Mouse thymic virus-mediated immunosuppression: association with decreased helper T cells and increased suppressor T cells

Mouse thymic virus (MTV) is a herpesvirus which, when administered to newborn mice, induces an extensive but temporary thymic necrosis associated with immunosuppression. In the present study, the T cell subsets in the thymus of MTV infected newborn C57Bl/6 mice were evaluated at 4, 7, 14, 28, 56, and 84 days after infection, using labeled monoclonal anti-CD4 and anti-CD8 antibodies with two-color flow cytometry. At 7 and 14 days, the percentages of CD4+8- and CD4+8+ cells were significantly decreased whereas the percentage of CD4-8+ cell was increased. At days 28 and 56 percentages had returned to normal. These results indicate that the virus has an affinity for CD4+ T cells (helper cells and their precursors). Increased percentage of CD4-8+ T cells (suppressor cells) is also associated with depressed immune functions in MTV infected newborn mice.     

Biology of Mouse Thymic Virus, a Herpesvirus of Mice, and the Antigenic Relationship to Mouse Cytomegalovirus

Mouse thymic virus (TA) is a herpesvirus which produces extensive necrosis of the thymus of newborn mice 7 to 14 days after infection. Infectious virus can be recovered from the thymus for only 10 days after infection, with highest titers occurring between days 5 and 7. In mice 5 days old or less, TA infects thymus cells and produces massive necrosis. TA also infects the salivary glands and persists as a chronic infection. Newborn mice infected with TA have no detectable humoral immune response. Infected adult mice respond, and humoral antibody is detected 7 days after infection. Titers are maintained for months thereafter. Regardless of the age of the mice inoculated with TA, persistent infection was established in the salivary glands, but no evidence for thymus involvement was observed when adults were infected. TA does not cross-react serologically by immunofluorescent, complement fixation, or virus neutralization tests with mouse cytomegalovirus; however, interestingly, the epidemiology of the two herpesviruses are similar. Both mouse cytomegalovirus and TA were isolated from the same animals in populations of laboratory and wild mice. Evidence of infection with mouse cytomegalovirus and TA were most apparent by virus isolations, since humoral antibody responses are rarely observed. All strains of mice tested were susceptible to TA infection. However, in some strains maximum necrosis occurred at 7 days, compared with 10 to 14 days for other strains. The difference in age susceptibility and the target tissue of thymus in newborn mice suggests that TA is a model herpesvirus for studying the effects of viral infections on humoral and cell-mediated immunological functions.     

Rapid diagnosis of human Crimean-Congo hemorrhagic fever and detection of the virus in naturally infected ticks.

An enzyme-linked immunosorbent assay (ELISA) was developed to detect Crimean-Congo hemorrhagic fever (CCHF) virus-specific immunoglobulin M (IgM) in human serum samples. For this test, a heat-inactivated antigen was prepared from the brains of suckling mice infected with CCHF virus. The IgM-capture ELISA proved more sensitive than indirect fluorescence tests for IgM to this virus. A human serum containing high-titer IgM to CCHF virus was used for an antigen-capture ELISA to detect this virus in heat-inactivated suspensions of virus-infected ticks. The antigen-capture ELISA appeared to be as sensitive as virus isolation in suckling mice. The studies described suggest that the IgM-capture ELISA and the antigen-detection ELISA should provide a rapid and sensitive diagnosis of human CCHF virus infection and should be useful in ecologic studies of this virus.     

Experimental parainfluenza virus infection in mice: Growth and spread of a highly pathogenic variant of parainfluenza 3 virus in the mouse brain

Summary We had previously showed that following intracerebral inoculation of newborn mice, the 910 N and M strains of bovine parainfluenza 3 virus induce a non-lethal hydrocephalus and a lethal diesease with marked thymic and splenic atrophy, respectively. Moreover, only the M virus was lethal for 2-week-old mice.In the present study, we demonstrate that the M virus multiplies and spreads in the mouse brain invading the thalamus, hypothalamus and brain stem beyond the ependyma whereas the 910 N virus causes only slight ependymitis. This growth and spread of M virus was blocked by passive immunization 3 days after infection. Mouse embryo brain cell cultures were infected with M and 910 N viruses, about 50 per cent became antigenpositive for M whereas only a small proportion of cells were positive for the 910 N virus. However, the latter did produce higher yields of infectious virus than M.With 3 Figures     

