key: cord-0896448-jx695xo8
authors: Smith, A. L.; de Souza, M. S.; Finzi, D.; Barthold, S. W.
title: Responses of mice to murine coronavirus immunization
date: 1992
journal: Arch Virol
DOI: 10.1007/bf01309627
sha: d20199d18fcefb351de46ebc1bb614920f31b654
doc_id: 896448
cord_uid: jx695xo8

Oral and/or intranasal inoculation of susceptible mouse genotypes with the JHM strain of mouse hepatitis virus (MHV-JHM) consistently results in T cell dysfunction as reflected by in vitro proliferative responses to mitogens or allogeneic cells. One approach to examining the mechanism responsible for the observed functional T cell suppression is to determine whether virus replication is required for its induction. To this end, mice were inoculated oronasally with MHV-JHM that was inactivated with short-wave ultraviolet light, betapropiolactone or psoralen. Mice were also inoculated with live MHV-JHM after recovery from homotypic or heterotypic MHV infection. Spleen cells from BALB mice inoculated oronasally with inactivated MHV-JHM yielded extremely variable in vitro proliferative responses after concanavalin A stimulation. MHV-susceptible mice exposed oronasally or intraperitoneally to virus inactivated by any of the minimum effective treatments failed to seroconvert. Immunization with psoralen-treated virus intraperitoneally in Freund's complete adjuvant or oronasally failed to protect from live virus challenge, but survivors had elevated virus-specific serum IgG antibody titers compared to mock-immunized controls at two weeks post-challenge. Spleen cells from mice that were challenged after recovery from homotypic live virus infection did not exhibit the profound in vitro T cell suppression normally observed during the acute stage of primary infection. In contrast, MHV-JHM challenge of mice vaccinated with heterotypic live MHV-S resulted in significantly depressed in vitro T cell function. The combined data suggest that either virus replication or exposure to more concentrated antigen may be required for induction of the dramatic T cell dysfunction that occurs as a consequence of MHV-JHM infection as well as for a detectable MHV-specific humoral response.

Mouse hepatitis virus (MHV) is a highly contagious coronavirus infecting laboratory mice worldwide at high prevalence [21, 22, 25, 38] , usually without clinical signs. The impact of infection on research involving mice is enormous, with several adverse effects on biological responses of mice having been demonstrated [3-5, 9, 12-14, 33, 34, 36] . Infection has the potential to render experimental results in a number of disciplines invalid. A major adverse effect of MHV infection is immunomodutation [9, 13, t4, 33, 36] .

Prior studies have shown that susceptible strains of mice, such as the BALB/ c, sustain transiently suppressed T cell function as a consequence of infection by a natural route with the JHM strain of MHV [13, 14, 33, 36] . An important issue pertaining to the mechanism responsible for this immunosuppression is whether virus replication is required for its induction. Studies with other viruses have yielded varying results. Measles virus-associated inhibition of mitogendriven proliferation and immunoglobulin production is eliminated when UVor heat-inactivated virus is used [24, 26] . The immunosuppressive variant of the parvovirus, minute virus of mice, did not inhibit the appearance of cytotoxic T cells in mixed lymphocyte cultures if used in the form of UV-inactivated virus or purified viral capsids [15] . Live Newcastle disease virus, which does not achieve a full replication cycle in murine cells, induced suppressed concanavalin A (Con A)-driven and allogeneic responses in mice, whereas UV-inactivated virus did not [11] . In contrast, inhibition of the poxvirus DNA polymerase with phosphonoacetic acid did not affect the ability of malignant rabbit fibroma virus to suppress lymphocyte proliferation or initiation of antibody production [37] . Proliferation of bovine peripheral blood mononuclear cells stimulated by either Con A or interleukin (IL) 2 was inhibited by bovine herpesvirus 1, although less than 0.1% of the cells were productively infected [8] . In a model system using human peripheral blood lymphocytes and vesicular stomatitis virus, Sendal virus, Friend leukemia virus, herpes virus types 1 and 2, or a battery of avian retroviruses known to be incapable of entering and infecting human lymphocytes, both live and UV-inactivated viruses abrogated proliferative responses both to mitogens (Con A and phytohemagglutinin) and allogeneic cells [27, 39, 40] . Thus, the ability of replication-defective virus to alter immune function is unpredictable.

