Effect of Murine Norovirus Infection on Mouse Parvovirus ... - CiteSeerX

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Susan R Compton,1 Frank X Paturzo,1 and James D Macy1,2. Enzootic infection with mouse ...... Gaertner DJ, Otto G, Batchelder M. 2007. Health delivery and.
Journal of the American Association for Laboratory Animal Science Copyright 2010 by the American Association for Laboratory Animal Science

Vol 49, No 1 January 2010 Pages 11–21

Effect of Murine Norovirus Infection on Mouse Parvovirus Infection Susan R Compton,1 Frank X Paturzo,1 and James D Macy1,2 Enzootic infection with mouse parvovirus (MPV) remains a common problem in laboratory colonies, and diagnosis of MPV infection is complicated by viral and host factors. The effect of an underlying viral infection on MPV infection has not previously been investigated. We assessed the effect of murine norovirus (MNV) infection, the most prevalent infectious agent in laboratory mice, on MPV shedding, tissue distribution and transmission. Fecal MPV shedding persisted longer in BALB/c mice infected with MNV 1 wk prior to MPV infection than in mice infected with MPV only, but transmission of MPV to soiled-bedding sentinels was not prolonged in coinfected mice. MPV DNA levels in coinfected BALB/c mice were higher in mesenteric lymph nodes and spleens at 1 and 2 wk after inoculation and in small intestines at 1 wk after inoculation compared with levels in mice infected with MPV only. In C57BL/6 mice, fecal shedding was prolonged, but no difference in soiled bedding transmission or MPV DNA levels in tissues was detected between singly and coinfected mice. MPV DNA levels in singly and coinfected SW mice were similar. MPV DNA levels were highest in SW, intermediate in BALB/c and lowest in C57BL/6 mice. MPV DNA levels in mesenteric lymph nodes of BALB/c and SW mice exceeded those in small intestines and feces, whereas the inverse occurred in C57BL/6 mice. In conclusion, MNV infection increased the duration of MPV shedding and increased MPV DNA levels in tissues of BALB/c mice. Abbreviations: MLN, mesenteric lymph nodes; MNV, murine norovirus; MPV, mouse parvovirus.

Mouse parvovirus, a small nonenveloped DNA virus, initially was isolated from cloned T cells and infects several lymphoid tissues including mesenteric and peripheral lymph nodes, Peyer patches, spleen, and thymus.22,35 Although MPV infections do not directly result in clinical disease, they have the ability to disrupt mouse-based research through the production of aberrant T-cell proliferative responses and acceleration of Tcell–mediated rejection of tumors, skin allografts, and syngeneic skin grafts.36,37 MPV infections are difficult to detect and control because the amount of virus shed is low and transmission can be inefficient, resulting in inconsistent seroconversion of sentinel mice.7,32 Eradication of MPV infection from enzootically infected colonies of mice can be achieved by embryo transfer or test-and-cull methods.6,15,51 During the acute phase of MPV infection (from 3 to 14 d after infection), transmission through contact or soiled bedding is reliable, and MPV DNA levels are consistently high in the intestine, mesenteric lymph nodes (MLN), and spleen.14,22,45,47,48 All ages and strains of mice can be infected with MPV, but the ease with which infection is induced is dependent on many factors, including the strain and age of the mouse, the dose and strain of MPV, the virus’s passage history in cultured cells, and the type of caging the mice are housed in. In 1 study, all 4- and 8-wk-old ICR mice inoculated with MPV1b seroconverted, whereas a 20fold greater dose of virus resulted in only 80% seroconversion in 12-wk-old ICR mice.7 Other studies have shown that C57BL/6 mice were infected less reliably than were BALB/c, C3H/HeN, and DBA/2 mice.7,43 Infection of mice with tissue-culture– adapted stocks of MPV was more difficult than was infection of mice with tissue stocks of MPV.14 Transmission of virus through Received: 02 Jul 2009. Revision requested: 10 Aug 2009. Accepted: 11 Aug 2009. 1Section of Comparative Medicine and 2Yale Animal Resources Center, Yale University School of Medicine, New Haven, Connecticut. *Corresponding Author. Email: [email protected]

soiled bedding was more reliable in mice housed in individually ventilated cages than in static isolation cages.48 During the chronic phase of MPV infection (from 4 to 24 wk after infection), transmission of MPV to contact or soiled bedding sentinels occurs infrequently, but low levels of viral DNA are frequently detected in the feces and MLN.22,47 The inconsistencies reported for the duration of MPV infections under various conditions may be due in part to differences in the methods used to detect infection, such as using functional assays of infectivity (for example, transmission) compared with molecular assays. For example, SW mice infected with MPV did not transmit virus to soiled bedding or contact sentinels after 2 wk but MPV DNA was detected in the MLN through 18 wk after infection.48 In addition, inconsistencies can be due to the dose of the virus and the sample assayed. In general, after exposure of mice to a high dose of MPV, virus persists in the MLN for longer than it does in the feces, whereas exposure to a low dose of MPV does not always result in dissemination to the MLN, and MPV can only be detected in the feces.48 Several studies have suggested that in most cases, the MPV DNA detected during the chronic phase of the infection is noninfectious or below the threshold required to initiate a productive infection.45,48 The 1 documented exception is SCID mice, which sustain high levels of MPV in the intestine, spleen, lymph nodes, thymus, and feces and reliably transmitted virus to sentinels for several months.4 Whereas MPV was the most prevalent viral agent in laboratory mice in 1996 (27% of SPF mice) through extensive prevention and control efforts, MPV is now the second to fourth most prevalent agent, with a prevalence of 1% to 1.86% in current mouse colonies.23,33,44 Even with this drop in prevalence, more than 90% of academic institutions and teaching hospitals surveyed in 2006 reported having MPV on campus within the last 3 y.9 Therefore, MPV remains a difficult infection to control, and the epidemiology of spread has some interesting caveats. For 11

Vol 49, No 1 Journal of the American Association for Laboratory Animal Science January 2010