Improved detection and quantification of mouse cytomegalovirus by real-time PCR

During latent cytomegalovirus (CMV) infection, viral presence cannot be detected by plaque assay. Therefore, we assessed the applicability of real-time PCR for temporal determination of virus dissemination in two different mouse strains. Eight-week-old BALB/c and C57BL/6J mice were infected with mouse CMV (MCMV) and sacrificed at 1, 2, 4, 6, 14 and 28 days post infection. Real-time PCR was used to determine MCMV copy number in the heart, bone marrow cells, aorta and blood. In lung, liver, salivary gland and spleen the presence of MCMV was determined both by plaque assay and real-time PCR. In analogy with the plaque assay, the real-time PCR technique revealed higher numbers of MCMV genomic copies in all organs obtained from BALB/c mice when compared with C57BL/6J mice, demonstrating the applicability of the technique. A significant correlation was observed between both assays when a positive test result was seen with both assays. Nonetheless, lower viral infectivity titers were found compared to real-time PCR data. Thus, the real-time PCR technique is more sensitive in detecting the presence of MCMV and is therefore well suited for (dose-response) intervention studies aimed at studying virus eradication.     

Neonatal impaired response to viral superantigen encoded by MMTV(SW) and Mtv-7

MMTV(SW) is an exogenous mouse mammary turnor virus that codesfor a superantigen sharing the same V_ß specificityas Mtv-7 (Mis-1^a). Neonatal mice infected by suckling-infectedmilk show a deletion of the CD4⁺ V_ß6⁺ T cell subsetwithin 8 weeks. In contrast, adult mice infected by injectionof the virus in the footpad have a much faster deletion, whichoccurs within 2 weeks. In the present work, we investigatedpossible mechanisms for the different kinetics of deletion inthe adult and newborn mice. To find out if the route of infectioncould be responsible for this discrepancy, we infected 5-day-oldand adult mice by injection in the footpad. Our results demonstratethat the route of infection is not responsible for the delayedkinetics of reactive T cell deletion since newborn mice injectedwith the virus show similar kinetics to neonates infected bymaternal milk. To exclude differences in viral spreading betweenthe two models, we used a PCR assay to detect proviral DNA.Spreading of the virus was shown to occur at a similar rateor even more rapidly in neonates than in adults. We also comparedthe activation induced by MMTV(SW) or Mis-1^a spleen cells inthe draining lymph node in neonatal and adult mice and showedthat a poor local activation is induced in neonates comparedwith adults. In vitro, neonatal T cell reactivity to anti-V_ß6antibody was also impaired. Thus, the delay in clonal deletioncould be linked to impaired expression, presentation and/orresponse to the viral superantigen. Our results suggest thatthe initial response to MMTV(SW) could be of importance forthe kinetics of reactive T cell deletion.     

Susceptibility of mouse mammary glands to murine gammaherpesvirus 72 (MHV-72) infection: evidence of MHV-72 transmission via breast milk.

Murine gammaherpesvirus 72 (MHV-72) is a virus of wild rodents and serves as a convenient small animal model to understand the pathogenesis of Epstein-Barr virus (EBV) and human herpesvirus 8 (HHV8) infection. In laboratory mice MHV-72 causes an acute infection of lung epithelial cells and establishes the latency in B lymphocytes. In this study, we investigated athymic nude and immunocompetent mice for distribution of virus in organs after infection with MHV-72. Ten days following subcutaneous dorsal injection of nude mice, virus replicated in lungs, lymphoid organs, salivary glands and also in mammary glands. The virus titre decreased by day 21 post-infection in former tissues, but increased in mammary glands. Presence of virus DNA sequences was detected in the lymphoid and non-lymphoid tissues until the death of the animals (about 1 month post-infection). Infection of immunocompetent mice with MHV-72 induced replication of virus up to 42 days post-infection in mammary glands reaching the highest level of infectious virus at day 8 post-infection. These data show that there is latent infection in mice never detected before. Moreover, virus DNA was detected using nested PCR (by amplification of a portion of gp150 gene sequence) in the mammary glands and the milk of mouse mothers infected with MHV-72 2 days before delivery. We demonstrated the presence of virus DNA also in the milk removed from the stomach of non-infected newborn mice, which were nourished by infected mothers (wet-nurses) for 1 or 2 days. The failure to detect the virus DNA in newborn mice lungs confirmed that they did not become infected from wet-nurses by the intranasal route. This suggests that MHV may be naturally transmitted to newborn mice via breast milk.     

