and viral copies were plotted using heat map.