example, frequently only 1 of 2 cohoused sentinels seroconvert despite being exposed to the same dose of soiled bedding.48 In addition, transmission and spread appear to be more efficient in some MPV outbreaks and less so in others. Another factor that could affect MPV infection and its ability to be detected in colonies of mice that has not been investigated is the presence of underlying infection with another agent with a route of infection through the intestinal tract. Two recent serologic surveys have reported murine norovirus (MNV) as the most prevalent agent (32%) in laboratory mouse colonies.33,44 In addition, MNV RNA was detected in the feces of 64% of mouse strains tested.40 Therefore, we investigated the effect of an established infection with MNV, the most common viral agent, on MPV pathogenesis to determine whether coinfection could contribute to the unpredictable nature of MPV infections. Murine norovirus is a small nonenveloped RNA virus that was first identified in 2002.24 More than 60 strains of MNV have been identified, and multiple MNV strains have been reported to coexist in mouse populations and can be found within an individual mouse.17 Murine noroviruses cause subclinical infections in immunocompetent mice without significant tissue pathology, and infection is limited to the intestines, liver, spleen, lymph nodes, and lungs.19,24,41 MNV targets macrophages and dendritic cells, and dissemination to multiple organs is controlled by the innate immune response, as fatal disseminated disease occurs in mice with compromised innate immune systems (for example, Stat1−/− and IFNαβγR−/− mice).24,54 Mouse strain has not been reported to influence susceptibility to MNV infection but does affect MNV titers in intestines and spleens.10,30 Duration of infection ranges from less than 3 d to more than 6 mo and appears to be dependent on the history of the viral inocula.19,20,24 Infection can consistently be detected through serology or fecal RT-PCR.19,20 MNV is highly stable within the environment and in feces.2,3,8,34,42 Therefore fomite-based transmission can occur, and viral RNA remains detectable in feces for at least 2 wk.12,19 Eradication of infection from enzootically infected mice can be achieved through fostering of neonatal mice to uninfected dams or caesarean rederivation.1,12,16 The infectious dose for human norovirus is the lowest of any reported virus, with only a single Norwalk virus particle needed to initiate infection;49 therefore, it is assumed that very few MNV virions are needed to initiate an infection. Because MNV infections are chronic and difficult to eliminate from enzootically infected populations of mice, their presence is tolerated reluctantly in many contemporary mouse colonies. Given the enzootic nature of MNV, chronic infection of mice with MNV frequently may precede acute infection with MPV. Understanding the effect of underlying infections on MPV pathogenesis is essential to develop effective strategies to detect and eliminate MPV infections in mice. The potential for coinfections to affect the pathogenesis of intestinal infections has been demonstrated. For example, coinfection with H. hepaticus and mouse hepatitis virus and coinfection with MNV and H. bilus in immunodeficient mice have shown that coinfections can increase the severity of disease and alter the lesion distribution substantially compared with infections with these agents singly.13,29 The present studies were done to determine whether MNV infection alters MPV dissemination from the intestine, duration of shedding, or viral load in the feces and tissues. Changes in any of these measures could contribute to the variability seen in the course of MPV infections. We used BALB/c and C57BL/6 mice in these studies because they differ in their susceptibility to MPV. SW mice were utilized because they are routinely used as sentinels. 12

Materials and Methods

Animal care and use. Female Swiss Webster mice (Tac:[SW] or CRL:CFW[SW]; age, 4 wk) were obtained from Taconic Farms (Germantown, NY) or Charles River (Kingston, NY), and female BALB/CByJ and C57BL/6J mice were obtained from Jackson Laboratories (Bar Harbor, ME). Vendor reports indicated mice were seronegative for ectromelia virus, murine rotavirus, lymphocytic choriomeningitis virus, mouse hepatitis virus, MPV, minute virus of mice, MNV, pneumonia virus of mice, reovirus, Sendai virus, and Mycoplasma pulmonis and were free of bacterial and parasitic infections at time of shipment. Mice were housed in an animal room with a negative pressure differential relative to the corridor, room temperature of 22.2 ± 1.1 °C, room humidity of 50 ± 10%, a 12:12 h-light cycle, and 10 to 15 air changes per hour. Mice were housed in sterilized ventilated caging (Allentown, Allentown, NJ) on sterilized corncob bedding and were fed sterilized rodent chow (diet 5010, Purina Mills International, St Louis, MO) and hyperchlorinated water (8 to 12 ppm) ad libitum by water bottle. Cages were changed weekly in a class II biosafety cabinet within the animal room. Work space, instruments, gloves, and cage exteriors were disinfected with chlorine dioxide after handling mice. All animal care and experimental procedures were approved by the institutional animal care and use committee and were in accordance with all federal policies and guidelines governing the use of vertebrate animals. Viral infections. Mice were inoculated orally with 300 infectious doses (ID50; 20 µL of a 10% intestinal stock in Dulbecco media) of MPV1d,46 100 ID50 (20 µL of a 10% intestinal stock in Dulbecco media) of MNVG,12 20 µL media, or 20 µL 10% MLN homogenates from experimentally infected mice. Sample collection. A single fecal pellet was collected from the anus of each unanaesthetized mouse while it was restrained gently. Fecal pellets were frozen at –70 °C pending PCR and RT-PCR analysis. After euthanasia of mice by carbon dioxide overdose, organs were examined for gross lesions. Small intestine, colon, and liver were collected aseptically and were frozen at –70 °C for PCR analysis. Spleen and MLN were collected and processed for flow cytometric and PCR analysis. Serology. After euthanasia of mice by carbon dioxide overdose, blood was collected from all mice by cardiocentesis, and sera were tested for antibodies to MPV and MNV by using indirect immunofluorescence assays as previously described.12,47 PCR assays. Fecal pellets were homogenized in 400 µL (1 pellet), 600 µL (3 pellets), or 800 µL (5 pellets) of PBS, and tissues were homogenized in Dulbecco media. DNA was isolated from 200 µL of fecal homogenates or 30 µL of 10% (w/v) tissue homogenates by using DNeasy kits (Qiagen, Valencia, CA) according to the manufacturer’s instructions. PCR amplification was performed by using 5 µL of DNA, Roche PCR core kit (Indianapolis, IN), and primers specific for the MPV nonstructural gene as previously described.14,32 Quantitative PCR analysis was performed by using 3 µL of DNA, Dynamo SYBR Green qPCR kit (New England Biolabs, Ipswich, MA), and MPV nonstructural gene primers. The reaction conditions were: 2 min at 94 °C; 35 cycles of 30 s at 92 °C, 30 s at 56 °C, 60 s at 72 °C; and 5 min at 72 °C. RNA was isolated from 50 µL of fecal or tissue homogenates by using RNeasy kits (Qiagen) according to the manufacturer’s instructions. MNV RT-PCR analysis was performed by using 2 µL of RNA, Superscript One-Step RTPCR kits (Invitrogen, Carlsbad, CA), and primers specific for the MNV capsid gene as previously described.12 PCR primers were obtained from the WM Keck Foundation Biotechnology Resource Laboratory at Yale University. All assays included positive and negative controls.

