Cooperation between humoral factor(s) and Lyt-2+ T cells in effective clearance of Sendai virus from infected mouse lungs

The mechanism of cooperation between the L3T4+ and Lyt-2+ T cell subsets in effective clearance of Sendai virus from infected mouse lungs was studied by adoptive cell transfer using nude mice. Simultaneous transfer of a long-term-cultured Sendai virus-specific L3T4+ T cell line with L3T4+ cell-depleted immune spleen cell (L3T4-) fraction to infected nude mice could result in viral clearance, although single injection with either of these cells was not effective. Instead of the L3T4+ T cells, culture supernatants of the L3T4- T cell line or concanavalin A-stimulated mouse spleen cells and mouse serum immunized with the virus were also active in the cooperative viral clearance with L3T4- fraction. The role of the Sendai virus-sensitized L3T4- cell fraction in cooperative viral clearance with humoral factors could be replaced by neither T cell-deprived immune spleen cell fraction nor normal spleen cells. The 1,500 units of recombinant mouse interleukin 2 (IL-2), which was more than 12 times the IL-2 activity present in the supernatants of the T cell line or concanavalin A-stimulated spleen cells, failed to clear the virus in combination with the L3T4- fraction. Monoclonal antibodies to Sendai or mouse hepatitis viruses were also effective in the cooperative antiviral activity. IL-2 activity was not detected in these monoclonal antibodies and the mouse immune serum. Single injection of any humoral factors failed to clear the virus. These results indicate that Sendai virus-sensitized Lyt-2+ subset of T cells acts cooperatively with humoral factor(s) other than IL-2 or Sendai virus-specific antibody present in supernatants of the T cell line, of concanavalin A-stimulated spleen cells or hybridomas, and in mouse serum immunized with the virus.     

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

Mouse hepatitis virus and Sendai virus are among the most common viruses naturally infecting laboratory mice. Concanavalin A-stimulated in vitro proliferative responses of splenocytes were examined after infection of BALB/cByJ mice with the JHM strain of mouse hepatitis virus (MHV-JHM) or Sendai virus. Mice were exposed to these viruses by presumed natural routes (per os or intranasally). Immunodepression was marked but transient among BALB/cByJ mice exposed to MHV-JHM. Among mice exposed to Sendai virus and examined over a 21-day period, spleen cells from only one mouse, sacrificed 10 days postinoculation, exhibited a severely impaired ability to respond to concanavalin A. Lymphokine production by spleen cells from control and infected mice was then assessed. IL 2 was either absent or present at very low levels in culture supernates of concanavalin A-unresponsive spleen cells from MHV-JHM-infected mice. Spleen cells from the single Sendai virus-infected mouse also produced very low levels of IL 2. In contrast, IL 1 was detected in supernatants of all spleen cell cultures derived from control, MHV-JHM-infected, or Sendai virus-infected mice. There was not a clear correlation between concanavalin A responsiveness and the ability of spleen cells to produce interferon-gamma. These results stress the importance of using laboratory mice of known microbiological status for immunologic experiments.     

Temperature-sensitive virus derived from BHK cells persistently infected with HVJ (Sendai virus).

BHK-HVJ cells, a cell line of baby hamster kidney cells persistantly infected with HVJ (Sendai virus), started to produce infectious virus by shifting down the incubation temperature from 38 to 32 C. The virus derived from BHK-HVJ cells, designated as HJV-pB, was effectively neutralized with antibody against wild-type virus (HVJ-W) which was used for the establishment of BHK-HVJ cells. HVJ-pB replicated in eggs at 32 C, but not at 38 C, while HVJ-W grew equally well at both temperatures. When BHK cells infected with HVJ-PB were incubated at 38 C, production of infectious virus, hemagglutinin, and neuraminidase was markedly restrained, whereas a considerable amount of viral nucleocapisid and envelope antigens was detected in the cells by complement fixation tests. These viral activities became detectable immediately after temperature shift-down from 38 to 32 C even at the later stage of infection. HVJ-pB was indistinguishable from HJV-W with respect to particle size, density, and morphological characteristics, but appeared to possess a higher neuraminidase activity and was inactivated more rapidly at 50 C than HVJ-W. HVJ-pB was less cytocidal and could easily cause latent infection in BHK and mouse L cells.     

