Latency, without persistence, of murine cytomegalovirus in the spleen and kidney.

It is not known if murine cytomegalovirus (MCMV) establishes a state of molecular latency independent of low-level persistent infection. The presence of low levels of infectious MCMV distinguishes persistence from molecular latency. Thus, the distinction between persistence and latency has depended on the sensitivity of plaque assays for detecting low levels of infectious virus in tissue of previously infected mice. To determine whether MCMV establishes molecular latency or remains persistent, we developed two assays for detecting low levels of MCMV in tissue. Using prolonged in vitro culture of virus with either mouse embryonic fibroblasts or the murine 3T12 fibroblast cell line, we reproducibly detected a single PFU of MCMV. Inclusion of undiluted sonicated tissue in this assay decreased sensitivity by up to 100-fold. However, sensitivity was improved to 1 PFU of MCMV when sonicated tissue was appropriately diluted. Severe combined immunodeficient (SCID) mice were also used to detect MCMV in sonicated tissue. Infection of SCID mice with a single PFU of MCMV killed two of eight SCID mice, and the 50% lethal dose of MCMV in SCID mice was 2 to 3 PFU. Applying these two methods, we detected infectious virus in 0 of 34 spleens, 1 of 34 kidneys, and 0 of 37 salivary glands from latently infected mice. Spleens and kidneys assessed for persistent virus contained MCMV DNA by PCR and reactivated after 10 to 50 days in explant cultures. Latently infected kidney cells reactivated after adoptive transfer to SCID mice. Quantitation of the MCMV genome by PCR showed that latently infected spleens without detectable infectious MCMV contained about 3,000,000 copies of the MCMV genome. These results demonstrate that MCMV latency in spleen and kidney exists in the absence of low-level persistent infection. Use of assays with defined sensitivity for detection of MCMV in tissue provides a basis for evaluation of cytomegalovirus gene expression in the spleen and kidney during molecular latency.     

Detection of murine cytomegalovirus DNA in circulating leukocytes harvested during acute infection of mice.

We used virus assay and in situ hybridization with a cloned fragment of the murine cytomegalovirus (MCMV) genome to study MCMV infection of circulating leukocytes harvested from 3-week-old BALB/c, C57BL/6, and C3H mice infected with MCMV intraperitoneally. Infectious virus or MCMV DNA was detected in leukocytes on days 1 through 21 of infection in BALB/c mice and on days 3 through 7 in C57BL/6 mice. On days 5 and 7, MCMV DNA or infectious virus was detected in the leukocytes of 17 (94%) of 18 BALB/c mice and 10 (59%) of 17 C57BL/6 mice. In both strains infection peaked on days 5 and 7, when as many as 0.01 to 0.1% of the circulating leukocytes contained MCMV DNA. In C3H mice, however, infectious virus was rarely recovered from leukocyte fractions and MCMV DNA was detected in the circulating leukocytes of only one animal. Circulating leukocytes may have an important role in the dissemination of CMV infections in susceptible hosts.     

<Emphasis Type="Italic">Ly49h</Emphasis> is necessary for genetic resistance to murine cytomegalovirus

Natural killer (NK) cells play critical roles in antiviral immunity. While the importance of effector mechanisms such as interferons has been demonstrated through knockout mice, specific mechanisms of how viruses are recognized and controlled by NK cells are less well defined. Previous genetic studies have mapped the resistance genes for murine cytomegalovirus (MCMV), herpes simplex virus-1 (HSV-1), and ectromelia virus to the NK gene complex on murine chromosome 6, a region containing the polymorphic Ly49 and Nkrp1 families. Genetic resistance to MCMV in C57BL/6 has been attributed to Ly49H, an activation receptor, through susceptibility of the recombinant inbred strain BXD-8 that lacks Ly49h (also known as Klra8) but derived about half of its genome from its DBA/2 progenitor. However, it remained possible that epigenetic effects could account for the MCMV phenotype in BXD-8 mice. Herein, we report the generation of a novel congenic murine strain, B6.BXD8-Klra8 ( Cmv1-del )/Wum, on the C57BL/6 genetic background to evaluate the effect of deletion of a single NK activation receptor, Ly49H. Deletion of Ly49H rendered mice much more susceptible to MCMV infection. This increase in susceptibility did not appear to be a result of a difference in NK cell expansion or interferon-gamma (IFN-gamma) production between the C57BL/6 and the B6.BXD8 strains. On the other hand, the deletion of Ly49h did not otherwise affect NK cell maturation or Ly49D expression and had no effect on susceptibility to HSV-1 or ectromelia virus. In conclusion, Ly49h is necessary for genetic resistance to MCMV, but not HSV-1 or ectromelia virus.     

