key: cord-0004951-2oecs4kx authors: Garlinghouse, L. E.; Smith, A. L.; Holford, T. title: The biological relationship of mouse hepatitis virus (MHV) strains and interferon:In vitro induction and sensitivities date: 1984 journal: Arch Virol DOI: 10.1007/bf01309365 sha: 93714d0ddce6e1f2bafcde542139c708c1968f29 doc_id: 4951 cord_uid: 2oecs4kx Five prototype strains of mouse hepatitis virus (MHV) -1,-3, -S,- A59 and -JHM were analyzed for their ability to induce interferon (IFN) in seven cell lines of rodent origin. Induction of IFN by all of the prototype MHV strains was infrequent and unpredictable, while IFN was produced consistently by five cell lines treated with known inducers. Priming and/or aging of cells did not enhance IFN induction by the MHV strains except in the case of MHV-A59 which consistently induced moderate levels of IFN on L-cells which were both primed and aged. Kinetic studies of MHV-A59-induced IFN on primed and aged L-cells demonstrated that detectable levels of IFN were not produced until 24 hours post-inoculation (p.i.). Peak levels were attained at 30 hours p.i. with no additional IFN produced through 48 hours p.i. MHV-induced IFN was similar in composition and properties to Newcastle disease virus-induced IFN. The sensitivities of the five MHV strains to eight concentrations of preformed L-cell IFN were also assessed. All strains except MHV-S fit a linear model with MHV-3, MHV-A59 and MHV-JHM having similar slopes. At most concentrations MHV-3 was less sensitive than MHV-1, -A59 or -JHM to IFN. The response curve for MHV-S was non-linear. This strain was more sensitive to the antiviral effects of the pre-formed IFN except at the highest concentrations of IFN used. The mouse hepatitis viruses (MHV) are a group of enveloped, singlestranded RNA viruses whose genome is nonsegmented and infectious (23) . The prototype strains include mouse hepatitis virus (MHV)-I, MHV-2, MHV-3, MHV-S, MHV-A59 and MHV-JHM. Different tropisms and disease producing potentials have been ascribed to the prototype strains of MHV (18, 30) . These differences are dependent not only on virus strain, but also on host genotype (ii, i4), age (16, 28) and the route of inoculation (6) . Resistance or susceptibility has, in general, been attributed to the ability or inability of the virus to replicate in macrophages (2, 15) for the non-neurotropic strains or in neurons for the neurotropic strains (5, 11) . Despite a significant amount of recent information on MHV replicative strategies and natural history (18, 23) , little has been published on the interferon (IFN) inducing characteristics of the MHV strains or on the relative sensitivities of the prototype strains to pre-formed IFN. IFN induction is a property common to most I~NA viruses (25) , although the ease with which this can be demonstrated may depend on several factors, including host cell type and age, prior treatment with IFN (priming) and inducing virus strain (3) . The in "vitro characteristics of IFN induction by MttV strains were of interest to us because this was an aspect of MHV biology that had not been thoroughly investigated. The three reports on the subject indicate that MHV-JHM does not induce IFN production in cultured neuronal cells (7, 26, 27) . It was also of interest to determine if there was a differential sensitivity to IFN among the prototype strains of MHV. Mouse hepatitis virus strains and Sendai virus were obtained from the American Type Culture Collection (ATTC, Roekville, MD), Newcastle disease virus (Hickman strain), Vesicular Stomatitis virus (Indiana strain) and Sindbis virus (EgAr339) were obtained from the Yale Arbovirus Research Unit (New tqaven, CT). Mouse encephalomyelitis virus (GDVII) was isolated by the Section of Comparative Medicine (Yale University, New Haven, CT) from the brain of a naturally infected mouse. Propagation, quantification and storage were by standard methods (22) . ATTC. The history-and maintenance of mouse neuroblastoma cells (clone N 18) and rat glioma cells (clone C 6) have been described previously (24) . ViTira cells are a rat "fibreblast-like" line and were