MNV and MPV coinfection

Fluorescent cell sorting. Flow cytometry was performed on cells isolated from the spleen and MLN of uninfected control and virally infected mice. Spleens and MLN were homogenized in 200 µL ice-cold calcium- and magnesium free-PBS. Aliquots of cells (10 to15 µL; approximately 106 cells) were added to each 12 × 75 mm tube (BD Falcon, New Bedford, MA). Cells were washed in PBS containing 1% bovine serum albumin, decanted, and then resuspended in 50 µL antibody cocktail (Pharmingen, San Jose, CA) containing fluorescently labeled antiB220 (B cells), antiCD4 (T cells), antiCD8 (T cells), antiCD11b (macrophages), antiCD11c (dendritic cells), antiCD49b (NK cells), or antiCD69 (activated lymphocytes; Pharmingen). After a 30-min incubation on ice, the cells were washed, resuspended in 200 µL 2% paraformaldehyde, and then analyzed by using a FACSCalibur system (BD Biosciences, San Jose, CA). Statistics. Two-tailed t tests were done by using Microsoft Excel (Microsoft, Redmond, WA). Fisher exact probability tests were done by using http://faculty.vassar.edu/lowry/ fisher.html. Experimental design. Studies 1 and 2: MNV and MPV infections in BALB/c mice. Three groups of 4-wk-old female BALB/c mice (n = 25 each) were inoculated orally with: MNVG on day 0 and mock-infected on day 14 with media (MNV group); mock-infected on day 0 with media and inoculated with MPV1d on day 14 (MPV group); or inoculated on day 0 with MNVG and on day 14 with MPV1d (to simulate an acute MPV infection in mice enzootically infected with MNV; MNV+MPV group). The 15 mice used to determine shedding and transmission mice were housed 3 per cage in ventilated caging. At weekly cage changes from 0 to 7 wk after infection with MNV, fecal pellets were collected for PCR analysis. A single SW sentinel mouse was placed in each soiled cage 2 to 5 wk after infection. Bedding sentinels were housed singly in contact with soiled bedding for 1 wk, and then they were housed singly for 2 more weeks before they were tested for the presence of MPV and MNV antibodies. Ten mice (housed 5 per cage) were used to determine tissue distribution of MPV. The distribution of MPV DNA and MNV RNA in MLN, spleen, small intestine, and colon of 5 mice in each experimental group were assessed at 1 and 3 wk after MPV infection (4 and 6 wk after MNV infection) by quantitative PCR and RT-PCR analysis. Immune cell populations in the spleen and MLN were analyzed by using a fluorescent cell sorter to identify CD4+ T cell, CD8+ T cell, B cell, NK cell, macrophage, and dendritic cell response profiles. A follow-up experiment was attempted to determine whether the effects of MNV+MPV coinfection observed in BALB/c mice could be reproduced by using Helicobacter hepaticus infection followed by MPV infection. However, H. hepaticus infection could not be established, and the mice were used to repeat the MNV+MPV study with a few modifications (described following). Feces from H. hepaticus-inoculated mice were negative for H. hepaticus DNA at 2 and 3 wk after Helicobacter inoculation and at 1 and 4 wk after MPV inoculation. Forty 6-wk-old BALB/c mice were mock-infected on day 0 with media and inoculated with MPV1d on day 7 (MPV group) and 40 6-wk-old BALB/c mice were inoculated on day 0 with MNVG and on day 7 with MPV1d (MNV+MPV group). Half of the mice inoculated previously with noninfectious Helicobacter cultures were placed in each group. No differences in results were observed between mice that were or were not inoculated with Helicobacter cultures. MPV DNA and MNV RNA in MLN, spleen, small intestine, colon, and feces of 5 mice in each experimental group were assessed at 1 and 2 after MPV infection (3 and 4 wk after MNV

infection) by quanitative PCR and RT-PCR analysis; flow cytometry was not done. Study 3: MNV and MPV infections in C57BL/6 mice. Forty 6-wk-old female C57BL/6 mice were inoculated orally on day 0 with media and with MPV1d on day 7 (MPV group) and 40 6-wk-old C57BL/6 mice were inoculated on day 0 with MNVG and on day 7 with MPV1d (MNV+MPV group). Thirty mice per group (housed 3 per cage) were used to determine shedding and transmission. At weekly cage changes from 0 to 5 wk after infection with MNV fecal pellets were collected for PCR analysis, and 1 SW sentinel mouse was placed in each of the 10 soiled cages at 2 to 4 wk after infection. Bedding sentinels were housed singly in contact with soiled bedding for 1 wk and then they were held singly for 2 more weeks before they were tested for the presence of MPV and MNV antibodies. The remaining 10 mice in each group (housed 5 per cage) were used to determine tissue distribution of MPV. The distribution of MPV DNA and MNV RNA in MLN, spleen, small intestine, colon, and feces of 5 mice in each experimental group were assessed at 1 and 2 wk after MPV infection (2 and 3 wk after MNV infection) by quanitative PCR and RT-PCR analysis. Study 4: MNV and MPV infections in SW mice. Ten 5-wk-old female SW mice were inoculated orally on day 0 with media and on day 7 with MPV1d (MPV group), and 10 5-wk-old SW mice were inoculated on day 0 with MNVG and on day 7 with MPV1d (MNV+MPV group). The distribution of MPV DNA and MNV RNA in MLN, spleen, small intestine, colon, and feces of 5 mice in each experimental group were assessed at 1 and 2 wk after MPV infection (2 and 3 wk after MNV infection) by quanitative PCR and RT-PCR.

Results

Study 1: Effect of MNV infection on MPV infection in BALB/c mice. To determine whether an established infection with MNV affected MPV infection, fecal shedding and transmission by soiled bedding, BALB/c mice were inoculated with MNV alone (MNV group), MPV alone (MPV group), or MNV 2 wk prior to MPV infection (MNV+MPV group). Chronic MNV infection was verified by fecal RT-PCR analysis, and MNV RNA was detected in the feces of all mice in the MNV and MNV+MPV groups from 1 wk before MPV infection through 5 wk after MPV infection; all mice in these groups were MNV-seropositive at the end of the study. MPV DNA was detected in pooled feces of mice in the MPV group from only 71% of cages at 1 wk after infection and no cages at 2, 3, 4, and 5 wk after infection (Table 1). In contrast, MPV DNA was detected in pooled feces of mice in the MNV+MPV group from all cages at 1 and 2 wk after infection and no cages at 3, 4, and 5 wk after infection (Table 1). At 1 wk after infection, all mice in the MNV+MPV group were seropositive for MPV, whereas the mice in the MPV group were not seropositive (Table 2). All mice in the MPV and MNV+MPV groups were seropositive for MPV at 3 and 5 wk after infection (Table 2). To determine whether the amount of virus present in the soiled bedding was sufficient to transmit infection to naïve mice, 1 SW mouse was placed in each cage at weekly cage changes. All of the mice exposed to soiled bedding from the MPV or MNV+MPV groups at 1 wk after infection were seropositive for MPV, indicating that during acute infection MPV levels in soiled bedding are sufficient to transmit infection (Table 3). At 2 and 3 wk after infection, only 20% to 40% of mice exposed to soiled bedding from the MNV+MPV group were MPV-seropositive, whereas none of the mice exposed to soiled bedding from the MPV group were MPV-seropositive (Table 3). Although 13

Vol 49, No 1 Journal of the American Association for Laboratory Animal Science January 2010

Table 1. Cages of mice containing feces positive for MPV DNA Wk after infection Study

Strain

Group

1

2

3

4

5

1

BALB/c

MNV

0/7

0/6

0/5

0/5

0/5

1

BALB/c

MPV

5/7

0/6

0/5

0/5

0/5

1

BALB/c

MNV+MPV

2

BALB/c

MPV

7/7

6/6

0/5

0/5

0/5

12/12

0/11

0/10

0/10

0/10

2

BALB/c

MNV+MPV

12/12

11/11

10/10

9/10

3/10

3

C57BL/6

MPV

12/12

1/11

0/10

not done

not done

3

C57BL/6

MNV+MPV

12/12

8/11

0/10

not done

not done

Data are given as number fecal pools positive for MPV DNA / number of fecal pools tested. Table 2. MPV serology in MPV-inoculated mice Wk after infection MPV group MNV+MPV group

Study

Strain

1

BALB/c

1

1

BALB/c

3

5/5

5/5

1

BALB/c

5

15/15

15/15

2

BALB/c

1

1/5

4/5

2

BALB/c

2

5/5

5/5

2

BALB/c

6

30/30

30/30

3

C57BL/6

1

0/5

0/5

3

C57BL/6

2

0/5

1/5

3

C57BL/6

4

28/30

28/30

4

SW

1

5/5

5/5

4

SW

2

5/5

5/5

0/5a

5/5a

Data are given as number of mice seropositive for MPV/ number of mice tested. aValues significantly (P = 0.007) different between groups.