The major goal of this study was to determine whether the T cell dysfunction observed after MHV inoculation of a susceptible genotype by a natural route is due directly to active virus infection or indirectly to interaction with MHV antigens. The goal was addressed by characterizing the responses of mice that were exposed to virus inactivated by any of several physical or chemical methods and of mice that were challenged after recovery from live virus immunization.

MHV-JHM and MHV-S (American Type Culture Collection, Rockville, MD) were used in the fOnXl of infected infant mouse brain homogenates. Both virus stocks had titers of 10 7.3 intracerebral infant mouse LDs0 per ml. Mice inoculated oronasally received 10 5.6 icLDs0-equivalents, and mice inoculated intraperitoneally received 106.3 icLDs0-equivalents of virus.

Female BALB/cByJ (BALB) mice (The Jackson Laboratory, Bar Harbor, ME) were four weeks old at the time of initial virus exposure. Cr:ORL Sencar (hereafter referred to as Sencar) dams with litters and weanling female Sencar and NIH Swiss mice 'were obtained from the Animal Genetics and Production Branch, NCI (Frederick, MD). Randomly selected mice were free of antibody to common murine viruses on arrival. All mice were housed in micro-isolator cages (Lab Products, Maywood, NJ) and were given food and water ad libitum. Manipulations and husbandry were performed in a class II biological safety cabinet. An open-cage sentinel mouse seromonitoring program was in place during the course of the reported studies. Seroconversion to none of 11 common murine viruses or Mycoplasma pulmonis was detected, except among selected MHV-inoculated mice as detailed in the Results.

Beta-propiolactone (3-hydroxypropionic acid lactone; Sigma Chemical Co., St. Louis, MO) was used as a 1% stock solution. Psoralen (4'-aminomethyl-4,5'8-trimethylpsoralen hydrochloride; HRI Associates, Inc., Emeryville, CA) was prepared as a 1 mg/ml stock solution in 50% ethyl alcohol, stored at room temperature in a light-tight container and used at a final concentration of 10 gg/ml. Concanavalin A (Con A; Sigma Chemical Co.) was used at a final concentration of 2 gg/ml. Rat monoclonal antibodies to mouse IL2, designated $4B6 [28I, and IL4, designated l lBll [29] , were originally obtained from T. Mosmann ($4B6) and J. Ohara and W. E. Paul (11 B 11) and were kindly provided by Dr. Kim Bottomly (Section of Immunobiology and Howard Hughes Medical Institute, Yale University).

Short-wave ultraviolet (UV) irradiation results in the formation of pyrimidine dimers [17] and was accomplished by exposing a thin layer of stock virus in a glass petri dish within an ice bath to a GTE 30W germicidal lamp at a distance of six inches for 10min.

Beta-propiolactone (BPL) is an alkylating agent that reacts with guanidine residues of RNA and DNA and is considered moderately effective in maintaining antigenic structure [23] . BPL inactivation was accomplished by adding one volume of the 1% stock solution to nine volumes of stock virus. The mixture was incubated for 1 h in a 37 °C water bath, aliquoted and stored at -7 0 °C.