obtained from Dr. Samuel Baron, University of Texas-Medical Branch, Galvaston, Texas. Primary mouse embryo (PME) cells were cultured by the method of I-IsI~G (8) . All cells were propagated and maintained by standard methods (22) . All I F N assays were performed b y t h e c y t o p a t h i c effect (CPE) r e d u c t i o n assay of RUBE:~S~:EIN et al. (19) in 96 well cluster dishes c o n t a i n i n g confluent cell monolayers. Vesicular s t o m a t i t i s virus (VSV) was t h e i n d i c a t o r of a n t i v i r a l a~tivity. P u t a t i v e I F N p r e p a r a t i o n s derived from m o u s e cell lines were assayed on mouse L-cells, those from r a t cell lines on W i r a cells a n d those from h a m s t e r cells on B H K -2 1 cells. All samples were assayed in triplicate a n d c o m p a r e d to our l a b o r a t o r y s t a n d a r d . T h e l a b o r a t o r y s t a n d a r d (LS) used in these studies was p r e p a r e d b y i n o c u l a t i n g confluent m o n o l a y e r s of L-cells in 75 cm~ culture vessels w i t h l09 m e d i a n egg infectious doses of Newcastle disease virus (NDV). T h e culture m e d i u m was r e m o v e d after 24 hours, acidified to p H 2 w i t h 1N HC1, held a t 4 ° C for seven days, n e u t r a l i z e d w i t h 1N a n d 0.2N N a O H a n d clarified b y c e n t r i f u g a t i o n a t 80,000 × g for 60 m i n u t e s in a B e c k m a n S W 2 8 rotor. Residual infectivity was n o t detected in t h e LS which was r e p e a t e d l y c o m p a r e d to a n N A I A D , W H O i n t e r n a t i o n a l reference s t a n d a r d (G002-904-511). One I U / m l of our LS was equivalent to 1 I U / m l of t h e reference standm-d. T h e specific a c t i v i t y of our LS was 8 × 108 I U / m g of protein. T h e r a t I F N s t a n d a r d (RLS) was p r e p a r e d in t h e same m a n n e r as t h e LS except t h a t ~Vira cells were used for production. I n t h e absence of a reference s t a n d a r d , t h e R L S was assigned a n a r b i t r a r y a c t i v i t y in u n i t s w h i c h was equal to t h e reciprocal of t h e highest dilution a t which a n t i v i r a l a c t i v i t y was detected. tivity to 1 percent trypsin at 37 ° C for one hour, partial sensitivity to heat (56 ° C for 30 minutes) and stability at p H 2.0. Further characterization included measurement of activity before and after 36 hours of dialysis (500 volumes phosphate buffered saline p H 7.2), antivirM activity against more than one virus (the G D V I I strain of mouse encephalomyelitis virus), decreased activity on cells of heterologous species, and retention of activity after ultra-centrifugation (LS only). The LS and IFN-containing test samples were treated with mouse anti-~ I F N globulin to determine the proportion of ~ I F N . The anti-~ I F N globulin was obtained from LEE BioMolecular (San Diego, CA). The globulin was reported to be 99 percent pure anti-~ I F N and to have an I F N neutralizing activity of 1 × t04 IU/ml. I n our studies, 1 × i03 IU/ml or less of I F N were treated with 1000 IU/ml of anti-~ I F N globulin at 37 ° C for two hours. Rat I F N was not antigenicatly characterized. L-cell monolayers in 96-well cluster dishes were pre-treated at 37°C with serial 0.5 log10 dilutions of L-cell I F N . After 18 hours, the I F N was decanted and the cells were rinsed with Hanks' balanced salt solution (I-IBSS). Serial ten-fold dilutions of the MHV strain or VSV were then added to each pre-treated set of L-cells (0. t ml per well). After a 90 minute absorption period, the virus was removed and replaced with 0.I ml of minimal essential medium (Earle's salts) containing 2 percent fetal bovine serum. The cluster dishes were incubated at 37 ° C in a 5 percent CO2 atmosphere for 24 hours, fixed and stained with a 5 percent glutaraldehyde --1 percent crystal violet solution and evaluated macroscopieally (VSV) or microscopically (I~IHV) for virusinduced CPE. Virus titers at each I F N dilution were calculated by the method of REED and MVE~NC}t (17) . Results are expressed as log10 reduction in virus titer compared to the virus titer on diluent pre-treated