Pregnancy and parturition of mice latently infected with Pseudorabies virus.

In this study, the influence of pregnancy and parturition on mice in a mouse model of latent infection with Pseudorabies virus (PrV) was analyzed. Latently infected (LI) female mice were paired with mature uninfected males. The mating produced progeny without any clinical signs of Aujeszky's Disease. At weaning, both male and female progeny of LI mice showed significantly lower weight than control mice. PrV was not detected from nasal swab specimens of the female parent mice or the trigeminal ganglia of all mice, except 3 of 50 neonatal mice. These findings demonstrate that pregnancy and parturition induce little reactivation of latent PrV, but do affect the mother's body, as indicated by the decreased weight of progeny at weaning.     

Molecular survey of latent pseudorabies virus infection in nervous tissues of slaughtered pigs by nested and real-time PCR

In this study, the prevalence and quantity of a latent pseudorabies virus (PrV) infection in the nervous tissues of randomly selected pigs was determined via nested and real-time PCR. The nervous tissues, including the trigeminal ganglion (TG), olfactory bulb (OB), and brain stem (BS), were collected from the heads of 40 randomly selected pigs. The majority of the nervous tissues from the selected pigs evidenced a positively amplified band on nested PCR. In particular, nested PCR targeted to the PrV glycoprotein B (gB) gene yielded positive results in all of the BS samples. Nested PCR for either the gE or gG gene produced positive bands in a less number of nervous tissues (57.5% and 42.5%, respectively). Real-time PCR revealed that the examined tissues harbored large copy numbers of latent PrV DNA, ranging between 10(0.1) and 10(7.2) (1-1.58 x 10(7)) copies per 1 microg of genomic DNA. Real-time PCR targeted to the PrV gE gene exhibited an accumulated fluorescence of reporter dye at levels above threshold, thereby indicating a higher prevalence than was observed on the nested PCR (100% for BS, 92% for OB, and 85% for TG). These results indicate that a large number of farm-grown pigs are latently infected with a field PrV strain with a variety of copy numbers. This result is similar to what was found in association with the human herpes virus.     

Glycoprotein gp50-negative pseudorabies virus: a novel approach toward a nonspreading live herpesvirus vaccine.

Essential herpesvirus glycoproteins are involved in membrane fusion processes during infection, e.g., viral penetration and direct cell-to-cell transmission. We previously showed that the gD-homologous glycoprotein gp50 of pseudorabies virus (PrV) is essential for virus entry into target cells but proved to be dispensable for direct viral cell-to-cell spread in cell culture (I. Rauh and T. C. Mettenleiter, J. Virol. 65:5348-5456, 1991). For gp50-negative (gp50-) viruses, after phenotypic complementation necessary for primary infection, the only means of viral spread is by way of direct cell-to-cell transmission. In contrast, virus mutants lacking the essential gB-homologous glycoprotein gII after phenotypic complementation are only able to infect primary target cells and are blocked in further viral spread. To analyze how these in vitro phenotypes translate into virus replication in the animal, mice were infected intranasally with gp50- or gII- PrV mutants after prior phenotypic complementation by propagation on cell lines providing the essential glycoprotein in trans. Our results show that whereas the gII- mutants did not cause disease or any symptoms, gp50- mutants derived from two different PrV strains were fully virulent, with animals exhibiting severe symptoms ultimately leading to death. However, free infectious virus could not be recovered from either gp50- or gII- PrV-infected animals. We conclude that direct cell-to-cell transmission as the only means of viral spread of the gp50- mutants is sufficient for a full virulent phenotype in mice. After infection of pigs with phenotypically complemented gp50- PrV, only mild symptoms were observed, whereas the gII- mutant was totally avirulent. In both cases, shedding of infectious virus did not occur, in contrast to results with animals infected by gX- PrV that showed severe signs of disease and extensive virus shedding. After challenge infection with the highly virulent NIA-3 strain, the previously gII- PrV-infected animals exhibited severe symptoms, whereas the gp50- PrV-infected pigs showed a significant level of protection. In conclusion, vaccination with a PrV mutant lacking glycoprotein gp50, which is unable to spread between animals because of a lack of formation of free infectious virions, can confer on pigs protection against challenge infection. These results provide the basis for the development of new, nonspreading live herpesvirus vaccines based on gp50- PrV mutants.     