Psoralens are naturally occurring aromatic compounds found in many fruits and vegetables and are chemically inert until exposed to long-wave UV light resulting in direct photoreaction with nucleic acids [17] . The linear psoralen molecule intercalates in a double helical region and forms covalent adducts with pyrimidine bases, usually thymidine in DNA and uridine in RNA [10] . The bonds formed firmly link together and prevent the separation of double helical nucleic acid strands without disruption of protein structure [17] . For psoralen inactivation, 4'-aminomethyl-4,5',8-trimethylpsoralen hydrochloride was added at a final concentration of 10 Ixg/ml to a shallow layer of stock virus in a petri dish that was manually swirled during the irradiation periods and transferred to an ice bath during sampling periods. Ultraviolet irradiation from below was accomplished with a long-wave UV transilluminator (model TL33, UVP, Inc., San Gabriel, CA) emitting at a wavelength of 365 nm with an average intensity of 7000 ~tW/cm 2. Aliquots were removed after 0 (prior to UV light exposure), 1, 2, 3, 4, 5, 7 and 10 min and stored at -7 0 °C. 

Each virus preparation treated as described above was assayed by intracerebral inoculation of two-day-old Sencar mice that were observed daily for mortality. This infant mouse bioassay currently serves as the most sensitive test for MHV infectivity [13] . In addition, livers from BALB mice inoculated oronasally with either UV-inactivated or BPL-treated MHV and used for functional T cell assays were processed for histology. They were fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned at 5~tm and stained with hematoxylin and eosin. The concentration of infectious virus in the liver is very high during the acute phase of active infection [1, 32] , and there is a 100% correlation between liver histopathology and virus recovery from this major target organ (Smith and Barthold, unpubl, obs.). Inactivation of psoralen-treated MHV-JHM was further confirmed by the inability of primers specific for a segment of the M gene to amplify in polymerase chain reaction [19] .

Antigenicity of virus that was confirmed to be inactivated was tested by intraperitoneal and/or oronasal inoculation of weanling Sencar or BALB mice that were exsanguinated 21 or 28 days later. Sera were screened for MHV antibody by indirect immunofluorescence (IFA) using sera diluted 1 : 10 and bivalent MHV-JHM/MHV-S antigen and by an IgG enzyme immunoassay (EIA) using sera diluted 1 : 50 and MHV-JHM antigen as previously described [31, 35] . Antibody titers were determined by EIA.

Con A-stimulated proliferation was measured as previously described [14, 33, 36] . Briefly, spleen cells from individual BALB mice exposed oronasally to inactivated or live virus four, five or seven days earlier were suspended in 5 ml of RPMI 1640 medium (Gibco BRL, Gaithersburg, MD) containing 5% fetal bovine serum (Gibco BRL), 0.05 mM 2-mercaptoethanol (Sigma Chemical Co.) and 2 mM L-glutamine (Gibco BRL) [RPMI 95 -5] . Cell suspensions were diluted as needed in filtered normal saline containing 0.2% trypan blue and 1.7% ammonium chloride to determine viable nucleated cell counts. The cells were then washed, adjusted to 2 x l 0 6 viable cells per ml in RPMI 95-5, and 100~tl of cell suspension were added to well of 96-well cluster dishes. Each set of four or six replicate wells received 100 ~tl of RPMI 95-5 or 100 ~tl of Con A. The cells were incubated in a 37 °C, 5% CO2 humidified atmosphere and were labeled with 1 ~tCi per well of methyl [3H]thymidine (Dupont NEN, Boston, MA; 80 Ci per retool) 24 h prior to collection on glass fiber filters for scintillation counting. Cultures were harvested either after 72 h or at 24 h intervals from days 2 through 6 [14] . Results are expressed as [mean cpm for Con A-treated cells-mean cpm for RPMI 95-5-treated cells].

Supernates collected from spleen cells cultured with Con A or RPMI 95-5 for 24h were assyed on CTLL-2 cells [16, 41] as described earlier [14, 33, 36] . Supernates from Con Astimulated cultures that induced proliferation of CTLL-2 cells were serially diluted and retested with $4B6 (anti-IL2) and 11B11 (anti-IL4) monoclonal antibodies [14, 36] .