Induction of murine p30 by superinfecting herpesviruses.

The interaction of endogenous type C viruses with superinfecting herpes simplex virus type 2 (HSV-2) was investigated in two murine cell lines. Replication of HSV-2 was suboptimal in random-bred Swiss/3T3A cells and, in initial experiments, infection with a low virus-to-cell ratio resulted in carrier cultures with enhanced murine leukemia virus (MuLV) p30 expression. Immunofluorescence tests with Swiss/3T3A cells productively infected with HSV-2 also showed HSV-associated cytoplasmic antigens and enhanced MuLV p30 expression when compared with uninfected controls. Inactivation of HSV-2 with UV light did not abolish this reaction, although the number of cells expressing p30 was reduced. HSV-2 replicated more efficiently in a line of NIH Swiss cells (N c1 A c1 10). These cells are not readily inducible for type C expression by conventional methods; however, untreated and UV-inactivated HSV-2 induced both HSV-2-associated antigens and MuLV p30 in these cells. Although the Birch strain of human cytomegalovirus induced MuLV p30, neither mouse cytomegalovirus nor vesicular stomatitis virus induced MuLV p30 in either cell line.     

Involvement of an early human cytomegalovirus function in reactivation of quiescent herpes simplex virus type 2

We have previously described an in vitro system in which the function lacking for herpes simplex virus type 2 (HSV-2) replication can be induced by human cytomegalovirus (HCMV). The mechanism of this reactivation of quiescent HSV-2 by HCMV has been further defined. The HCMV function(s) responsible for HSV-2 stimulation was examined temporally, and the fraction of cells in quiescent cultures producing HSV-2 after superinfection was determined. Using independent biological, genetic and molecular techniques we have made the following observations. (i) As early as 12 h after HCMV superinfection, HSV-2 RNA was expressed in latently infected cells. (ii) At 24 h after HCMV superinfection, a time when newly synthesized HCMV was not yet apparent, infectious HSV-2 was produced by reactivated cultures. (iii) Four HCMV temperature-sensitive mutants, which are DNA-negative at nonpermissive temperature and represent four different complementation groups, induced reactivation of HSV-2 at 39.5 degrees C. (iv) Early after HCMV superinfection, 1.6% of quiescent cells could be induced to transcribe HSV-2 information. (v) Early after HCMV superinfection, 0.3% of cells in the quiescent cultures could be induced to yield infectious HSV-2. The finding that a significant interaction can occur between HCMV and quiescent HSV-2 in an in vitro model is noteworthy in light of the knowledge that both of these herpesviruses often reside simultaneously in the human host.     

Synthesis and antiviral activity of 6-deoxycyclopropavir, a new prodrug of cyclopropavir

Synthesis of 6-deoxycyclopropavir (10), a prodrug of cyclopropavir (1) and its in vitro and in vivo antiviral activity is described. 2-Amino-6-chloropurine methylenecyclopropane 13 was transformed to its 6-iodo derivative 14 which was reduced to prodrug 10. It is converted to cyclopropavir (1) by the action of xanthine oxidase and this reaction can also occur in vivo. Compound 10 lacked significant in vitro activity against human cytomegalovirus (HCMV), human herpes virus 1 and 2 (HSV-1 and HSV-2), human immunodeficiency virus type 1 (HIV-1), human hepatitis B virus (HBV), Epstein-Barr virus (EBV), vaccinia virus and cowpox virus. In contrast, prodrug 10 given orally was as active as cyclopropavir (1) reported previously [Kern, E. R.; Bidanset, D. J.; Hartline, C. B.; Yan, Z.; Zemlicka, J.; Quenelle, D. C. et al. Antimicrob. Agents Chemother. 2004, 48, 4745] against murine cytomegalovirus (MCMV) infection in mice and against HCMV in severe combined immunodeficient (SCID) mice.     