Pseudorabies virus expressing bovine herpesvirus 1 glycoprotein B exhibits altered neurotropism and increased neurovirulence

Herpesvirus glycoproteins play dominant roles in the initiation of infection of target cells in culture and thus may also influence viral tropism in vivo. Whereas the relative contribution of several nonessential glycoproteins to neurovirulence and neurotropism of Pseudorabies virus (PrV), an alphaherpesvirus which causes Aujeszky's disease in pigs, has recently been uncovered in studies using viral deletion mutants, the importance of essential glycoproteins is more difficult to assess. We isolated an infectious PrV mutant, PrV-9112C2, which lacks the gene encoding the essential PrV glycoprotein B (gB) but stably carries in its genome and expresses the homologous gene of bovine herpesvirus 1 (BHV-1) (A. Kopp and T. C. Mettenleiter, J. Virol. 66:2754-2762, 1992). Apart from exhibiting a slight delay in penetration kinetics, PrV-9112C2 was similar in its growth characteristics in cell culture to wild-type PrV. To analyze the effect of the exchange of these homologous glycoproteins in PrV's natural host, swine, 4-week-old piglets were intranasally infected with 10(6) PFU of either wild-type PrV strain Kaplan (PrV-Ka), PrV-9112C2, or PrV-9112C2R, in which the PrV gB gene was reinserted instead of the BHV-1 gB gene. Animals infected with PrV-Ka and PrV-9112C2R showed a similar course of disease, i.e., high fever, marked respiratory symptoms but minimal neurological disorders, and excretion of high amounts of virus. All animals survived the infection. In contrast, animals infected with PrV-9112C2 showed no respiratory symptoms and developed only mild fever. However, on day 5 after infection, all piglets developed severe central nervous system (CNS) symptoms leading to death within 48 to 72 h. Detailed histological analyses showed that PrV-9112C2R infected all regions of the nasal mucosa and subsequently spread to the CNS preferentially by the trigeminal route. In contrast, PrV-9112C2 primarily infected the olfactory epithelium and spread via the olfactory route. In the CNS, more viral antigen and significantly more pronounced histological changes resulting in more severe encephalitis were found after PrV-9112C2 infection. Thus, our results demonstrate that replacement of PrV gB by the homologous BHV-1 glycoprotein resulted in a dramatic increase in neurovirulence combined with an alteration in the route of neuroinvasion, indicating that the essential gB is involved in determining neurotropism and neurovirulence of PrV.     

Glycoprotein D-independent infectivity of pseudorabies virus results in an alteration of in vivo host range and correlates with mutations in glycoproteins B and H