Virus that was exposed to psoralen and long-wave UV light for 3 rain was used to vaccinate weanling mice. BALB mice were twice vaccinated oronasally at two week intervals. NIH Swiss mice were inoculated intraperitoneally with killed virus emulsified in Freund's com-plete adjuvant and boosted oronasally two weeks later. Control mice were similarly inoculated with sterile culture medium or uninfected infant mouse brain homogenate emulsifted in Freund's complete adjuvant. Two weeks after the booster dose, a drop of blood was collected from the tip of the tail for serology, and the mice were challenged intranasally with 1053icLDs0 of live MHV-JHM. The mice were observed for mortality and exsanguinated at 14 days post-challenge.

To determine the effect of prior vaccination with live virus on in vitro T cell function after subsequent challenge, BALB mice were inoculated oronasally with sterile culture medium, MHV-JHM or MHV-S. After four weeks, an interval in which mice fully recover from oronasat MHV infection, small blood samples were collected to verify seroconversion, and all of the mice were challenged oronasally with MHV-JHM. Con A-induced proliferation and IL2 production by spleen cells collected four days after challenge were measured as described above.

Based on the infant mouse bioassay, MHV-JHM was completely inactivated by exposure to a germicidal UV lamp for ten minutes. Weanling BALB mice exposed oronasally to this material and used for functional T cell assays had histologically normal livers at four and seven days post-inoculation, further confirming inactivation of the inoculum. These mice also-failed to seroconvert. Mice similarly exposed to live virus seroconverted by seven days post-inoculation and had moderate hepatitis on day 4 that increased in severity by day 7. Con A-stimulated proliferative responses of spleen cells from five of six mice assayed at four days after exposure to UV-inactivated MHV were significantly reduced compared to control responses (Table 1) . Spleen cells from one mouse were hyper-responsive. At day 7, cells from four of six mice that received UVinactivated M H V proliferated poorly compared to controls and cells from two mice were hyper-responsive. Cells from actively infected mice were still functionally suppressed at this interval, with proliferative values of 9 to 15% of control (Table 1) . IL2 production by cultured spleen cells was generally correlated with proliferative capacity ( Table 1 ). The data shown for UV-inactivated virus represent one of three replicate experiments, all of which yielded similarly highly variable responses for cells from mice given killed MHV-JHM.

BALB mice given BPL-inactivated MHV-JHM oronasally 7 or 14 days prior to necropsy had histologically normal livers, confirming virus inactivation. These mice failed to seroconvert by day 21. Mice similarly exposed to live virus had severe hepatitis on day seven and were seropositive on day 14. Con Astimulated proliferative responses of spleen cells from mice given BPL-inactivated M H V seven days earlier were consistently elevated compared to control responses, whereas responses of cells from actively infected mice ranged from 16 to 47% of control proliferation (Table 1) . Mouse-to-mouse variability in 

All Sencar pups (8 per group) exposed intracerebrally to virus treated with psoralen for two minutes or longer survived (Fig. 1) . Virus collected at each interval exceeding two minutes was inoculated intraperitoneally into groups of four weanling Sencar mice. All survived for three weeks, and none developed M H V antibody. Five B A L B mice were exposed oronasally to virus treated for three minutes with psoralen and long-wave UV light, and spleen cell proliferative responses were measured five days later (Fig. 2) . Proliferation of ceils cultured for two to six days was essentially identical to that of cells from three naive control mice. In contrast, proliferation of spleen cells derived from four actively infected mice was significantly reduced with peak proliferation oc, curring later than that of control cells, as reported earlier [14] . The immunogenicity of psoralen-inactivated MHV-JHM was tested in three vaccination-challenge experiments ( Table 2 ). Data pooled from two experiments in which BALB mice were twice exposed oronasally to psoralen-treated virus yielded identical mortality rates and average survival times (7.9 days) for vaccinated and control mice. Both experiments yielded modestly elevated post- (Table 2 ). NIH Swiss mice exposed intraperitoneally to psoralen-treated virus emulsified in Freund's complete adjuvant and boosted oronasally also were not protected from challenge, but had significantly elevated post-challenge virus-specific IgG antibody titers ( Table 2) . None of 50 mice tested on the day of live virus challenge were MHV seropositive.