Enzyme-linked immunosorbent assay to detect antigens of Dirofilaria immitis (Spirunida: Filariidae) larvae in Aedes albopictus and Culex tritaeniorhynchus (Diptera: Culicidae)

An enzyme-linked immunosorbent assay (ELISA) method was developed for detecting the larval antigens of Dirofilaria immitis (Leidy) in Aedes albopictus (Skuse) and Culex tritaeniorhynchus Giles to replace the conventional dissection method. The assay was sensitive enough to detect 21.3 ng/ml (2.13 ng per microplate well) or more of the soluble antigen obtained from adult worms and an antigen amount corresponding to at least one larva at any developing stage. No inhibitory effect of host tissues was observed on the ELISA reaction even when the homogenate of 20 mosquitoes was included in the 1-ml ELISA diluent. Moreover, third-stage larval antigen could be detected readily when mixed with up to 100 mosquito heads. The ELISA method has been effective in field surveys of mosquito populations with low filarial infection rates.     

The pathogenesis of infection in mouse brain by Mount Elgon bat virus

Mount Elgon bat virus given intracerebrally readily killed mice up to 12 days of age (1 to 4 p.f.u/LD50). Virus in doses of 10 to 10(6) p.f.u. killed 40 to 80% of weanling mice of both sexes and up to 20% of adult females and 40 to 80% of adult males. Clearance of brain infectivity in the resistant mice coincided with the appearance of circulating virus neutralizing antibody which occurred earliest in the adult female mice. Immunosuppression of adult mice with cyclophosphamide resulted in the death of all the virus-inoculated mice of both sexes. Virus in these mice reached tenfold higher titres and their brains contained 30- to 60-fold more interferon than the brains of infected mice not given cyclophosphamide. Interferon was apparently without effect on the outcome of infection in the brains of resistant mice and its synthesis reflected the extent of virus growth.     

Evaluation of enzyme-linked immunosorbent assay and reversed passive hemagglutination for detection of Crimean-Congo hemorrhagic fever virus antigen.

Enzyme-linked immunosorbent assay (ELISA) and a reversed passive hemagglutination (RPHA) test were evaluated for rapid detection of Crimean-Congo hemorrhagic fever (CCHF) virus antigens. Both RPHA and ELISA detected CCHF antigen in the brains of infant mice 2 to 3 days after infection, several days before the animals sickened and died. Antigen was also detected after 1 to 2 days in infected cell culture extracts and after 2 to 4 days in culture supernatant fluids. Both tests detected CCHF antigen at threshold values of approximately 2.5 log10 tissue culture infective doses per ml and were more sensitive than complement fixation, immunodiffusion, or immunofluorescence. In a comparative study on specimens from CCHF patients, virus was isolated from 38 of 49 sera and 23 of 28 patients. Antigen was detected in 20 of 49 sera (15 of 28 patients) by RPHA and in 29 of 49 sera (18 of 28 patients) by ELISA. Antigenemia was detected more frequently in fatal cases (9 of 11) than in nonfatal cases (9 of 17). Although the antigen detection assays offered a more rapid approach than infectivity assays for diagnosing CCHF, the latter test was more sensitive. The results suggest that RPHA and ELISA may be of use in rapid diagnosis of CCHF infection, particularly in severe cases, in which the danger of nosocomial spread is greatest.     

Intrauterine transmission of Sendai virus in inbred mouse strains.

A study of Sendai virus infection in adult mice (2 to 3 months of age) showed that the inbred strains were more susceptible to infection than randomly bred Swiss white mice and that virus could be isolated from inbred strains for as long as 21 days postinfection. For this reason, these mouse strains (C57B1/6J [black] and C57Br [brown]) were selected for the study of intrauterine transmission of virus. The major effect of infection was a decreased weight of both embryos at 16 days of gestation and newborn mice. Virus was isolated from 17 to 20% of the embryos and at least 20 to 30% of the newborns from intravenously infected mothers. Fluorescent-antibody studies showed that the virus was widely distributed in the tissues of both embryos and newborns, including the central nervous system.     

Identification of antigenic peptides derived from B-cell epitopes of nucleocapsid protein of mouse hepatitis virus for serological diagnosis

Mouse hepatitis virus (MHV) infection is found commonly in laboratory mice and this virus has been known to cause various diseases such as subclinical infection, enteritis, hepatitis, and encephalitis. Serological tests are used commonly to diagnose MHV infection. Complete MHV virions have been used primarily as antigens for serological diagnosis to date. To develop an antigen that is more specific, more sensitive, and easier to prepare for serological diagnosis, the antigenic sites in the MHV-nucleocapsid (N) protein were screened in this study. Sixteen antigenic linear sequences in the N protein were found using antisera obtained from mice infected naturally with MHV and a peptide array containing overlapping 10-mer peptides covering the entire N protein. From these antigenic sequences, two synthesized peptides, ILKKTTWADQTERGL and RFDSTLPGFETIMKVL, which were consistent with positions 24-38 and 357-372 of the N protein respectively, were used as antigens in ELISA. Evaluation of ELISA with these peptides revealed that both peptides were specific to anti-MHV antisera. Furthermore, ELISA performed using these peptides was more sensitive than commercial ELISA used for a screening sera from mice infected accidentally to MHV maintained in cages, suggesting that these peptides are useful for serological diagnosis of MHV infection.     