the mice in the MNV+MPV group shed MPV longer than did the MPV group (2 wk and 1 wk, respectively), differences in transmission to soiled bedding sentinels were not statistically significant between the 2 groups. Only 1 mouse exposed to soiled bedding from the MNV group and 3 mice exposed to soiled bedding from the MNV+MPV group were seropositive for MNV (Table 3), indicating that although all mice in these groups seroconverted to MNV and were shedding MNV in their feces, the levels shed during the chronic phase of the infection were below the amount necessary to transmit infection in most cages. MNV RNA and antibodies were not detected in any of the mice in the MPV group, and MPV DNA and antibodies were not detected in any of the mice in MNV group, thus confirming that cross-contamination did not occur. Tissues from 5 mice in each group were analyzed at 1 and 3 wk after MPV infection to determine how MPV and MNV infections affect immune cells populations and to characterize the tissue distribution of MPV in singly and dually infected mice. Flow cytometry of cells isolated from MLN and spleen revealed no differences in the percentages of CD4+, CD8+, CD11b+, CD11c+, CD49b+, and CD69+ cells in the uninfected controls and 3 groups of infected mice (data not shown). Quantitative PCR analysis was performed on DNA extracted from aliquots of cells from the MLN and spleen isolated for flow cytometry, from fecal homogenates, and from 10% (w/v) colonic homogenates. MPV levels were significantly (P < 0.03) higher at 1 wk after infection in MLN, feces, spleens, and colons and at 3 wk after infection in MLN of MNV+MPV infected mice compared with mice infected with MPV only (Table 4). At 3 wk after MNV infection, MNV RNA was detected in the feces, MLN, and colons, but 14

not the spleens, of all of the mice in both the MNV+MPV and MNV groups. Study 2: Effect of MNV infection on MPV DNA levels in BALB/c mice. We performed a second study to confirm that MNV infection alters the course of MPV infection in BALB/c mice and to more accurately determine the MPV DNA levels in tissues of singly and dually infected mice. MNV RNA was detected in the feces of all mice in the MNV+MPV group from 1 wk before MPV infection through 4 wk after MPV infection, and all mice in the MNV+MPV group tested at 2, 3, and 5 wk after infection were MNV-seropositive. MPV DNA was detected in pooled feces of mice in the MPV group from all cages at 1 wk after infection and no cages at 2 to 5 wk after infection (Table 1). In contrast, MPV DNA was detected in pooled feces of mice in the MNV+MPV group from all cages at 1, 2, and 3 wk after infection; 90% of cages at 4 wk after infection; and 30% of cages at 5 wk after infection (Table 1). MPV was shed for a longer period of time from the mice in the MNV+MPV group in this study as compared with the MNV+MPV group in study 1 (5 versus 2 wk). Collectively, these results confirm that MNV infection can extend the period of shedding of MPV in feces for 1 to 4 wk. The MPV PCR signal was substantially weaker at 4 and 5 wk after infection than at 1, 2, and 3 wk after infection, suggesting that MPV levels in the feces in each mouse were decreasing or that not all mice in the cage were shedding MPV. This result was confirmed by quantitative PCR analysis of fecal pools DNA from the MNV+MPV group, which showed that MPV DNA levels were 8600 to 40,000 fg/µL at 1 wk after infection, 10 to 300 fg/µL at 2 and 3 wk after infection, and 0 to 3 fg/µL at 4 and 5 wk after infection. A single mouse in the MPV group and 4 mice in the MNV+MPV group were seropositive for MPV at 1 wk after infection, and all mice in both groups were seropositive for MPV at 2 and 6 wk after infection (Table 2). When combined with those of study 1, these results demonstrate that mice in the MNV+MPV group seroconverted to MPV faster than mice in the MPV group. Most of the mice exposed to soiled bedding from the MPV group at 1 and 2 wk after infection were seropositive for MPV, whereas none of mice exposed to soiled bedding from the MPV group at 3 and 4 wk after infection were seropositive for MPV (Table 3). All of the mice exposed to soiled bedding from the MNV+MPV group at 1 and 2 wk after infection were seropositive for MPV, a single mouse exposed to soiled bedding from the MNV+MPV group at 3 wk after infection was seropositive for MPV, and none of the mice exposed to soiled bedding from the MPV group at 4 wk after infection were seropositive for MPV (Table 3). Similar to the results of study 1, the mice in the MNV+MPV group shed MPV longer than the MPV group (5 wk compared with 1 wk) but differences in transmission to soiled bedding sentinels were not statistically significant between the 2 groups in either

MNV and MPV coinfection

Table 3. MPV- and MNV-seropositive sentinels exposed to soiled bedding from BALB/c or C57BL/6 mice Wk of viral inoculation

MPV-seropositivity at indicated wk after inoculation

MNV-seropositivity at indicated wk after inoculation

Study

Strain

MNV

MPV

1

2

3

4

2

3

4

5

1

BALB/c

0

nd

0/5

0/5

0/5

nd

0/5

1/5

0/5

nd

1

BALB/c

nd

2

5/5

0/5

0/5

nd

0/5

0/5

0/5

nd

1

BALB/c

0

2

5/5

2/5

1/5

nd

1/5

2/5

0/5

nd

2

BALB/c

nd

1

8/10

7/10

0/10

0/10

0/10

0/10

0/10

0/10

2

BALB/c

0

1

10/10

10/10

1/10

0/10

6/10

3/10

2/10

2/10

3

C57BL/6

nd

1

5/10

0/10

0/10

nd

0/10

0/10

0/10

nd

3

C57BL/6

0

1

7/10

1/10

1/10

nd

0/10

1/10

0/10

nd

nd, not done Data are given as number of sentinel mice seropositive for MPV or MNV/ number of mice tested Table 4. MPV DNA (fg/µL ) in tissues of infected BALB/c mice Group

Wk after inoculation

MLN 1719a

Feces 68b

(341–3007)

MPV

1

MNV+MPV

1

33190a (19420–54175)

MPV

3

MNV+MPV

3

(14–165)

Spleen 53c

(32–74)

Colon 36d

(17–61)

4212b (1881–4588)

3455c (2357–4496)

873d (486–1427)

129e (0–215)

0

nd

nd

5876e (3100–10264)

34 (0–113)

nd

nd

nd, not done Data are given as mean (range) of 5 mice per group and sample type. aValue significantly (P = 0.010) different between groups for the same sample and time point. bValue significantly (P = 0.027) different between groups for the same sample and time point. cValue significantly (P = 0.001) different between groups for the same sample and time point. dValue significantly (P = 0.013) different between groups for the same sample and time point. eValue significantly (P = 0.009) different between groups for the same sample and time point.

study. Transmission of MNV to soiled bedding sentinels was more efficient in study 2, in that 60% of mice exposed to soiled bedding from the MNV+MPV group at 2 wk after infection were seropositive for MNV and transmission from soiled bedding also occurred at 3, 4, and 5 wk after infection (Table 3). MNV RNA and antibodies were not detected in any of the mice in MPV group confirming that cross-contamination did not occur. Mesenteric lymph nodes, small intestines, feces, spleens, and colon from 5 mice in each group were analyzed at 1 and 2 wk after infection to confirm that MPV levels were higher in MNV+MPV-infected mice as compared with mice infected with MPV only. MPV levels were significantly (P < 0.04) higher at 1 wk after infection in the MLN, small intestines, and spleens of mice in the MNV+MPV group as compared with the MPV group (Table 5, Figure 1). MPV levels tended to be higher at 1 wk after infection in the feces and colons of mice in the MNV+MPV group as compared with the MPV group (P = 0.1; Table 5, Figure 1). MPV levels were lower in all tissues at 2 wk after infection, but MPV levels were still significantly (P < 0.03) higher in the MLN and spleens of mice in the MNV+MPV group as compared with the MPV group (Table 5, Figure 1). At 2 wk after MNV infection, MNV RNA was detected in all feces, all MLN, 3 colons, and 1 small intestine of mice in the MNV+MPV group. At 3 wk after MNV infection, MNV RNA was detected in all feces, 3 MLN, 2 colons, and 1 small intestine of mice in the MNV+MPV group. These 2 studies in BALB/c mice indicated that MNV infection can increase MPV levels in several tissues and could increase the probability of detecting an MPV infection by MLN or fecal PCR but that these increases in MPV DNA levels do not translate into a significant rise in transmission to soiled bedding sentinels.