Spleen cells from four mock-vaccinated BALB mice that were challenged with MHV-JHM proliferated at levels that were 1 to 5% of control spleen cell proliferation (Table 3) . These cells also produced negligible concentrations of IL2. In contrast, cells from four mice immunized with homologous (MHV-JHM) virus yielded proliferative responses that were 39 to 67% of control responses (Table 3) . Spleen cells from six of 12 mice vaccinated with heterologous (MHV-S) virus proliferated at a level that was less than 10% of control proliferation ( Table 3) . Proliferation of cells from the remaining six mice ranged from 11 to 42% of control proliferation. IL2 production was generally correlated with the magnitude of the proliferative response (Table 3) , and serial dilutions yielded the expected dose-response curve (data not shown).

IFA testing of sera from MHV-JHM-and MHV-S-immunized mice revealed that all of the mice were MHV antibody-positive at the time of MHV-JHM challenge, whereas sera from controls or mice that were mock-vaccinated did not contain detectable antibody. At four days after MHV-JHM challenge, sera from control mice and mock-vaccinated mice did not contain MHV-JHMreactive EIA antibody (Table 3) . Mice vaccinated with MHV-JHM had high titered serum IgG antibody (geometric mean EIA titer = 1 : 22,400). Sera from 

The was seen by seven days after inoculation, with cells from two of six mice being hyper-responsive at that interval. Cells from four of four mice given BPLinactivated virus and tested seven days later were hyper-responsive. Responses of cells from mice given UV-treated MHV-JHM were quite variable in three separate experiments, despite the fact that confirmation of virus inactivation was obtained for each inoculated mouse. Spleen cells from mice given psoralen-treated virus yielded Con A-induced proliferative responses that were essentially identical to those of cells from naive control mice. Mouse-to-mouse variation was minimal among animals that received psoralen-treated MHV-JHM. Of the inactivation methods used, psoralen treatment is the least harsh and therefore least likely to cause conformational changes in structural proteins [ 17] . The lack of a detectable virus-specific humoral response even after two exposures to psoralen-treated virus, including one parenteral injection in Freund's complete adjuvant, renders interpretation and conclusions difficult. It could be argued, however, that the immune systems of mice twice immunized with psoralen-inactivated virus were stimulated, since these mice had consistently, albeit marginally in the case of oronasal inoculation, elevated antibody titers compared to mock-vaccinated mice after live virus challenge.

Mice given MHV-JHM inactivated by any of the methods described clearly did not receive enough antigen to induce seroconversion. It may also be argued that oronasal exposure to killed virus may be less likely to stimulate an immune response than parenteral inoculation, with or without adjuvant. However, our research program has historically been devoted to studies of MHV as a natural pathogen of laboratory mice. We have, therefore, always used natural routes of exposure. The studies in which immunosuppression by live MHV-JHM were documented [13, 14, 33, 36] were performed with mice infected orally, intranasally or by the combined routes. It therefore seemed reasonable, if not essential, to expose mice to killed virus by the same natural routes in attempts to determine whether such virus could induce immune dysfunction.

The accumulated data from studies using three inactivation methods also provide support for the notion that virus replication or exposure to more antigen is required for induction of a detectable antibody response to and protection from challenge with MHV-JHM. This contrasts with reports for other viruses. For instance, arthropod-borne viruses and ecologically related viruses that cause high mortality among peripherally inoculated mice are frequently BPL-inactivated in order to prepare immune sera [7] . Reports on the immunogenicity of psoralen-treated virus are rare; however, psoralen-inactivated crude Pichinde virus, without adjuvant, induced seroconversion among intramuscularly injected guinea pigs within two weeks [17] . Guinea pigs given two doses of virus that was 25-fold more dilute, again without adjuvant, also seroconverted [17] . In addition to the methods reported here, we also evaluated the ability of formalininactivated MHV-JHM to induce an antibody response, using a modification of a recently published technique [18] . In that study, inoculation of mice with Responses to mouse hepatitis virus immunization 49 lactic dehydrogenase elevating virus treated with 0.06% formalin at 37 °C for as long as 5 h yielded hybridomas that produced virus neutralizing antibody [18] . Treatment of MHV-JHM with 0.05% formalin at 37 °C for 25 rain (minimum effective treatment time) resulted in complete loss of infectivity. However, Sencar mice inoculated oronasally or intraperitoneally with that material had not seroconverted when tested 21 days later. Formalin seemed a reasonable treatment choice, since higher concentrations do not destroy MHV antigens [6, 35] . The possibility that an antibody response could be stimulated by parenteral inoculation of mice with inactivated MHV-JHM in a potent adjuvant, perhaps with several boosters, cannot be excluded. However, as stated above, the major goal of the reported studies was to characterize T cell function of mice given killed virus by a natural route.