Infection of cells by herpesviruses is initiated by the interaction of viral envelope glycoproteins with cellular receptors. In the alphaherpesvirus pseudorabies virus (PrV), the causative agent of Aujeszky's disease in pigs, the essential glycoprotein D (gD) mediates secondary attachment of virions to target cells by binding to newly identified cellular receptors (R. J. Geraghty, C. Krummenacher, G. H. Cohen, R. J. Eisenberg, and P. G. Spear, Science 280:1618-1620, 1998). However, in the presence of compensatory mutations, infection can also occur in the absence of gD, as evidenced by the isolation in cell culture of an infectious gD-negative PrV mutant (PrV-gD(-) Pass) (J. Schmidt, B. G. Klupp, A. Karger, and T. C. Mettenleiter, J. Virol. 71:17-24, 1997). PrV-gD(-) Pass is replication competent with an only moderate reduction in specific infectivity but appears to bind to receptors different from those recognized by wild-type PrV (A. Karger, J. Schmidt, and T. C. Mettenleiter, J. Virol. 72:7341-7348, 1998). To analyze whether this alteration in receptor usage in vitro influences infection in vivo, the model host mouse and the natural host pig were intranasally infected with PrV-gD(-) Pass and were compared to animals infected by wild-type PrV. For mice, a comparable progress of disease was observed, and all animals infected with mutant virus died, although they exhibited a slight delay in the onset of symptoms and, correspondingly, a longer time to death. In contrast, whereas wild-type PrV-infected pigs showed clinical signs and histological and histopathological findings typical of PrV infection, no signs of disease were observed after infection with PrV-gD(-) Pass. Moreover, in these animals, virus-infected cells were not detectable by immunohistochemical staining of different organ samples and no virus could be isolated from nasal swabs. Mutations in glycoproteins B and H were found to correlate with, and probably contribute to, gD-independent infectivity. In conclusion, although PrV-gD(-) Pass is virulent in mice, it is apparently unable to infect the natural host, the pig. This altered host range in vivo correlates with a difference of receptor usage in vitro and demonstrates for the first time the importance of gD receptors in alphaherpesvirus infection of an animal host.     

Adaptability in herpesviruses: glycoprotein D-independent infectivity of pseudorabies virus.

Initial contact between herpesviruses and host cells is mediated by virion envelope glycoproteins which bind to cellular receptors. In several alphaherpesviruses, the nonessential glycoprotein gC has been found to interact with cell surface proteoglycans, whereas the essential glycoprotein gD is involved in stable secondary attachment. In addition, gD is necessary for penetration, which involves fusion between virion envelope and cellular cytoplasmic membrane. As opposed to other alphaherpesvirus gD homologs, pseudorabies virus (PrV) gD is not required for direct viral cell-to-cell spread. Therefore, gD- PrV can be passaged in noncomplementing cells by cocultivating infected and noninfected cells. Whereas infectivity was found to be strictly cell associated in early passages, repeated passaging resulted in the appearance of infectivity in the supernatant, finally reaching titers as high as 10(7) PFU/ml (PrV gD- Pass). Filtration experiments indicated that this infectivity was not due to the presence of infected cells, and the absence of gD was verified by Southern and Western blotting and by virus neutralization. Infection of bovine kidney cells constitutively expressing PrV gD interfered with the infectivity of wild-type PrV but did not inhibit that of PrV gD- Pass. Similar results were obtained after passaging of a second PrV mutant, PrV-376, which in addition to gD also lacks gG, gI, and gE. Penetration assays demonstrated that PrV gD- Pass entered cells much more slowly than wild-type PrV. In summary, our data demonstrate the existence of a gD-independent mode of initiation of infection in PrV and indicate that the essential function(s) that gD performs in wild-type PrV infection can be compensated for after passaging. Therefore, regarding the requirement for gD, PrV seems to be intermediate between herpes simplex virus type 1, in which gD is necessary for penetration and cell-to-cell spread, and varicella-zoster virus (VZV), which lacks a gD gene. Our data show that the relevance of an essential protein can change under selective pressure and thus demonstrate a way in which VZV could have evolved from a PrV-like ancestor.     

Pseudorabies: experimental studies in raccoons with different virus strains

Raccoons (Procyon lotor) were infected by the nasal/oral route with as little as 10(2) plaque forming units (PFU) of pseudorabies virus (PrV). There was no apparent difference in the susceptibility of raccoons to infection with either of two virulent field strains or with the naturally avirulent K strain which has been used in modified live virus vaccines. Each of these three viruses was transmitted by contact to uninfected raccoons. All raccoons that were infected with virulent field strains died; however only two of 11 (18%) raccoons that were infected with the K strain died. One of four raccoons that survived infection with the K strain survived superinfection with virulent virus. This finding was significant because it could be a mechanism by which virulent PrV can be introduced and persist in the raccoon population. The possibility of this event occurring is increasing because of the widespread prevalence of PrV and the use of modified live virus vaccines for controlling clinical pseudorabies in swine. Virus neutralizing activity was found in five of 47 serums collected from raccoons that were trapped in PrV endemic areas. This observation implies that a herpesvirus, possibly PrV, may be present in the wild raccoon population.     