Selective vulnerability of neural cells and age-related susceptibility to OC43 virus in mice

Suckling CD 1 mice infected intracerebrally or extraneurally with OC43 virus developed a lethal neurotropic infection with high titres of virus in the brain. Examination of infected brain by routine H & E staining revealed no necrosis even in extensively infected tissue. Resistance to infection developed with increasing age, and by 20 days of age mice were completely insusceptible to i.c. inoculation. Virus replication was also demonstrable by FA staining, in spinal cord, dorsal root ganglia and retina. All other tissues were insusceptible and in particular, macrophages from both susceptible and resistant mice were found to be resistant to infection both in vivo and in vitro. Immunosuppression rendered 15 day old mice more susceptible to infection but adult mice remained insusceptible. The transfer of immune or non immune spleen cells from resistant mice did not confer resistance to newborn mice. Treatment of resistant mice with anti interferon globulin (AIG) did not render them more susceptible. These results indicate that the immune response is partially responsible for the development of resistance to OC43 infection but that it is only partially protective and other factors must also be required. The basis for the unique susceptibility of neural tissues in suckling mice is being investigated.     

Evaluation of an enzyme-linked immunosorbent assay for the detection of ectromelia (mousepox) antibody.

Ectromelia virus, an orthopoxvirus that can cause extensive morbidity and mortality (mousepox) in colonized mice, has been epizootically responsible for serious disruption of biomedical research since 1930. The lack of a sensitive and specific serological assay for infection with this virus became apparent during outbreaks of mousepox at the National Institutes of Health, Bethesda, Md., and other biomedical research institutions in 1979 and 1980. To fill this need, we evaluated an enzyme-linked immunosorbent assay. Sucrose gradient-purified ectromelia and vaccinia viruses were compared as antigens in tests on approximately 1,000 mouse sera from experimentally infected mice and conventional colonies of uninfected mice. A statistical analysis based on the frequency distribution of the absorbance values for 152 mouse sera (free of ectromelia antibody) gave 0.22 as a value to differentiate ectromelia-positive sera from ectromelia-negative sera. When enzyme-linked immunosorbent assay results were compared with those obtained by an indirect immunofluorescence assay, the former was found to be at least 10-fold more sensitive. With the procedures employed, including the use of purified vaccinia virions as antigen, the enzyme-linked immunosorbent assay proved to be highly sensitive and specific for detecting antibodies to ectromelia and vaccinia viruses. False-positive results were not encountered. False-negative results were observed in 3% of 108 separate tests of a known positive serum. Although data indicated that ectromelia antibody can be differentiated from vaccinia antibody with homologous and heterologous antigen, this procedure probably cannot be generally used because of unavailability of ectromelia antigens.     

Pathogenesis and antibody response to a cytomegalovirus infection in newborn rats

The present study described the kinetics of Rat cytomegalovirus (RCMV) infection in newborn rats by monitoring infectious virus and viral antigens in various organs, viral DNA in the blood (DNAemia) and antibody response. These parameters were evaluated quantitatively using double-antibody sandwich ELISA (DAS-ELISA), real-time PCR, indirect ELISA and virus infectivity assay. For the first time DAS-ELISA was used for detection of RCMV antigen directly from organ samples. The relationships between the presence of viral antigens in the infected organs and antibody levels were established by the Spearman's rank test. It was found that the virus was present in the blood, spleen, liver, lungs, and kidneys earlier than in the salivary glands. Furthermore, the early immunity of the newborn rats led to a delayed seroconversion. We suggested that the prolonged presence of the virus in salivary glands could augment the antibody response that conversely might be responsible for a reduction of viremia. This study expanded our understanding of RCMV pathogenesis leading to improved therapeutic and preventive treatment regimens particularly for the neonatal Human cytomegalovirus (HCMV) infections. Additionally, the detection procedures developed in this study such as DAS-ELISA and real-time PCR could serve as alternative techniques for rapid screening of large number of samples.     