L-cells. Each determination was based on three experiments with three replicates per dilution. Straight line models were generated for each virus by linear regression using the least squares method. The goodness of fit for the linear model was determined by analysis of variance. The individual slopes for the viruses where a linear model was appropriate were compared using an F test for parallelism. If the slopes did not differ significantly, intercepts were compared by analysis of eovariance. The calculations were performed using PROC GL~I in the statistical package, SAS (20) . To determine if the cells used were responsive only t o / q D V , i.e., essentially poor producers, or whether the M H V strains are poor inducers, the cells were pre-treated with two other k n o w n viral inducers of IF/q, the synthetic polynucleotide poly I--C, or D E A E -d e x t r a n plus poly I --C to induce I F N production. All of the cell lines tested except B H K -2 t were capable of producing I F N in response to one or more of the inducers ( Table 1) . Three of six cell lines (L-cells, C 6 and Wira) produced I F N in response to a t least four known inducers. Two cell lines (N18 and B H K ) were poor producers./qCTC 1469 cells were intermediate. SendM virus and N D V were used to induce IF/q at MOI ranging from 100 to 0.0001. Maximum I F N concentrations were induced at MOI of 1 t h r o u g h 100 (12,000--18,000 IU/ml). A n approximate 204old decrease in I F N concentration was observed at an MOI of 0.1 (600--1000 IU/ml), a 100-fold decrease at an MOI of 0.01 ( t 0 0 --5 0 0 IU/ml) and no I F N production was observed at MOI of 0.001 or 0.0001 (~4 IU/mt). [6] 81,000) [6] 270,000) [6] I~esults are given as geometric mean IFN concentration in IU per ml (range) [number of positive samples used to calculate mean]; --is less than 4 IU per ml; three replicate determinations per assay N D V consistently induced I F N production regardless of monolayer age or pre-treatment status. Cells primed or aged produced substantially more I F N t h a n those t h a t were u n t r e a t e d prior to induction. Combined priming and aging further increased the a m o u n t of I F N produced in response to NDV. This effect appeared to be more t h a n additive since there was a fourfold increase in I F N production in primed and aged cells compared to production b y cells which were either primed or aged. The characteristics and properties of I F N induced b y the MHV strains and other agents fit the basic criteria for I F N . MHV-induced I F N did not differ significantly in the proportion of ~ I F N compared to I F N produced b y L-cells induced b y NDV. NCTC cells induced b y N D V appeared to produce about 10-fold more ~ I F N t h a n did L-cells. Differences of 10--20 percent are difficult to evaluate using our methods due to small variations in the I F N assay. In all cases however, ~ ] F N accounted for 70 percent or more of the I F N produced in these studies. Mouse encephalomyelitis virus seemed to be somewhat more sensitive to MHV-induced I F N t h a n was VSV although this, too, m a y reflect assay varia$ion. The kinetics of M t t V I F N induction were studied using MHV-A59 as the inducing agent on primed and aged L-cells. A t t e m p t s at using other M H V prototypes as the inducing agents were unsuccessful due to the inconsistent nature of I F N induction b y these strains of MHV. The induction scheme was t h a t described in Methods with the modification t h a t putative IFN samples were removed and assayed after time intervals of 0--48 hours after virus inoculation. IFN was not produced during the first 18 hours after virus inoculation. Measurable IFN was evident at 24 hours post-inoculation (42 IU/ml:~16 IU/ml). In a second study, these results were confirmed and peak IFN concentrations were achieved by 30 hours postinoculation (120 IU/ml). Additional IFN production could not be detected between 30 and 48 hours. .0001]; however, there were no significant differences between MIlV-A59 and MHV-JHM. MHV-3 had a Y-intercept that was significantly lower than those for each of the other viruses, while