Figure 1. Mean concentration (fg/µL) of MPV DNA in tissues from 5 BALB/c mice at 1 and 2 wk after MPV inoculation. Blue bars represent mice inoculated with MPV only, and red bars indicate mice inoculated with MNV and MPV. Error bars indicate ± 1 SD. *, P ≤ 0.05 between MPV and MNV+MPV groups.

Study 3: Effect of MNV infection on MPV infection in C57BL/6 mice. We designed a study in C57BL/6 mice to determine whether the increase in MPV DNA levels produced by coinfection with MNV also occurred in a second inbred mouse strain. C57BL/6 mice were chosen because they are the most commonly used inbred strain of mice. C57BL/6 mice are also much less susceptible than BALB/c mice to MPV infection, presumably due to their bias toward a Th1 rather than a Th2 immune response.7,39,43 MNV RNA was detected in the feces of all mice in the MNV+MPV group from 1 wk before MPV infection through 4 wk after MPV infection; all mice in the MNV+MPV group tested at 2, 3, and 4 wk after infection were MNV seropositive, although MNV was transmitted to only a single mouse exposed 15

Vol 49, No 1 Journal of the American Association for Laboratory Animal Science January 2010

Table 5. MPV DNA (fg/µL) in tissues of infected BALB/c, C57BL/6, and SW mice Strain

Group

Wk after inoculation

MLN

Small intestine

Feces

Spleen

Colon

647a

11c

15f

0d

4g (0–16)

BALB/c

MPV

1

BALB/c

MNV+MPV

BALB/c

(261–1519)

(2–27)

(2–35)

1

16 648a (6035–25602)

1339c (972–1840)

1941f (343–5122)

100d (14–183)

71g (20–185)

MPV

2

119b (30–190)

0.8 (0–2)

0

0e

0

BALB/c

MNV+MPV

2

2184b (1045–4636)

51 (11–130)

1.4 (1–2)

49e (23–86)

5 (3–11)

C57BL/6

MPV

1

0

10 (5–14)

1.4 (1–2)

0

0

C57BL/6

MNV+MPV

1

0

13 (2–45)

32 (4–135)

0

1.4 (0–7)

C57BL/6

MPV

2

0

0

0

0

0

C57BL/6

MNV+MPV

2

0

0

0

0

0

SW

MPV

1

42133 (22202–65845)

3857 (1190–6890)

333 (95–672)

518 (80–856)

84 (54–122)

SW

MNV+MPV

1

46,756 (16404–67646)

3316 (1780–4162)

1054 (35–3183)

964 (326–1228)

327 (20–847)

SW

MPV

2

10454 (3889–16127)

250 (156–318)

1.2 (1–2)

50 (27–90)

10 (6–14)

SW

MNV+MPV

2

10571 (6348–15461)

151 (75–357)

1.2 (1–2)

122 (15–375)

17 (5–30)

Data given as mean (range) of 5 mice per group and sample type. aValue significantly (P = 0.0111) different between groups for the same sample and time point. bValue significantly (P = 0.0325) different between groups for the same sample and time point. cValue significantly (P = 0.0022) different between groups for the same sample and time point. dValue significantly (P = 0.0378) different between groups for the same sample and time point. eValue significantly (P = 0.0091) different between groups for the same sample and time point. fP = 0.0997 between groups for the same sample and time point. gP = 0.1052 between groups for the same sample and time point.

to soiled bedding at 3 wk after infection (Table 3). MPV DNA was detected in pooled feces of mice from all cages at 1 wk after infection for both the MPV and MNV+MPV groups (Table 1). At 2 wk after infection, MPV DNA was detected in pooled feces from 9% of cages of mice in the MPV group and 73% of cages of mice in the MNV+MPV group (Table 1). MPV DNA was not detected in pooled feces of mice from 3 wk after infection for both the MPV and MNV+MPV groups (Table 1). None of the mice in the MPV and MNV+MPV groups were seropositive for MPV at 1 wk after infection, and only 1 mouse in the MNV+MPV group was seropositive at 2 wk after infection (Table 2). Therefore, the propensity for early seroconversion in the MNV+MPV-infected BALB/c mice was not observed in the MNV+MPV-infected C57BL/6 mice. At 4 wk after infection, all 10 cages in both groups contained at least 2 mice that were seropositive for MPV, with a total of 28 of 30 mice seropositive for MPV in each group (Table 2). These data indicate that not all mice inoculated with MPV became infected, and transmission from infected mice to uninfected cage mates was inefficient. Half of the mice exposed to soiled bedding from the MPV group at 1 wk after infection and none of the mice exposed to soiled bedding from the MPV group at 2 and 3 wk after infection were seropositive for MPV (Table 3). Whereas 70% of the mice exposed to soiled bedding from the MNV+MPV group at 1 wk after infection, 10% of the 16

mice exposed to soiled bedding from the MNV+MPV group at 2 and 3 wk after infection were seropositive for MPV (Table 3). As with the BALB/c mice, fecal shedding was prolonged at a low level in dually infected C57BL/6 mice, but there was no significant difference in transmission of MPV by soiled bedding. MNV RNA and antibodies were not detected in any of the mice in MPV group. Feces, MLN, small intestines, spleens, and colons from 5 mice in each group were analyzed at 1 and 2 wk after infection to determine whether MPV DNA levels differed between the mice in the MPV and MNV+MPV groups. The amount of MPV DNA detected in C57BL/6 mice ranged from 0 to 135 fg/µL at 1 wk after infection, and MPV DNA was not detected in any of the tissues at 2 wk after infection from either group (Table 5). At 2 and 3 wk after MNV infection, MNV RNA was detected in all feces, all MLN, all small intestines, and 3 colons of mice in the MNV+MPV group. This study confirmed that C57BL/6 mice are difficult to infect with MPV and that MPV levels are substantially lower in C57BL/6 mice as compared with BALB/c mice. These data also indicate that the increased levels of MPV DNA observed in BALB/c mice coinfected with MNV and MPV as compared with mice infected with MPV only did not occur in C57BL/6 mice.