Replication of mouse cytomegalovirus in thymidine kinase-deficient mouse cells.

In an attempt to determine whether mouse cytomegalovirus (MCMV) requires thymidine kinase (TK) for replication and whether it induces TK, TK-deficient mouse cells were isolated and used as host cells for MCMV. Mutant cells resistant to 200 μg/ml of 5-Bromodeoxyuridine (BUdR) were selected from SV40-transformed mouse cells, mks-A TU-7, by propagating the cells in the presence of varying concentrations of BUdR graded by serial 2-fold increments. The mutant cells, designated as TU-7 BU, showed a very low TK activity (less than 1/20 that of mks-A TU-7). Herpes simplex virus type 1 (HSV-1) replicated in starved as well as in unstarved TU-7 BU, whereas MCMV could replicate only in growing TU-7 BU and could not form plaques in monolayers of mks-A TU-7 or TU-7 BU. HSV-1 infection enhanced TK activity equally in both mks-A TU-7 and TU-7 BU. In contrast, TK activity of MCMV-infected mks-A TU-7 was lower than that of uninfected cells or cells inoculated with UV-inactivated virus. In addition, TK activity of the MCMV-infected TU-7 BU remained minimal without showing any increase. The replication of HSV-1 was completely inhibited in the presence of BUdR (10 μg/ml), whereas MCMV could replicate even in the presence of 50 μg/ml of BUdR. The results indicate that MCMV neither requires TK nor induces TK activity in the infected cells.     

Prolongation of herpes simplex virus latency in cultured human cells by temperature elevation.

Treatment of herpes simplex virus type 2 (HSV-2)-infected human fibroblast cells with cytosine arabinoside (ara-C) at 25 microgram/ml resulted in complete inhibition of virus replication. Removal of ara-C after 7 days of treatment ultimately resulted in renewed virus replication, but after a delay of at least 5 days. If however, the temperature was elevated from 37 degrees C to 39.5 to 40 degrees C at the time of ara-C reversal, infectious HSV-2 did not reappear. As long as the cultures were maintained at 39.5 to 40 degrees C (up to at least 128 days), HSV-2 was latent and infectious virus was undetectable. If the temperature was reduced to 37 degrees C at any time during the latent period, infectious virus was always reactivated, but only after a period of incubation at 37 degrees C of a least 11 days. Infectious-center assays performed with latent cultures indicated that only a very small fraction of cells could reactivate virus. The infectious-center titer did not show significant changes during much of the period of latency. This seemed to argue against the possibility that the latent cultures were synthesizing very small amounts of infectious virus. Additional studies were aimed at determining the minimum incubation period at 37 degrees C required to reactivate infectious HSV-2. Latent cultures reduced from 39.5 to 40 degrees C to 37 degrees C for less than 96 h did not yield infectious HSV-2, but those incubated at 37 degrees C for 96 h or more did.     

Quantitative assay for transformation of 3T3 cells by herpes simplex virus type 2.

The interaction of herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) with Swiss/3T3 cells was investigated. Virus-induced cytopathic effects developed in the absence of production of infectious virus. HSV-2 inactivated with UV light (2, 4, 6, and 8 min) also induced cell death in the absence of virus replication. Cell death was not detectable after infection by HSV-2 that had been inactivated by UV irradiation for 10, 12, and 14 min. 3T3 cells infected with UV-inactivated virus (10 and 12 min) continued to replicate past the contact-inhibited monolayer normally associated with these cells. Infection of 3T3 cells with UV-irradiated USV-2 also induced the development of transformed foci. Transformed cells with an epithelioid of fibroblastoid morphology were identified and isolated. All HSV-2-transformed cell lines contained HSV-2-specific antigens detectable by immunofluorescence techniques. The maximum frequency of HSV-2-induced transformation was 3 times 105 PFU per transformed focus, and the observed transformation could be inhibited by pretreatment of the virus with specific antiserum. No type C particles were detected within five cell culture passages after transformation by HSV-2. Type C virus particles were detected after 10 cell culture passages of the HSV-2-transformed cell lines.     