Enumeration of antigen-presenting cells in mice infected with Sendai virus

Substantial progress has been made in understanding Ag presentation to T cells; however, relatively little is known about the location and frequency of cells presenting viral Ags during a viral infection. Here, we took advantage of a highly sensitive system using lacZ-inducible T cell hybridomas to enumerate APCs during the course of respiratory Sendai virus infection in mice. Using lacZ-inducible T cell hybridomas specific for the immunodominant hemagglutinin-neuraminidase HN421-436/I-Ab and nucleoprotein NP324-332/Kb epitopes, we detected APCs in draining mediastinal lymph nodes (MLNs), in cervical lymph nodes, and also in the spleen. HN421-436/I-Ab- and NP324-332/Kb-presenting cells were readily detectable between days 3 and 9 postinfection, with more APCs present in the MLN than in the cervical lymph nodes. Interestingly, no infectious virus was detected in lymphoid tissue beyond day 6, suggesting that a depot of noninfectious viral Ag survives, in some form, for 2-3 days after viral clearance. Fractionation of the MLN demonstrated that APC frequency was enriched in dendritic cells and macrophages but depleted in the B cell population, suggesting that B cells do not form a large population of APCs during the primary response to this virus.     

Delayed clearance of Sendai virus in mice lacking class I MHC-restricted CD8+ T cells.

The role and interdependence of CD8+ and CD4+ alpha beta-T cells in the acute response after respiratory infection with the murine parainfluenza type 1 virus, Sendai virus, has been analyzed for H-2b mice. Enrichment of CD8+ virus-specific CTL effectors in the lungs of immunologically intact C57BL/6 animals coincided with the clearance of the virus from this site by day 10 after infection. Removal of the CD4+ T cells by in vivo mAb treatment did not affect appreciably either the recruitment of CD8+ T cells to the infected lung, or their development into virus-specific cytotoxic effectors. In contrast, depletion of the CD8+ subset delayed virus clearance, although most mice survived the infection. Transgenic H-2b F3 mice homozygous (-/-) for a beta 2 microglobulin (beta 2-m) gene disruption, which lack both class I MHC glycoproteins and mature CD8+ alpha beta-T cells, showed a comparable, delayed clearance of Sendai virus from the lung. Virus-specific, class II MHC-restricted CTL were demonstrated in both freshly isolated bronchoalveolar lavage populations and cultured lymph node and spleen tissue from the beta 2-m (-/-) transgenics. Treatment of the beta 2-m (-/-) mice with the mAb to CD4 led to delayed virus clearance and death, which was also the case for normal mice that were depleted simultaneously of the CD4+ and CD8+ subsets. These results indicate that, although classical class I MHC-restricted CD8+ cytotoxic T cells normally play a dominant role in the recovery of mice acutely infected with Sendai virus, alternative mechanisms involving CD4+ T cells exist and can compensate, in time, for the loss of CD8+ T cell function.     

Sendai virus stimulates chemiluminescence in mouse spleen cells.

Sendai virus stimulates chemiluminescence within a few seconds after it is added to a suspension of mouse spleen cells. Virus rendered non infectious by irradiation with ultraviolet light induces a similar burst of chemiluminescence. Heating or pronase treatment of the virus abrogate this reaction, as does sonication of the cells before the addition of the virus. The ability of the virus to stimulate chemiluminescence is correlated with its hemagglutination, neuraminidase, cell fusion and hemolytic properties. It is suggested that Sendai virus-induced chemiluminescence is initiated by the interaction of the virus envelope spike glycoproteins with the cell membrane.     

An attempt for eradication of Sendai virus from a mouse breeding colony

Eradication of Sendai virus from an infected mouse breeding colony was successfully made by sacrificing all offspring younger than 5 weeks of age at a time and then newborns as well as severely affected breeder mice for successive 2 weeks. By this method, number of breeders decreased of two third of those in the original colony but recovered again about 2 months later.     

Respiratory syncytial virus infection in C57BL/6 mice: clearance of virus from the lungs with virus-specific cytotoxic T cells.