MNV and MPV coinfection

Study 4: Effect of MNV infection on MPV infection in SW mice. In previous studies, outbred mice sustained the most robust infection with MPV, shedding virus for more than 1 mo.32 We selected SW mice for this study to determine whether the high levels of MPV DNA produced during MPV infection of outbred mice could be increased further by underlying infection of the mice with MNV. At 2 wk after MNV infection, MNV was detected in only 4 of 5 mice, with detectable MNV RNA present in 2 feces, 2 MLN, 3 colons, and 2 small intestines of mice in the MNV+MPV groups. At 3 wk after MNV infection, MNV RNA was detected in all feces, all 5 MLN, 3 colons, and 1 small intestine in the MNV+MPV group. Only 2 mice tested at 2 wk after infection and all of the mice tested at 3 wk after infection in the MNV+MPV group were MNV seropositive. These data suggest that not all mice inoculated with MNV became infected initially, but all mice became infected by their cage mates by 3 wk after infection. MNV RNA and antibodies were not detected in any of the mice in MPV group, confirming that cross-contamination did not occur. No significant differences were observed in MPV DNA levels in feces, MLN, small intestines, colons or spleens between the MPV and MNV+MPV groups (Table 5). At 1 and 2 wk after infection, MPV DNA levels in all tissues from SW mice infected with MPV alone were higher (P < 0.05) than those in BALB/c or C57BL/6 mice infected with MPV alone (Table 5). Tissue-specific patterns of MPV DNA in SW mice were similar to those in BALB/c mice, in that MLN had the highest amount of MPV DNA for both the MPV and MNV+MPV groups and colons had the least. To determine whether MLN contained infectious virus or only viral genomes in the process of being degraded, MLN homogenates from 9 SW and 7 BALB/c mice that had MPV DNA levels ranging from 190 to 67646 fg/µL were inoculated into SW mice to correlate MPV DNA levels with levels of infectious virus. All 16 MLN homogenates contained infectious virus, because all SW mice had detectable MPV DNA in their feces at 1 wk after infection and were seropositive for MPV at 3 wk after infection. Of the 8 MLN homogenates from mice in the MNV+MPV group, only 1 contained infectious MNV as measured by fecal RT-PCR at 1 and 3 wk after infection and seroconversion at 3 wk after infection.

Discussion

We hypothesized that an underlying infection with MNV could alter the time to seroconversion, duration of shedding, the amount of virus shed, or the tissue distribution of MPV, all of which could affect the ability to diagnose MPV infections and confound epidemiologic interpretations. Coinfection with MNV significantly decreased the time to seroconversion to MPV, from 2 to 1 wk, in BALB/c mice. A change to time of seroconversion was not detected in C57BL/6 mice. All MPV-infected and -coinfected C57BL/6 mice were seronegative at 2 wk after infection. Mice were not retested until the end of the study at 4 wk after infection, when 93% of both groups of mice were seropositive; perhaps a difference might have been detected had the mice been tested at 3 wk after infection. A change to time of seroconversion was not detected in SW mice as all MPV-infected and -coinfected SW mice were seropositive at 1 wk after infection. Time to MPV seroconversion has been reported to range from 1 wk after infection to more than 4 wk after infection, depending on mouse age and viral strain.5,7,27 Our data indicate that time to seroconversion also is influenced by mouse genotype and, in BALB/c mice, by MNV infection. No difference in fecal shedding of MPV was observed at 1 wk after infection, because feces from almost all cages tested by PCR

were positive for MPV DNA in both the mice infected with MPV only and those coinfected with MNV. But coinfection with MNV increased the efficiency of fecal PCR-based detection of MPV at 2 wk after infection in both BALB/c and C57BL/6 mice (from 0% to 9% to 73% to 100%; P < 0.0075) and allowed for detection of MPV at 3 to 5 wk after infection in BALB/c mice. Although MPV DNA levels in feces varied widely, a trend toward higher MPV DNA levels in feces was observed in BALB/c mice coinfected with MNV at 1 wk after infection. Therefore, MNV coinfection has the potential to increase the probability of detecting an MPV infection directly by serology or PCR analysis in individual mice or cages of mice during acute infection. Transmission of MPV from infected mice through contact or soiled bedding is often inefficient. However, detection of infectious virus by soiled bedding sentinels has been shown to have a similar sensitivity to that of detection by contact sentinels as long as a sufficient quantity of bedding is used.4,47 Soiled bedding sentinels are a standard quality-assurance method to detect infectious viruses and bacteria and were used in the present studies as the means to measure shedding of infectious virus. The presence of contact sentinels in a cage can confound results in that feces of the index mice might contain ‘pass-through’ MPV that is not a result of the initial infection but rather due to ingestion of feces from the recently infected contact sentinels. To optimize transmission by soiled bedding, sentinels were exposed to a whole cage’s worth of soiled bedding. Parvoviruses are considered to be highly stable in the environment, with rat virus stable for 3 to 5 wk,55 but the amount of infectious virus in soiled bedding decreases over time (4 to 8 wk) to levels below the threshold required to induce productive infection in mice.4,5 Sentinels in the current studies were exposed to soiled bedding that was at most 1 wk old, and therefore a substantial decrease in infectivity of MPV in the bedding should not have occurred. Many MPV infections result in levels of shedding of infectious virus that are close to or just below the threshold necessary for transmission of infection to another mouse.45,48 We postulated that because MNV coinfection increased the amount of MPV DNA being shed in feces at 1 wk after infection, it might also increase transmission if the amount of MPV that accumulated in the bedding between 1 and 2 wk after infection was greater than the threshold needed to initiate infection. Our studies indicated that the increase in MPV DNA levels detected in individual fecal pellets from coinfected BALB/c mice did not translate into a significant increase (47% versus 80%, P = 0.13) in transmission to soiled bedding sentinels. The level of MPV DNA in feces from individual mice in the MPV-only group varied greatly and, given that 3 mice were housed in each cage, it appears that many cages of MPV-infected mice housed 1 or more mice shedding levels of virus sufficient to initiate infection in the sentinel. Once the threshold of virus needed to initiate an infection is reached in the cage, the presence of additional mice shedding levels of MPV substantially above the threshold needed for infection (as occurred in the coinfected BALB/c mice) did not increase the probability of transmission. Pooled feces from all cages of coinfected BALB/c mice were positive for MPV at 2 wk after infection, but very low levels of MPV DNA were detected in single feces from individual mice, and transmission occurred in only 13% of sentinels placed on soiled bedding at 3 wk after infection (accumulation of waste occurred between 2 and 3 wk after infection). Differences in transmission to soiled bedding sentinels between the mice infected with MPV only and those coinfected with MPV and MNV might have become evident had sentinels been exposed to less soiled bedding or if the soiled bedding was mixed with bedding from cages housing uninfected 17

Vol 49, No 1 Journal of the American Association for Laboratory Animal Science January 2010