Efficacy of methylenecyclopropane analogs of nucleosides against herpesvirus replication in vitro

We have reported previously that purine methylenecyclopropane analogs are potent agents against cytomegaloviruses. In an attempt to extend the activity of these compounds, the 2-amino-6-cyclopropylaminopurine analog, QYL-1064, was selected for further study by modifying the purine 6 substituent. A total of 22 analogs were tested against herpes simplex virus types 1 and 2 (HSV-1, HSV-2), varicella zoster virus (VZV), human cytomegalovirus (HCMV), murine cytomegalovirus (MCMV), Epstein-Barr virus (EBV), human herpesvirus type 6 (HHV-6) and human herpesvirus type 8 (HHV-8). Ten of the analogs had activity against at least one of the viruses tested. One compound had moderate activity against HSV-1 and six had activity against VZV. All but one compound was active against HCMV with a mean EC50 of 2.1 +/- 0.6 microM, compared with a mean EC50 of 3.9 +/- 0.8 microM for ganciclovir. Of special interest was the fact that eight of the ten compounds were active against both HHV-6A and HHV-6B with mean EC50 values of 6.0 +/- 5.2 mciroM and <2.4 +/- 1.5 microM, respectively. Only two compounds had activity against EBV, whereas all but one compound was active against HHV-8 with a mean EC50 of 3.1 +/- 1.7 microM. These results indicate that members of this series of methylenecyclopropane analogs are highly active against HCMV, HHV-6, and HHV-8 but are less active against HSV, VZV, and EBV.     

Serological evidence of herpesvirus infection in gibbons

Background  

Herpesviruses are not only infectious agents of worldwide distribution in humans, but have also been demonstrated in various non-human primates as well. Seventy-eight gibbons were subjected to serological tests by ELISA for herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), Epstein-Barr virus (EBV) and cytomegalovirus (CMV).     

The enantiomers of carbocyclic 5'-norguanosine: activity towards Epstein-Barr virus

(-)-5'-noraristeromycin (1) has shown antiviral activity towards, particularly cytomegalovirus, vaccinia virus and measles while its (+)-enantiomer (2) is effective towards hepatitis B virus. To determine if the antiviral characteristics of 1 and 2 extended to the guanine analogues (3 and 4), these enantiomers were prepared and evaluated against herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2), cytomegalovirus (CMV), varicella zoster virus (VZV), Epstein-Barr virus (EBV), human herpes virus type 6 (HHV-6), human herpes virus type 8 (HHV-8), vaccinia virus (VV), cowpox virus (CV), vesicular stomatitis virus (VSV), respiratory syncytial virus (RSV), hepatitis B virus (HBV), and human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2). The only activity found for 3 was for Epstein-Barr virus in VCA Elisa (EC50 0.78 microg/mL), immunofluorescence assay for VCA or gp 350/250 (1.8-4.0 microg/mL) and DNA hybridization (EC50 0.82 microg/mL) assays with no accompanying toxicity seen in the host Daudi cells. No activity was noted for 4.     

Herpes Simplex Virus Types 1 and 2 Completely Help Adenovirus-Associated Virus Replication

In addition to adenoviruses, which are capable of completely helping adenovirus-associated virus (AAV) multiplication, only herpesviruses are known to provide any AAV helper activity, but this activity has been thought to be partial (i.e., AAV DNA, RNA, and protein syntheses are induced, but infectious particles are not assembled). In this study, however, we show that herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) are in fact complete AAV helpers and that AAV type 2 (AAV2) infectivity yields can approach those obtained when coinfections are carried out with a helper adenovirus. AAV helper activity was demonstrated in KB cells with two HSV-1 strains (11124 and 17MP) and an HSV-2 strain (HG52). Each herpesvirus supported AAV2 multiplication with comparable efficiency. AAV2 multiplication was similarly efficient in HSV-1 coinfections of HeLa cells, whereas lower yields were obtained in HEp-2 and primary human embryonic kidney cells. HSV-1 also supported AAV1 multiplication in HeLa cells but, at corresponding multiplicities of infection, AAV1 grew less efficiently than AAV2. Comparisons of the time courses of AAV2 DNA, RNA, and protein syntheses after coinfection with either adenovirus type 5 or HSV-1 revealed that, in each case, the onset of synthesis and attainment of maximal synthesis rate occurred earlier in coinfections with HSV-1. These findings demonstrate the linkage of AAV macromolecular synthesis to an event(s) in the helper virus cycle. Aside from this temporal association, helper-related differences in AAV macromolecular synthesis were not apparent.     