Enhanced multiplication and transmission of Streptococcus pneumoniae in guinea pigs by Sendai virus infection

It was demonstrated that the transmission S. pneumoniae in guinea pigs was remarkably promoted by the combined infection with Sendai virus in the following experiments. When guinea pigs infected with S. pneumoniae alone (infector) were cagemated with non-treated guinea pigs (contact) for 2 and 4 weeks, only 2 of 30 contacts were infected with the organism. On the contrary, when the contact guinea pigs were infected with Sendai virus and immediately cage-mated with the infectors, the pneumococcal infection occurred in 25 of 30 contacts during 2 to 4 weeks period. In the experiment in which 30 non-treated contacts were cage-mated with pneumococcal infectors for 4 weeks and then infected with Sendai virus, no pneumococcal infection was demonstrated in the contacts, suggesting no presence of latent infection of the organism in the contact guinea pigs. Twenty-five of 30 contacts suffered from pneumococcal infection when they were exposed to Sendai virus for 2 weeks and then cage-mated with infectors. The multiplication of S. pneumoniae in the respiratory tract of the guinea pigs was remarkably enhanced by combined infection with Sendai virus. Namely, a 1000-fold increase in the number of organism resulted in the guinea pigs suffered from combined infection as compared with that in the animals received pneumococcal single infection.     

Role of IL-6 and IL-1beta in reactivation by acetylcholine of latently infecting pseudorabies virus.

We previously reported that the latently infecting Pseudorabies virus (PrV) could be reactivated by injection of swine or mice with acetylcholine. However, the mechanism of the reactivation was not clear yet. In this study, we analyzed the kinetics of cytokines related to stress to clarify the relationship between virus reactivation by acetylcholine and the immune system. IL-6 and IL-1beta were detected in mice after stimulation with acetylcholine. This shows that acetylcholine induced physiological stress conditions. However, there seemed to be no relationship between the kinetics of the cytokine levels and PrV excretion. Moreover, neither IL-6 nor IL-1beta alone could reactivate latently infecting PrV. Thus, acetylcholine causes the reactivation of latent PrV via a mechanism not involving these immunological factors.     

Establishment and maintenance of gammaherpesvirus latency are independent of infective dose and route of infection

Gammaherpesviruses such as Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus are important human pathogens that establish long-term latent infections. Understanding of the initiation and maintenance of latent infections has important implications for the prevention and treatment of gammaherpesvirus-related diseases. Although much is known about gammaherpesvirus pathogenesis, it is unclear how the infectious dose of a virus influences its ability to establish latent infection. To examine the relationship between the infectious dose and gammaherpesvirus latency, we inoculated wild-type mice with 0.01 to 10(6) PFU of murine gammaherpesvirus 68 (gammaHV68) and quantitatively measured latency and acute-phase replication. Surprisingly, during latency, the frequencies of ex vivo reactivation were similar over a 10(7)-fold range of doses for i.p. infection and over a 10(4)-fold range of doses for intranasal infection. Further, the frequencies of cells harboring viral genome during latency did not differ substantially over similar dose ranges. Although the kinetics of acute-phase replication were delayed at small doses of virus, the peak titer did not differ significantly between mice infected with a large dose of virus and those infected with a small dose of virus. The results presented here indicate that any initiation of infection leads to substantial acute-phase replication and subsequent establishment of a maximal level of latency. Thus, infections with doses as small as 0.1 PFU of gammaHV68 result in stable levels of acute-phase replication and latent infection. These results demonstrate that the equilibrium level of establishment of gammaherpesvirus latency is independent of the infectious dose and route of infection.     