The role of cellular susceptibility in the declining severity of respiratory influenza of ferrets with age

A comparison was made, both in vivo and in organ culture, between newborn (1-day-old) and suckling (15-day-old) ferrets of lower respiratory tract tissue infected with a virulent strain (clone 7a) of influenza virus. Newborn ferrets were killed by influenza virus following intranasal inoculation but suckling ferrets were almost as resistant as adult ferrets. In newborn ferrets there was a rapid, severe and progressive infection of lung tissue with infection of alveolar cells as well as those of bronchial and bronchiolar epithelium (assessed by monitoring virus infectivity and by fluorescent antibody staining). In suckling ferrets, as previously shown for adult animals, the lung infection was less severe, less persistent and confined to the epithelium of bronchi with only a small bronchiolar involvement and even less alveolar cell infection. These differences observed in vivo were repeated in organ cultures obtained from various areas of the lung. i.e. alveolar and airway epithelial cells of newborn ferrets exhibited a greater susceptibility than those of older ferrets. Thus, it appears that one factor determining the greater susceptibility of the lower respiratory tract of newborn ferrets is a greater inherent susceptibility of alveolar and airway epithelial cells to infection with influenza virus. Other factors may also be involved and have yet to be investigated.     

Infectivity of human coronavirus strain 229E.

The replication of human coronavirus strain 229E was observed by using indirect immunofluorescence in infected monolayers of MRC continuous cells. By 8 h after infection, bright cytoplasmic fluorescence was detected in cells infected with human coronavirus 229E. Discrete foci of infection were observed from 8 to 16 h after infection in cells infected with high dilutions of human coronavirus 229E; each fluorescent focus corresponded to a single virus infection. A fluorescent focus assay is described, using indirect immunofluorescence, which is more sensitive than the established techniques of tube titration and plaque assay. Particle/infectivity ratios for unpurified and purified virus preparations revealed a considerable drop in infectivity on purification.     

The role of cellular susceptibility in the declining severity of respiratory influenza of ferrets with age.

A comparison was made, both in vivo and in organ culture, between newborn (1-day-old) and suckling (15-day-old) ferrets of lower respiratory tract tissue infected with a virulent strain (clone 7a) of influenza virus. Newborn ferrets were killed by influenza virus following intranasal inoculation but suckling ferrets were almost as resistant as adult ferrets. In newborn ferrets there was a rapid, severe and progressive infection of lung tissue with infection of alveolar cells as well as those of bronchial and bronchiolar epithelium (assessed by monitoring virus infectivity and by fluorescent antibody staining). In suckling ferrets, as previously shown for adult animals, the lung infection was less severe, less persistent and confined to the epithelium of bronchi with only a small bronchiolar involvement and even less alveolar cell infection. These differences observed in vivo were repeated in organ cultures obtained from various areas of the lung. i.e. alveolar and airway epithelial cells of newborn ferrets exhibited a greater susceptibility than those of older ferrets. Thus, it appears that one factor determining the greater susceptibility of the lower respiratory tract of newborn ferrets is a greater inherent susceptibility of alveolar and airway epithelial cells to infection with influenza virus. Other factors may also be involved and have yet to be investigated.     

Coxsackie A7 virus infections of rodents

Summary Coxsackie A7 virus injected by various routes caused fatal paralytic disease in newborn mice, adult merions and adult cotton rats. In all these species, and also in adult mice and lemmings with silent infections, brown fat was infectious during the first few days after inoculation. Virus titres of brown fat were higher than those of other organs and gross and microscopic evidence of necrosis was seen in cotton rats infected with virus strains isolated in Scotland but not with the Russian ABIV strain. Brown fat is susceptible to many neurotropic viruses, and it may act to increase the severity of the infection or provide a model of the infectious process suitable for study.     

Electron microscopy of mouse thymic virus

Summary Thymuses of mice infected with mouse thymic virus (MTV), a herpesvirus, were examined with the electron microscope. Damage was manifested by distortions of cells, nuclei, and other organelles, with cellular aggregation apparent at advanced stages of necrosis. Infected cells also contained MTV particles and filaments. Negative staining showed that naked MTV capsids were icosahedral in shape with diameters ranging from 95 to 110 nm. Enveloped capsids appeared spherical and ranged in diameter from 125 to 165 nm. Immunogold labeling revealed the presence of antigenic sites around both naked and enveloped capsids, and also on the 10 nm filaments. The use of electron microscopy and immunohistochemical techniques as diagnostic tools to demonstrate MTV infection could prove valuable for the detection of the infection and the study of its pathogenesis.