that for VSV was significantly higher, implying that MHV-3 was less sensitive and VSV more sensitive to the antiviral effects of IFN. MHV-S was more sensitive to IFN than were the other strains at most of the IFN concentrations used. MHV-JHM and MHV-A59 were equivalent in IFN sensitivity and were intermediate to MHV-S and MHV-3. There have been few reports regarding IFN induction by MHV strains. MHV-JHM did not induce IFN in cultured neuronal cells (27) or in the spleens of mice injected intraperitoneally or intracranially (29) . However, MHV-3 given to mice intraperitoneally did induce detectable IFN in the spleen (21, 29) . Because of the difficulty associated with interpreting results of studies in which different host systems or routes of inoculation were used, we have done a systematic study of the in vitro capacities of several MHV strains to induce 1FN production in several host types. Our results strongly suggest that MHV strains are poor IFN inducers in otherwise untreated cells, regardless of the host cell used. At least one of the known chemical or viral IFN inducers used in these studies resulted in IFN production, except with BHK cells which are known to be poor IFN producers (10) . Manipulation of L cells by priming or aging had no consistent effect on IFN induction by the MHV strains, but increased the IFN yield in response to NDV by 5-and 8-fold, respectively. Combined priming and aging resulted in consistent low-level IFN induction only by MHV-A 59. The same treatment increased NDV-associated IFN production by almost 30-fold over control values. There was no indication during these studies that IFN production by any of the cell lines in response to MHV strains was directly correlated with the relative abilities of the cells to support virus replication. The kinetics of IFN induction were studied using MHV-A59-infected primed and aged L cells. IFN production began and peaked later than reported (4, 13) and found in these studies (data not shown) for NDVinduced IFN. One potential explanation for this finding is a requirement for a threshold concentration of virus, achieved by virus replication and below which IFN induction does not occur. However, since MHV-3, -S, -A59 and -JHM all achieved titers of ~5.5 logio TCIDs0 per ml within 24 hours after infection of L cells, this explanation may be simplistic. [['he sensitivities of the MHV strains to pre-formed I F N were highly variable. MHV-S was most sensitive and displayed a non-linear response pattern. In contrast, the sensitivity patterns of the other four MHV strains fit a linear model. The lines generated for MHV-A59 and MttV-JHM were indistinguishable. The line generated for MHV-3 had a slope identical to that for MHV-A59 and MHV-JHM, but higher I F N concentrations were required to inhibit replication of MHV-3. MHV-1 was more sensitive to the anti-viral effects of high concentrations of IFN than were the other MHV strains. Mouse hepatitis virus strains are common contaminants of laboratory mice. After inoculation by a natural route, MHV infection results in a spectrum of clinical manifestations ranging from no visible disease to death. The outcome of infection is dependent on several factors which include host age and genotype and strain of infecting virus (i2). In an effort to extend the findings reported here and to learn more about the basic biology of MtIV, current studies are directed toward evaluating the relative role of I F N in determining the outcome of infection. 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Adherent cellmediated protection Natural killer ceil activity during mouse hepatitis virus infection: Response in the absence of interferon Neuropathological effects of persistent infection of mice by mouse hepatitis virus This research was supported by NIt-I grant R R 00393 from the Division of t~eseai-eh Resources. L. E. Garlinghouse, jr., was supported by a National t~escarch Service Award (5-T 32-NF 07036) from the NationM Institute of Neurological and Communicative Diseases and Stroke, NIH. The authors thank Ms. Janice A. Halpin for preparation of the manuscript.