mice. Both of these practices could have lowered the level of virus in the MPV-only cages to below the level of virus needed to initiate infection while the level of virus in the coinfected cages might have remained above the infection threshold. Nevertheless, our data indicate that MNV coinfection is not likely to affect the ability of a soiled-bedding sentinel-based quality assurance program to detect enzootic MPV infections. Transmission studies were not done in SW mice, but given that MPV DNA levels the feces in SW mice infected with MPV only or coinfected with MNV and MPV were similar to those in coinfected BALB/c mice, we expect that all cages housing infected SW mice would have had sufficient virus within the soiled bedding to transmit infection to sentinel mice at 1 wk after infection, most would have had sufficient virus in soiled bedding to transmit infection to sentinel mice at 2 wk after infection, and a few would have had sufficient virus in soiled bedding to transmit infection to sentinel mice at 3 wk after infection. Coinfection with MNV and MPV resulted in significantly higher levels of MPV DNA in MLN, small intestine, and spleen at both 1 and 2 wk after infection in BALB/c mice as compared with mice infected with MPV only. Increases in MPV DNA levels in colons and feces were observed in the coinfected BALB/c mice, but due to the high variability in MPV DNA levels measured among samples, the increases were not statistically significant. Increases in MPV DNA levels were not observed in coinfected C57BL/6 and SW mice. Similar to fecal PCR results, tissue MPV DNA levels were greatest in SW mice, intermediate in BALB/c mice, and lowest in C57BL/6 mice infected with MPV only. To our knowledge, this study is the first to demonstrate MPV-infected outbred mice have significantly higher levels of MPV DNA in feces and tissues than inbred strains of mice (Table 5, Figure 2). The mechanism that causes this mouse strain-related difference in MPV DNA levels is unclear. In BALB/c and SW mice, MPV levels were greater in the small intestine than in the colon, in agreement with previous in situ hybridization data.22 The distribution of MPV in lymphoid tissues differed among the 3 strains of mice. In SW and BALB/c mice, MLN had significantly higher levels of MPV DNA than other tissues, indicating efficient dissemination of MPV to local lymph nodes and confirming that MLN are a good choice of tissue to use for PCR-based detection of MPV infections in BALB/c and SW mice. Coinfected SW and BALB/c mice and SW mice infected with MPV only also had systemic dissemination to the spleen, but MPV DNA levels were less than those detected in the MLN and small intestine. The only change in tissue distribution that was observed in coinfected BALB/ mice was in the spleen. Apparently the higher MPV DNA levels in the MLN of coinfected BALB/c mice, as compared with BALB/c mice infected with MPV only, allowed for dissemination of MPV to the spleen. In contrast, dissemination to MLN and spleen was not detected in coinfected or MPV-infected C57BL/6 mice (inoculated at 4 wk of age, tested at 1 and 2 wk after infection). In a previous study, MPV DNA was detected 4 wk after infection in the MLN of most C57BL/6 mice inoculated at 12 wk of age with MPV.7 This difference in the spread of MPV to MLN is probably the result of the inocula used: the previous study used 100,000 tissueculture ID50 of MPV1b, whereas we used 300 SW mouse ID50 of MPV1d. The ID50 for C57BL/6 mice is 10- to 100-fold more than that for BALB/c mice; our C57BL/6 mice were infected with the equivalent of 30 C57BL/6 ID50 or less, and spread to MLN is likely to be inefficient in mice inoculated with this low viral dose.7,43 Our results indicate that caution should be used when considering PCR analysis of the MLN as a diagnostic 18

Figure 2. Mean concentration (fg/µL) of MPV DNA in mesenteric lymph nodes from BALB/c, C57BL/6, and SW mice (n = 5 each strain) at 1 and 2 wk after MPV inoculation. Blue bars represent mice inoculated with MPV only, and red bars indicate mice inoculated with MNV and MPV. Error bars indicate ± 1 SD. *, P ≤ 0.05 between MPV and MNV+MPV groups.

method in C57BL/6 mice during natural infection, where mice frequently are exposed to a low infectious dose. In general, molecular methods (in situ hybridization and PCR analysis) are considerably more sensitive than mouse infectivity assays for detecting MPV,4,5,32,48 as we confirmed in the present study. Because MLN had the highest levels of MPV, we compared PCR detection of MPV DNA in MLN (representing both infectious and noninfectious forms of MPV) with infection of SW mice inoculated with MLN homogenates. All MLN containing at least 190 fg/µL MPV DNA produced a productive infection. A past study showed MPV DNA was detected in MLN from SW mice at 6 to 18 wk after infection, but infectious virus was present only at 4 and 6 wk after infection.48 Although some of the MPV DNA detected in MLN is probably present in a noninfectious form in immune cells involved in viral clearance, viral replication and the production of infectious virus is occurring in the MLN at 1 and 2 wk after infection because all of the MLN tested contained infectious MPV. Had the samples been tested, MLN with lower levels of MPV DNA might also have contained infectious virus. There are 2 types of MNV strains which differ in their virulence and duration of infection. The prototype high-virulence strain of MNV (MNV1) causes an acute infection in most strains of mice. Immunocompetent mice develop mild intestinal inflammation and diarrhea, whereas infection in Stat1−/− and IFNαβγR−/− mice is lethal.24,30,41 The interaction of the immune system with MNV1 has been studied in great detail. A rapid and strong innate immune response controls MNV1-induced disease in most mouse strains.24 Persistent subclinical infection with MNV1 occurs in RAG−/− mice, indicating a role for the adaptive immune response in viral clearance. MNV1 targets dendritic cells and macrophages and causes an increase in macrophage and B cells in the spleen at 3 d after inoculation.41,54 AntiMNV antibodies effectively control early MNV replication in the ileum and MLN and clear MNV1 infection from the MLN.11 CD4+ T cells are important for efficient MNV1 clearance in the distal ileum, especially at 3 to 5 d after infection, whereas their role is small in viral clearance from MLN. CD8+ T cells are important for efficient MNV clearance in the distal ileum and MLN, especially at 6 and 7 d after infection.10 We used a low-virulence MNV strain (MNVG) that causes chronic infection in both immunocompetent and immunocompromised strains of mice. With low-virulence MNV strains, most infections are subclinical, but 2- to 3-mo-old immunodeficient mice naturally infected with MNV developed weight loss, ruf-

MNV and MPV coinfection

fled fur, and hunched backs.52 Whether low-virulence MNV strains elicit immune responses that are similar to those induced by MNV1 is unclear. Although a strong antibody response that is capable of neutralizing all murine norovirus strains is elicited, it is insufficient to resolve persistent infection. Low-virulence MNV strains persist in the small intestine, cecum, MLN, and spleen for 6 mo or more.19,26,50,52 In our studies, all BALB/c and C57BL/6 mice inoculated with MNVG were seropositive at 2 to 5 wk after infection, yet MNV persisted in several tissues. MNV RNA was detected in all feces collected from MNV-inoculated BALB/c and C57BL/6 mice tested at 1 to 7 wk after infection and in 93% of MLN, 87% of small intestines, 77% of colons, and 50% of spleens from BALB/c and C57BL/6 mice tested at 2 to 3 wk after infection. In the present study, MNV infection was not as easily established in SW mice, in that only 2 of 5 mice were seropositive and 4 of 5 mice had detectable MNV RNA in 1 or more tissues (MLN, small intestines, feces, and colons) at 2 wk after infection. By 3 wk after infection, cohousing of the 5 mice resulted in infection of the remaining mouse, in that all feces and MLN were positive for MNV RNA and all mice were MNV seropositive. A significant difference in tissue distribution of MNV was not detected between mouse strains in these studies. It has been reported that 129 mice have higher MNV1 titers in the distal intestine compared with the proximal intestine, whereas the reverse is observed in C57BL/6 mice.10 In our study, MNV RNA was measured only in a homogenate containing a portion of the duodenum, ileum, and jejunum and not in individual regions of the intestine. Increased MNV1 titers in the spleen of 129 mice as compared with C57BL/6 mice have been reported.30 We detected MNV RNA only rarely in the spleens of MNVG-infected mice of all mouse strains, in agreement with previous results that detected MNV RNA in only 11% of spleens from NOD-scid and NOD/ShiLtJ mice chronically infected with MNV5 and MNV6.26 Although the mechanism of how MNV infection affected MPV infection differently in BALB/c and C57BL/6 mice was not determined, one possibility could be linked to the type of T cell response generated. C57BL/6 mice generate a Th1-dominant response characterized by strong cytolytic responses (secretion of IL2, TNFα, and interferon γ), whereas BALB/c mice generate a Th2-dominant immune response characterized by stronger antibody responses (secretion of IL4 and IL10 and antibody production).39 In C57BL/6 mice infected with MPV only, the strong interferon γ response seems to control the initial MPV replication within the intestine effectively, thereby limiting spread of the virus to the MLN and spleen. This concept was confirmed in that MPV DNA levels were significantly lower in the small intestine, colon, and feces of C57BL/6 mice at 1 and 2 wk after infection than in BALB/c mice and MPV DNA was not detected in spleen or MLN of C57BL/6 mice. The rapid control of MPV in the intestine also resulted in less-efficient transmission of MPV from C57BL/6 mice to sentinels. A second factor that could contribute to the difference in susceptibility to MPV infection between BALB/c and C57BL/6 is a difference in the resting populations of immune cells in these mouse strains. Compared with 8-wk-old female C57BL/6J mice, 8-wk-old female BALB/cByJ mice have more B and NK cells in their spleens.21 T cells traditionally have been considered the primary target for MPV,22,35 but by using flow cytometry, we noted that B cells and NK cells in MLN consistently are infected by MPV.31 Coinfected C57BL/6 mice were expected to control MPV infection better than C57BL/6 mice infected with MPV only, because MNV infection would have generated a strong interferon γ response before inoculation with MPV, but because MPV levels