In view of the high prevalence of MHV infection and the rapidity with which the virus is transmitted throughout a colony, control by means of elimination of the agent, as has been the case with ectromelia (mousepox) virus [30] , seems an unlikely possibility. One strategy is development of a vaccine that might provide protection without the functional immune deficits documented after live virus administration. Since protection from challenge was not afforded by psoralen-inactivated virus, immune function of mice challenged after live virus vaccination was evaluated. The design of the live virus vaccination-challenge experiments reported here was identical to that described in an earlier report [2] , in which complete resistance to homotypic challenge was demonstrated. In the earlier study, BALB mice that were challenged 30 days after an immunizing infection with MHV-JHM had histologically normal livers and noses four days after challenge, and virus was not detected in livers at that interval. This was strong evidence that homotypic live virus vaccination protected against infection upon challenge. In contrast, mice immunized with biologically similar, but antigenically distinct, MHV-S were fully susceptible to MHV-JHM challenge, based on the same criteria [2] . In the present study, spleen cells from mice challenged with MHV-JHM after recovery from homotypic infection yielded Con A-induced proliferative responses that were suppressed, but not to the extent observed for cells from mice undergoing primary infection (Mock/ JHM in Table 3 ). Function of cells from mice immunized with live MHV-S and challenged with MHV-JHM was suppressed in a manner similar to that of cells from mice sustaining primary infection. These data affirm by different methodology the earlier report of highly strain-specific challenge immunity to murine coronaviruses [2] , with both studies strongly suggesting that a polyvalent vaccine would be required to protect mice against infection with the many MHV strains that circulate in animal colonies. The current experiments suggest a caveat, however; even a polyvalent vaccine might not eliminate the deleterious effects of MHV on in vitro assays of immune function, since function of cells from mice challenged with homotypic virus was depressed, albeit not to the extent seen after mock vaccination and challenge. Therefore, efficacy testing of any candidate vaccine should include studies to determine whether vaccination (Table 3) was unexpected, since the M H V -J H M EIA has been used to detect MHV-S antibody in sera from mice injected intraperitoneally with infected mouse brain homogenate [35] . The sera from MHV-Simmunized mice did contain M H V antibody based on the IFA test with bivalent antigen. The EIA uses as antigen infected cell monolayers in which about 75% of the cells are fused [35] . Syncytium formation by M H V relies on the presence of the surface E2 or S glycoprotein which is also the protein against which neutralizing and protective antibodies are directed. Mature virus particles released into the culture medium may also be precipitated when formalin is added to inactivate the virus prior to assay. Thus, this modified enzyme immunoassay may, under certain conditions, be more likely to detect strain-specific antibody than assays that use immobilized soluble antigen. Whether route of inoculation plays a role is currently unknown. However, high MHV-JHM-specific antibody titers were positively correlated in this study with relative, but not complete, protection from MHV-JHM-induced immune dysfunction.