We describe respiratory syncytial virus (RSV)-specific cytotoxic T-cell (CTL) lines and clones developed from the spleens of C57BL/6 and BALB/c mice. Line 7 and clones derived from it were H-2Kb restricted, whereas line 12 had both Kb and Db components. Both lines, and all the clones except one, could lyse targets infected with either strain A or strain B RSV. Line 7 or 7-11E1 cells (8 x 10(6) to 10 x 10(6) given intravenously cleared RSV from the lungs of infected mice. There was no morbidity or mortality in any of the infected mice whether or not they received T cells. The C57BL/6 mouse is a useful model system in which to study the role of the CTL response in protective immunity to RSV. CTL lines and clones can mediate clearance of RSV from the lungs of normal mice without producing any associated morbidity.     

Expression of five proteins from the Sendai virus P/C mRNA in infected cells.

The polycistronic P/C mRNA of Sendai virus is translated under cell-free conditions into five proteins (P, C', C, Y1, and Y2) from overlapping reading frames. In this study, we showed that in addition to the P, C', and C proteins, Y1 and Y2 were expressed by six different Sendai virus strains in infected cells. The Y proteins exhibited strain-specific variation in their gel mobility which corresponds to the variation seen in the cognate C proteins. While the relative levels of the P, C', and C proteins were consistent among various cell lines, the levels of Y1 and Y2 proteins varied among the cell lines used for viral infection.     

Growth of poliovirus in HeLa cells persistently infected with HVJ (Sendai virus)

The growth of poliovirus in a HeLa cell culture persistently infected with the hemagglutinating virus of Japan (HVJ, the Sendai strain of parainfluenza 1 virus) (HeLaHVJ) was studied. Plaques produced by poliovirus on HeLaHVJ cell monolayers were hazier, smaller and fewer than those on HeLa cells. HeLaHVJ cells were indistinguishable from normal HeLa cells with respect to adsorption rate and penetration efficiency of poliovirus. Extracellular yields of poliovirus in HeLaHVJ cells were lower, and the cytopathic changes were less than those in normal HeLa cells, while cell-associated virus growth in HeLaHVJ cells was nearly equal to that in HeLa cells. HeLaHVJ cells responded more effectively to the action of magnesium chloride, which facilitates virus release from infected cells, resulting in an cytopathic effects. No reduction in poliovirus yield could be detected in HeLa cells acutely infected with HVJ. The relationship between the inhibition of the release of poliovirus from HeLaHVJ cells and the persistent infection of the cells with HVJ is discussed.     

Polyoma virus early and late mRNAs in productively infected mouse 3T6 cells.

We mapped polyoma virus-specific mRNAs isolated from productively infected mouse 3T6 cells on the viral genome by analyzing nuclease S1-resistant RNA-DNA hybrids. The polyoma early mRNAs, which code for the three T antigens, have several 5' ends near 73 map units (m.u.). During the late phase of infection an additional 5' end is found near 71 m.u. All of the major early mRNAs have common 3' ends at 26.01 m.u. There is a minor species of early mRNA with a 3' end at 99.05 m.u. There are two proximal and two distal splice junctions in the early region which are used to generate three different spliced early mRNAs. There are three late mRNAs encoding the three virion proteins, VP1, VP2, and VP3. The late mRNAs have common 3' ends at 25.34 m.u. The late mRNAs have heterogeneous 5' leader sequences derived from the region between 65.53 and 68.42 m.u. The leader sequences are joined to the bodies of the messages coding for VP2, VP3, and VP1 at 66.59, 59.62, and 48.57 m.u., respectively. These results confirm and extend previous analyses of the fine structure of polyoma mRNAs.     