Novel class of thiourea compounds that inhibit herpes simplex virus type 1 DNA cleavage and encapsidation: resistance maps to the UL6 gene

In our search for novel inhibitors of herpes simplex virus type 1 (HSV-1), a new class of thiourea inhibitors was discovered. N-(4-[3-(5-Chloro-2,4-dimethoxyphenyl)-thioureido]-phenyl)-acetamide and its 2-fluoro-benzamide derivative inhibited HSV-1 replication. HSV-2, human cytomegalovirus, and varicella-zoster virus were inhibited to a lesser extent. The compounds acted late in the replication cycle by impairing both the cleavage of concatameric viral DNA into progeny genome length and the packaging of the DNA into capsids, indicative of a defect in the encapsidation process. To uncover the molecular target of the inhibition, resistant HSV-1 isolates were generated, and the mutation responsible for the resistance was mapped using marker transfer techniques. Each of three independent isolates had point mutations in the UL6 gene which resulted in independent single-amino-acid changes. One mutation was located in the N terminus of the protein (E121D), while two were located close together in the C terminus (A618V and Q621R). Each of these point mutations was sufficient to confer drug resistance when introduced into wild-type virus. The UL6 gene is one of the seven HSV-1 genes known to play a role in DNA packaging. This novel class of inhibitors has provided a new tool for dissection of HSV-1 encapsidation mechanisms and has uncovered a new viable target for the treatment of herpesviral diseases.     

Regulation of persistent infection with herpes simplex virus in vitro by hydrocortisone.

About 1% of Raji cells showed sensitivity to herpes simplex virus type 2 (HSV-2) infection when tested by infectious center assays or immunofluorescence tests, and the percentage did not change during cell passage. The addition of hydrocortisone to Raji cells persistently infected with HSV-2 caused a marked increase in virus production and in the number of HSV-producing cells. In the case of HSV-1, it was shown that the addition of hydrocortisone was required to maintain persistent infection. These observations suggest that control of replication of HSV-1 and HSV-2 in these cells is regulated by different mechanisms.     

Natural Killer Cell Dependent Within-Host Competition Arises during Multiple MCMV Infection: Consequences for Viral Transmission and Evolution

It is becoming increasingly clear that many diseases are the result of infection from multiple genetically distinct strains of a pathogen. Such multi-strain infections have the capacity to alter both disease and pathogen dynamics. Infection with multiple strains of human cytomegalovirus (HCMV) is common and has been linked to enhanced disease. Suggestions that disease enhancement in multi-strain infected patients is due to complementation have been supported by trans-complementation studies in mice during co-infection of wild type and gene knockout strains of murine CMV (MCMV). Complementation between naturally circulating strains of CMV has, however, not been assessed. In addition, many models of multi-strain infection predict that co-infecting strains will compete with each other and that this competition may contribute to selective transmission of more virulent pathogen strains. To assess the outcome of multi-strain infection, C57BL/6 mice were infected with up to four naturally circulating strains of MCMV. In this study, profound within-host competition was observed between co-infecting strains of MCMV. This competition was MCMV strain specific and resulted in the complete exclusion of certain strains of MCMV from the salivary glands of multi-strain infected mice. Competition was dependent on Ly49H⁺ natural killer (NK) cells as well as the expression of the ligand for Ly49H, the MCMV encoded product, m157. Strains of MCMV which expressed an m157 gene product capable of ligating Ly49H were outcompeted by strains of MCMV expressing variant m157 genes. Importantly, within-host competition prevented the shedding of the less virulent strains of MCMV, those recognized by Ly49H, into the saliva of multi-strain infected mice. These data demonstrate that NK cells have the strain specific recognition capacity required to meditate within-host competition between strains of MCMV. Furthermore, this within-host competition has the capacity to shape the dynamics of viral shedding and potentially select for the transmission of more virulent virus strains.     