Recognition of EBV plasma membrane protein expressed on murine cells after gene transfer

The immune response to B lymphocytes infected with Epstein-Barr virus (EBV) prevents their overgrowth in normal humans. A murine model is now described for analyzing the T cell immune response to Epstein-Barr virus genes expressed in murine lymphoblasts by gene transfer. In mice, a 60,000 dalton virus-encoded protein characteristically found in the plasma membrane of latently infected human lymphocytes readily induces both proliferative and cytolytic T lymphocytes specific for both the EBV protein and murine major histocompatibility proteins. Longterm cultures of L3T4+ cells, some of which were cytolytic, were found to be restricted by H-2I-Ed and the latent membrane protein. Similarly, Lyt-2+ cells were cytolytic and were restricted by H-2Ld and the lymphocyte membrane protein gene product. The similarity in murine and human effector cell responses suggests that this is a useful experimental model, and the EBV latent infection membrane protein may be an important antigen in the immune restriction of growth transformed latently infected lymphocytes.     

Involvement of membrane-bound viral glycoproteins in adhesion of pseudorabies virus-infected cells.

Cell-associated spread of pseudorabies virus (PrV) plays an important role in the pathogenesis of the disease. Besides the already known direct cell-to-cell spread of the virus in monolayers, adhesion and subsequent fusion of suspended PrV infected cells to monolayers of uninfected cells are thought to occur. To study the adhesion of PrV-infected cells, an in vitro model was developed in SK-6 cells. Specific adhesion of PrV-infected cells to an uninfected monolayer started 5 h after infection of the cells and reached a maximum 6 h later. A correlation was found between the surface expression of PrV glycoproteins on the infected cells and the adhesion of these cells. PrV hyperimmune serum completely inhibited binding of the infected cells. To investigate which PrV envelope glycoproteins were responsible for the cell adhesion, the infected cells were incubated with antisera against glycoproteins gII, gIII, and gp50. Antiserum against either gII or gIII inhibited cell adhesion, and antisera against gII and gIII together had a cooperative effect. Antiserum against gp50 had no effect on binding when used alone but enhanced the inhibition induced by gII and gIII antisera. Heparin and neomycin inhibited adhesion, showing that the receptor for adhesion was a heparinlike substance. SK-6 cells infected with a gIII deletion mutant of PrV exhibited a much lower adhesion. This binding was heparin and neomycin independent and was not blocked by anti-gII serum. Nevertheless, it was completely inhibited with PrV hyperimmune serum and with anti-gp50 serum. This finding demonstrates that the ligand for adhesion of gIII(-)-infected cells is glycoprotein gp50. These results strongly suggest that the mechanism for adhesion of a PrV-infected cell to an uninfected monolayer is similar to the mechanism of adsorption and penetration of a PrV virion to a host cell.     

Identification and characterization of pseudorabies virus dUTPase.

Sequence analysis within the long segment of the pseudorabies virus (PrV) genome identified an open reading frame of 804 bp whose deduced protein product of 268 amino acids exhibited homology to dUTPases of other herpesviruses. The gene was designated UL50 because of its colinearity with the homologous gene of herpes simplex virus type 1. An antiserum raised against a bacterially expressed fragment of PrV UL50 specifically detected a 33-kDa protein in lysates of infected cells, which is in agreement with the predicted molecular mass of the PrV UL50 protein. A UL50-negative PrV mutant (PrV UL50-) was constructed by the insertion of a beta-galactosidase expression cassette into the UL50 coding sequence. A corresponding rescuant (PrV UL50resc) was also isolated. The interruption of the UL50 gene led to the disappearance of the 33-kDa protein, whereas restoration of UL50 gene expression restored detection of the 33-kDa protein. Enzyme activity assays confirmed that UL50 of PrV codes for a dUTPase which copurifies with nuclei of infected cells. PrV UL50- replicated with an only slightly reduced efficiency in epithelial cells in culture compared with that of its parental wild-type virus strain. Our results thus demonstrate that UL50 of PrV encodes a protein of 33 kDa with dUTPase activity which copurifies with nuclei of infected cells and is dispensable for replication in cultured epithelial cells.     