Response of genetically susceptible and resistant mice to intranasal inoculation with mouse heptatitis virus JHM

Virus strain specificity of challenge immunity to coronavirus

Western equine encephalitis mimicking herpes simplex encephalitis

Peritoneal macrophage alterations caused by naturally occurring mouse hepatitis virus

Viral hepatitis associated with transplantable mouse leukemia

Mouse hepatitis virus immunofluorescence in formalin-or Bouin's-fixed tissues using trypsin digestion

Lassa fever, a new virus disease of man from West Africa. III. Isolation and characterization of the viurs

Inhibition of T-lymphocyte mitogenic responses and effects on cell functions by bovine herpesvirus 1

Suppression of immune response induction in Peyer's patch lymphoid cells from mice infected with mouse hepatitis virus

Pgoralens as photoactive probes Responses to mouse hepatitis virus immunization 51 of nucleic acid structure and function: organic chemistry, photochemistry, and biochemistry

Inhibition of lymphocyte mitogenesis in mice infected with Newcastle disease virus: viral interference with the interleukin system

Effect of inapparent murine hepatitis virus infections on macrophages and host resistance

Characterization of accessory cell function during acute infection of BALB/cByJ mice with mouse hepatitits virus (MHV), strain JHM

Infection of BALB/cByJ mice with the JHM strain of mouse hepatitis virus alters in vitro splenic T cell proliferation and cytokine production

Inhibition ofT cell-mediated functions by MVM(i), a parvovirus closely related to minute virus of mice

T cell growth factor: parameters of production and a quantitative microassay for activity

Inactivation of viruses for use as vaccines and immunodiagnostic reagents

Formalin inactivation of the lactate dehydrogenaseelevating virus reveals a major neutralizing epitope not recognized during natural infection

Detection of rodent coronaviruses in tissues and cell cultures using polymerase chain reaction

Derivation of a T cell line that is highly responsive to IL-4 and IL-2 (CT.4R) and of an IL-2 hyporesponsive mutant of that line (CT.4S)

Diagnosis of murine infections in relation to test methods employed

Prevalence of viral and mycoplasmal infections in laboratory rodents

Antigenicity of beta-propiotactone-inactivated virus vaccines

Activation of measles virus from silently infected human lymphocytes

Prevalence of natural virus infection in laboratory mice and rats used in Canada

Suppression of T lymphocyte function by measles virus is due to cell cycle arrest in G1

Non-specific inhibition by virus particles of human lymphocyte mitogenesis

Two types ofmurine T helper cell clones. I. Definition according to profile of lymphokine activities

B cell stimulatory factor BSF-I: production of a monoclonal antibody to and molecular characterization of B-cell stimulatory factor-1

Responses to mouse hepatitis virus immunization

Prevention and control of mousepox

An immunofluorescence test for detection of serum antibody to rodent coronaviruses

The role of gamma interferon in infection of susceptible mice with murine coronavirus, MHV-JHM

Altered splenic T cell function of BALB/ cByJ mice infected with mouse hepatitis virus or Sendai virus

Antigenic characterization of Tettnang virus: Complications caused by passage of the virus in mice from a colony enzootically infected with mouse hepatitis virus

Two enzyme immunoassays for the detection of antibody to rodent coronaviruses

In vitro splenic T cell responses of diverse mouse genotypes after oronasal exposure to mouse hepatitis virus, strain JHM

Inhibition of virus replication does not alter malignant rabbit fibroma virus-induced immunosuppression

Serological survey of laboratory rodents for infection with Sendai virus, mouse hepatitis virus, reovirus type 3 and mouse adenovirus

Viral inhibition of lymhocyte mitogenesis. I. Evidence for nonspecificity of the effect

Viral inhibition of lymphocyte proliferative responsiveness in patients suffering from recurrent lesions caused by herpes simplex virus

Biochemical and biological characterization of lymphocyte regulatory molecules. I. Purification of a class of murine lymphokines

This work was supported by NIH grant RR04507. The authors thank Deborah Winograd, Debby Beck and George Hansen for technical assistance and Dr. Kim Bottomly for providing cytokine-specific monoclonal antibodies and CTLL-2 cells.

Received October 21, 1991