Induction of natural killer cell responses by ectromelia virus controls infection

Natural killer (NK) cells play a pivotal role in the innate immune response to viral infections, particularly murine cytomegalovirus (MCMV) and human herpesviruses. In poxvirus infections, the role of NK cells is less clear. We examined disease progression in C57BL/6 mice after the removal of NK cells by both antibody depletion and genetic means. We found that NK cells were crucial for survival and the early control of virus replication in spleen and to a lesser extent in liver in C57BL/6 mice. Studies of various knockout mice suggested that gammadelta T cells and NKT cells are not important in the C57BL/6 mousepox model and CD4+ and CD8+ T cells do not exhibit antiviral activity at 6 days postinfection, when the absence of NK cells has a profound effect on virus titers in spleen and liver. NK cell cytotoxicity and/or gamma interferon (IFN-gamma) secretion likely mediated the antiviral effect needed to control virus infectivity in target organs. Studies of the effects of ectromelia virus (ECTV) infection on NK cells demonstrated that NK cells proliferate within target tissues (spleen and liver) and become activated following a low-dose footpad infection, although the mechanism of activation appears distinct from the ligand-dependent activation observed with MCMV. NK cell IFN-gamma secretion was detected by intracellular cytokine staining transiently at 32 to 72 h postinfection in the lymph node, suggesting a role in establishing a Th1 response. These results confirm a crucial role for NK cells in controlling an ECTV infection.     

Efficacy of Methylenecyclopropane Analogs of Nucleosides Against Herpesvirus Replication In Vitro

Abstract

We have reported previously that purine methylenecyclopropane analogs are potent agents against cytomegaloviruses. In an attempt to extend the activity of these compounds, the 2-amino-6-cyclopropylaminopurine analog, QYL-1064, was selected for further study by modifying the purine 6 substituent. A total of 22 analogs were tested against herpes simplex virus types 1 and 2 (HSV-1, HSV-2), varicella zoster virus (VZV), human cytomegalovirus (HCMV), murine cytomegalovirus (MCMV), Epstein-Barr virus (EBV), human herpesvirus type 6 (HHV-6) and human herpesvirus type 8 (HHV-8). Ten of the analogs had activity against at least one of the viruses tested. One compound had moderate activity against HSV-1 and six had activity against VZV. All but one compound was active against HCMV with a mean EC₅₀ of 2.1 ± 0.6 µM, compared with a mean EC₅₀ of 3.9 ± 0.8 µM for ganciclovir. Of special interest was the fact that eight of the ten compounds were active against both HHV-6A and HHV-6B with mean EC₅₀ values of 6.0 ± 5.2 µM and <2.4 ± 1.5 µM, respectively. Only two compounds had activity against EBV, whereas all but one compound was active against HHV-8 with a mean EC₅₀ of 3.1 ± 1.7 µM. These results indicate that members of this series of methylenecyclopropane analogs are highly active against HCMV, HHV-6, and HHV-8 but are less active against HSV, VZV, and EBV.     

Laboratory strains of murine cytomegalovirus are genetically similar to but phenotypically distinct from wild strains of virus

Murine cytomegalovirus (MCMV) is widely used to model human cytomegalovirus (HCMV) infection. However, it is known that serially passaged laboratory strains of HCMV differ significantly from recently isolated clinical strains of HCMV. It is therefore axiomatic that clinical models of HCMV using serially passaged strains of MCMV may not be able to fully represent the complexities of the system they are attempting to model and may not fully represent the complex biology of MCMV. To determine whether genotypic and phenotypic differences also exist between laboratory strains of MCMV and wild derived strains of MCMV, we sequenced the genomes of three low-passage strains of MCMV, plus the laboratory strain, K181. We coupled this genetic characterization to their phenotypic characteristics. In contrast to what is seen with HCMV (and rhesus CMV), there were no major genomic rearrangements in the MCMV genomes. In addition, the genome size was remarkably conserved between MCMV strains with no major insertions or deletions. There was, however, significant sequence variation between strains of MCMV, particularly at the genomic termini. These more subtle genetic differences led to considerable differences in in vivo replication with some strains of MCMV, such as WP15B, replicating preferentially in otherwise-MCMV-resistant C57BL/6 mice. CBA mice were no more resistant to MCMV than C57BL/6 mice and for some MCMV strains appeared to control infection less well than C57BL/6 mice. It is apparent that the previously described host resistance patterns of inbred mice and MCMV are not consistently applicable for all MCMV strains.