Early establishment of gamma-herpesvirus latency: implications for immune control

The human gamma-herpesviruses, EBV and Kaposi's sarcoma-associated herpesvirus, infect >90% of the population worldwide, and latent infection is associated with numerous malignancies. Rational vaccination and therapeutic strategies require an understanding of virus-host interactions during the initial asymptomatic infection. Primary EBV infection is associated with virus replication at epithelial sites and entry into the circulating B lymphocyte pool. The virus exploits the life cycle of the B cell and latency is maintained long term in resting memory B cells. In this study, using a murine gamma-herpesvirus model, we demonstrate an early dominance of latent virus at the site of infection, with lung B cells harboring virus almost immediately after infection. These data reinforce the central role of the B cell not only in the later phase of infection, but early in the initial infection. Early inhibition of lytic replication does not impact the progression of the latent infection, and latency is established in lymphoid tissues following infection with a replication-deficient mutant virus. These data demonstrate that lytic viral replication is not a requirement for gamma-herpesvirus latency in vivo and suggest that viral latency can be disseminated by cellular proliferation. These observations emphasize that prophylactic vaccination strategies must target latent gamma-herpesvirus at the site of infection.     

Alpha/beta interferons regulate murine gammaherpesvirus latent gene expression and reactivation from latency

Alpha/beta interferon (IFN-alpha/beta) protects the host from virus infection by inhibition of lytic virus replication in infected cells and modulation of the antiviral cell-mediated immune response. To determine whether IFN-alpha/beta also modulates the virus-host interaction during latent virus infection, we infected mice lacking the IFN-alpha/beta receptor (IFN-alpha/betaR(-/-)) and wild-type (wt; 129S2/SvPas) mice with murine gammaherpesvirus 68 (gammaHV68), a lymphotropic gamma-2-herpesvirus that establishes latent infection in B cells, macrophages, and dendritic cells. IFN-alpha/betaR(-/-) mice cleared low-dose intranasal gammaHV68 infection with wt kinetics and harbored essentially wt frequencies of latently infected cells in both peritoneum and spleen by 28 days postinfection. However, latent virus in peritoneal cells and splenocytes from IFN-alpha/betaR(-/-) mice reactivated ex vivo with >40-fold- and 5-fold-enhanced efficiency, respectively, compared to wt cells. Depletion of IFN-alpha/beta from wt mice during viral latency also significantly increased viral reactivation, demonstrating an antiviral function of IFN-alpha/beta during latency. Viral reactivation efficiency was temporally regulated in both wt and IFN-alpha/betaR(-/-) mice. The mechanism of IFN-alpha/betaR action was distinct from that of IFN-gammaR, since IFN-alpha/betaR(-/-) mice did not display persistent virus replication in vivo. Analysis of viral latent gene expression in vivo demonstrated specific upregulation of the latency-associated gene M2, which is required for efficient reactivation from latency, in IFN-alpha/betaR(-/-) splenocytes. These data demonstrate that an IFN-alpha/beta-induced pathway regulates gammaHV68 gene expression patterns during latent viral infection in vivo and that IFN-alpha/beta plays a critical role in inhibiting viral reactivation during latency.     