Expression of ets genes in mouse thymocyte subsets and T cells

The cellular ets genes (ets-1, ets-2, and erg) have been identified by their sequence similarity with the v-ets oncogene of the avian erythroblastosis virus, E26. Products of the ets-2 gene have been detected in a wide range of normal mouse tissues and their expression appears to be associated with cell proliferation in regenerating liver. In contrast, the ets-1 gene was previously shown to be more highly expressed in the mouse thymus than in other tissues. Because the thymic tissue contains various subsets of cells in different stages of proliferation and maturation, we have examined ets gene expression in fetal thymocytes from different stages of development, in isolated subsets of adult thymocytes, and in peripheral T lymphocytes. Expression of the ets-1 gene was first detected at day 18 in fetal thymocytes, corresponding to the first appearance of CD4+ (CD4+, CD8-) thymocytes, and reaches maximal/plateau levels of expression in the thymus at 1 to 2 days after birth. The ets-2 gene expression is detected at least 1 day earlier, coinciding with the presence of both double-positive (CD4+, CD8+) and double-negative (CD4-, CD8-) blast thymocytes and reaches maximal/plateau levels 1 day before birth. In the adult thymus, ets-1 and ets-2 mRNA expression is 10- to 8-fold higher respectively in the CD4+ subset than in the other subsets examined. Higher levels of p55 ets-1 protein were also shown to exist in the CD4+ subset. Because the CD4+ thymic subset is the pool from which the CD4+ peripheral, helper/inducer T cells are derived, the ets gene expression was examined in lymph node T cells. Both the CD4+ and the CD8+ T cells subsets had lower ets RNA levels than the CD4+ thymocytes. These results suggest that ets-2 and more particularly ets-1 gene products play an important role in T cell development and differentiation and are not simply associated with proliferating cells, which are observed at a higher frequency in fetal thymocytes, or dull Ly-1 (low CD5+), and double-negative (CD4-, CD8-) adult thymocytes. Selectively enhanced expression of ets-1 gene may be observed in thymic CD4+ thymocytes because these cells have uniquely encountered MHC class II or other Ag in the thymic environment. These cells may have been subsequently stimulated to activate the ets genes in conjunction with their differentiation of helper/inducer function(s) and expression of mature TCR.(ABSTRACT TRUNCATED AT 400 WORDS)     

Sequential onset of permeability changes in mouse ascites cells induced by Sendai virus

The addition of haemolytic Sendai virus to cells induces membrane changes in the following sequence: (i) Increased permeability to ions, (ii) increased permeability to low molecular weight metabolites, (iii) increased permeability to proteins. The consequences of an increased permeability to ions are: (a) alteration of membrane potential, (b) net changes in intracellular cations and (c) cell swelling, in that order. Depending on virus: cell ratio, Ca2+ concentration and temperature, it is possible to observe ion leakage without metabolite or protein leakage, and ion and metabolite leakage without protein leakage. A model for the induction of permeability changes is presented.     

RNA interference-mediated virus clearance from cells both acutely and chronically infected with the prototypic arenavirus lymphocytic choriomeningitis virus

Several arenaviruses, including Lassa fever virus, cause severe, often lethal hemorrhagic fever in humans. No licensed vaccines are available in the United States, and currently there is no efficacious therapy to treat this viral infection. Therefore the importance of developing effective antiviral approaches to combat pathogenic arenaviruses is clear. Moreover, the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) is an important model for the study of viral persistence and associated diseases, as well as for exploring therapies to treat viral chronic infections. The use of small interfering RNAs (siRNAs) to downregulate gene expression via RNA interference (RNAi) has emerged as a powerful genetic tool for the study of gene function. In addition, the successful use of siRNAs to target a variety of animal viruses has led us to consider RNAi as a potential novel antiviral strategy. We have investigated the use of RNAi therapy against LCMV. Here, we show that siRNAs targeting sequences within the viral L polymerase and Z mRNAs inhibit LCMV multiplication in cultured cells. Unexpectedly, the antiviral efficacy of RNAi-based therapy against LCMV was highly dependent on the method used to deliver effector siRNA molecules. Thus, transfection of chemically synthesized siRNA pools to L and Z was ineffective in preventing virus multiplication. In contrast, targeting of the same viral L and Z gene products with siRNAs produced inside cells using a replication-deficient recombinant adenovirus expression system inhibited LCMV multiplication very efficiently. Notably, transduction with the replication-deficient recombinant adenovirus expression system to Z and L effectively cured persistently LCMV-infected cells, suggesting the feasibility of using RNAi therapy to combat viral chronic infections by riboviruses.     

Immunofluorescence for detection of viral antigen in mice infected with Sendai virus

Sendai virus infection transmitted by contact from cagemates was followed by virus titration and immunofluorescence. The virus grew in the respiratory tract and caused macroscopic lesions in all contact mice. The virus grew to a higher titer in the lung than in the trachea. Tracheal smears, however, were found to be the most suitable for the diagnosis of Sendai virus infection by immunofluorescence, since they contained a large number of cells with intense fluorescence. Diagnosis of Sendai virus infection was made by immunofluorescence within a few hours after autopsy made at early stages of infection.