in MPV-only infected C57BL/6 mice were so low, the additional control generated by MNV infection did not result in a dramatic decrease in MPV DNA levels in the coinfected C57BL/6 mice. Similar to our studies in which infection of C57BL/6 mice with MNV had no effect on MPV infection, others have shown that persistent infection of C57BL/6 mice with MNV-CR6 has no effect on T and antibody cell responses, morbidity, or mortality induced by infection with influenza virus or vaccinia virus.18 The mechanism by which MNV increases MPV replication in BALB/c mice is unclear. Although a few MNV strains have been isolated from BALB/c β2M-deficient mice,50 all published MNV pathogenesis studies have been done in outbred mice or mice of the C57BL/6 or 129 lineages. We originally hypothesized that because MNV1 infection induces mild inflammation,41 MNV infection might increase the population of MPV-susceptible cell in the intestine or lymphoid tissues. In the current study, we did not observe an increase in T-, B- or NK-cell populations in the MLN of BALB/c mice infected with MNVG. Although MNVG infection did not appear to cause an increase in susceptible cells in MLN, further studies are needed to determine whether the population of MPV-susceptible cells in the intestine is increased by MNV infection. Even though MNV antigen has been detected in the lamina propria and epithelia cells of the small intestine of mice infected with low-virulence MNV strains, the cell types that are infected in the intestines are unknown.52 A strong innate immune response is necessary for prevention of MNV-induced pathology, and BALB/c and C57BL/6 mice differ in the type of innate response generated by interferon γ and bacterial infections.24,38,53 The pathways activated by interferon γ in macrophages from BALB/c mice are conducive to cell replication and healing (low antimicrobial activity), whereas those activated by interferon γ in macrophages from C57BL/6 mice are conducive to cell destruction (high antimicrobial activity).38 In addition, in response to bacterial infections, BALB/c mice elicit a greater influx of macrophages into the infection site than do C57BL/6 mice.53 Further experiments are needed to determine whether the type and magnitude of innate immune response generated within the intestine of BALB/c mice in response to MNV infection results in an increase in MPV-susceptible cells (lymphocytes and macrophages). No substantial differences were observed between the BALB/c mice infected with MNV 1 and 2 wk prior to MPV infection, indicating that the changes produced by MNV infection last for at least 2 wk. Although RT-PCR readily detected MNV RNA, MNV transmission through soiled bedding was low. This result was somewhat unexpected, given the high environmental stability of MNV and the low infectious dose of noroviruses.2,3,8,34,42,49 However, our finding of inefficient transmission of MNV through soiled bedding is in agreement with several previous studies.17,29,34 One study showed that 1 wk of exposure to soiled bedding from MNV4-infected mice collected at 1 wk after infection resulted in transmission to sentinels in only 1 of 5 cages, and after weekly soiled bedding exposures for 10 to 12 wk, mice in only 4 of 5 cages were seropositive.34 Yet MNV RNA was detected through 12 wk after infection in the feces from all of the infected mice that contributed waste to the soiled bedding.34 MNV may not be stable in bedding, and therefore transmission to soiled bedding sentinels may be less effective than expected.17 Although fecal MNV RNA was detectable after 2 wk at room temperature,34 the infectious titer of MNV1 deposited onto gauze and then stored at 18 or 30 °C decreased more than 1000fold in a single day whereas MNV RNA titers did not drop.28 In addition, the infectious titer of MNV dried on stainless steel and then stored at room temperature decreased approximately 19

Vol 49, No 1 Journal of the American Association for Laboratory Animal Science January 2010

1000-fold in a single day.8 In addition, the age of the sentinels may play a role in the efficiency of transmission of MNV1 to sentinels: when 12- and 24-wk-old SW sentinels were cohoused on soiled bedding, all of the younger sentinels but only 54% of the of the older sentinels seroconverted to MNV.25 Only 1 of 8 MLN homogenates from coinfected mice, which contained detectable MNV RNA, produced a productive infection when inoculated orally into mice, indicating again that MNV RT-PCR analysis is much more sensitive than are measures of infectivity such as soiled bedding transmission and inoculation with tissue homogenates. These data indicate that the infectious dose needed to initiate an infection with MNV may be much higher than that for human noroviruses.30,49 The dose of MNV needed to cause seroconversion is reported to be 1 to 100 PFU, while the dose of MNV needed to initiate an infection in MLN is 250 PFU and to initiate an intestinal infection is 800 PFU indicating a differential susceptibility of tissues within the mouse.30 Therefore soiled-bedding sentinels should be used with caution to monitor for MNV, because this method may miss some cages containing MNV-infected mice. In addition, the detection of MNV RNA in feces by RT-PCR analysis does not always indicate that mice can transmit virus to other mice efficiently. The current studies have added to our understanding of 2 factors, mouse strain and underlying viral infection, that influence the efficiency of transmission of MPV to sentinels and to differences between the sensitivity of infectivity and molecular-based methods for detecting MPV. Because dually infected BALB/c mice seroconvert to MPV more rapidly and have substantially higher MPV DNA levels in several tissues and feces, thereby increasing the efficiency of fecal PCR-based detection at 2 to 5 wk after infection, MNV coinfection can improve the reliability of detection of MPV infection in BALB/c mice by serologic and PCR-based methods. However, MNV infection does not appear to increase the transmission efficiency of MPV, and therefore coinfection does not appear to create an increased risk for spread of MPV within a colony. Because levels of MPV in tissues of coinfected SW mice were not altered, the reliability of detection of MPV infection by using contact or soiled bedding sentinels in colonies infected with both MNV and MPV should not be altered. Our findings show that MPV DNA levels are greatest in SW mice, intermediate in BALB/c mice, and lowest in C57BL/6 mice. The results also indicate that in C57BL/6 mice, MPV infections may remain localized, such that PCR analysis of MLN or spleen may yield negative results in mice that are positive for MPV DNA in the feces.

Acknowledgments

This research was funded by a grant from the American College of Laboratory Animal Medicine and was supported by an educational donation provided by Amgen. We thank Roy Capper, Sandra Maher, Savas Sidiropoulas, and Jean Wilson for technical assistance.

References

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