The UL7 gene of pseudorabies virus encodes a nonessential structural protein which is involved in virion formation and egress

Homologues of the UL7 gene of herpes simplex virus type 1 are conserved in alpha-, beta-, and gammaherpesviruses. However, little is known about their functions. Using a monospecific rabbit antiserum raised against a bacterial fusion protein, we identified the UL7 gene product of the neurotropic alphaherpesvirus pseudorabies virus (PrV). In Western blot analyses of infected cells and purified PrV particles the serum specifically detected a 29-kDa protein, which matches the calculated mass of the 266-amino-acid translation product of PrV UL7. For functional analysis, UL7 was deleted by mutagenesis of an infectious full-length clone of the PrV genome in Escherichia coli. The obtained recombinant PrV-DeltaUL7F was replication competent in rabbit kidney cells, but maximum virus titers were decreased nearly 10-fold and plaque diameters were reduced by ca. 60% compared to wild-type PrV. Electron microscopy of infected cells revealed that in the absence of UL7, formation and nuclear egress of nucleocapsids were not affected, whereas secondary envelopment of cytoplasmic nucleocapsids appeared to be delayed and release of mature virions was less efficient. The observed replication defects were corrected by repair of the viral UL7 gene or by propagation of PrV-DeltaUL7F in UL7-expressing cells. PrV-DeltaUL7F was moderately attenuated in mice. Compared to wild-type virus, mean survival times were prolonged from 2 to 3 days after intranasal infection. However, neuroinvasion and transneuronal spread of PrV were not abolished in the absence of UL7. Thus, UL7 encodes a virion protein of PrV, which plays a role during virion maturation and egress both in vitro and in vivo.     

Cytokine GM-CSF genetic adjuvant facilitates prophylactic DNA vaccine against pseudorabies virus through enhanced immune responses

Granulocyte/macrophage colony-stimulatory factor (GM-CSF) is an attractive adjuvant for a DNA vaccine on account of its ability to recruit antigen-presenting cells (APCs) to the site of antigen synthesis as well as its ability to stimulate the maturation of dendritic cells (DCs). This study evaluated the utility of GM-CSF cDNA as a DNA vaccine adjuvant for glycoprotein B (gB) of pseudorabies virus (PrV) in a murine model. The co-injection of GM-CSF DNA enhanced the levels of serum PrV-specific IgG with a 1.5-to 2-fold increase. Moreover, GM-CSF co-injection inhibited the production of IgG2a isotype. However, it enhanced production of an IgG1 isotype resulting in humoral responses biased to the Th2-type against PrV antigen. In contrast, the co-administration of GM-CSF DNA enhanced the T cell-mediated immunity biased to the Th1-type, as judged by the significantly higher level of cytokine IL-2 and IFN-gamma production but not IL-4. When challenged with a lethal dose of PrV, the GM-CSF co-injection enhanced the resistance against a PrV infection. This suggests that co-inoculation with a vector expressing GM-CSF enhanced the protective immunity against a PrV infection. This immunity was caused by the induction of increased humoral and cellular immunity in response to PrV antigen.     

Murine cytomegalovirus gene amplification and culture after submaxillary salivary gland biopsy.

An important target tissue for murine cytomegalovirus (CMV) infection is the submaxillary salivary gland. Submaxillary salivary gland biopsy specimens from BALB/c mice latently infected with murine CMV were examined for murine CMV DNA by in vitro enzymatic amplification using the polymerase chain reaction preceding oligonucleotide hybridization. The amplified sequence was a 152-base pair segment from within the immediate early gene of murine CMV. Biopsy and whole gland specimens from acutely infected BALB/c mice and latently infected, immunosuppressed BALB/c mice were compared for active murine CMV infection. After acute infection with murine CMV, virus was recovered in all cultures of both biopsy and whole salivary gland specimens but from none of the latently infected animals. Reactivated virus was detected by culture of both biopsy (90%) and whole salivary gland specimens (100%) from latently infected mice that received antithymocyte serum. Viral nucleic acid was detected in 90% of biopsy specimens from latently infected animals. Hence, active murine CMV infection can be detected in biopsy specimens from mice with acute and reactivated infection and murine CMV DNA can be amplified and detected in salivary gland biopsy specimens from latently infected animals. Biopsy of this or other target tissues can be useful for obtaining tissue for viral studies where the survival of the animal is important and it is useful to distinguish latent from acute or reactivated infection.