Inactivation of Cytomegalovirus in Breast Milk Using Ultraviolet-C Irradiation: Opportunities for a New Treatment Option in Breast Milk Banking.

Pasteurized donor human milk is provided by milk banks to very preterm babies where their maternal supply is insufficient or unavailable. Donor milk is currently processed by Holder pasteurization, producing a microbiologically safe product but significantly reducing immunoprotective components. Ultraviolet-C (UV-C) irradiation at 254 nm is being investigated as an alternative treatment method and has been shown to preserve components such as lactoferrin, lysozyme and secretory IgA considerably better than Holder pasteurization. We describe the inactivation of cytomegalovirus, a virus commonly excreted into breast milk, using UV-C irradiation. Full replication was ablated by various treatment doses. However, evidence of viral immediate early proteins within the cells was never completely eliminated indicating that some viral gene transcription was still occurring. In conclusion, UV-C may be a safe alternative to pasteurisation for the treatment of human donor milk that preserves the bioactivity. However, our data suggests that CMV inactivation will have to be carefully evaluated for each device designed to treat breast milk using UV-C irradiation.

Murine cytomegalovirus strains co-replicate at multiple tissue sites and establish co-persistence in salivary glands in the absence of Ly49H-mediated competition.

Infection with multiple genetically distinct strains of pathogen is common and can lead to positive (complementation) or negative (competitive) within-host interactions. These interactions can alter aspects of the disease process and help shape pathogen evolution. Infection of the host with multiple strains of cytomegalovirus (CMV) occurs frequently in humans and mice. Profound, NK-cell-mediated (apparent) competition has been identified in C57BL/6 mice, and prevented the replication and shedding of certain co-infecting CMV strains. However, the frequency of such strong competition has not been established. Other within-host interactions such as complementation or alternative forms of competition remain possible. Moreover, high rates of recombination in both human CMV and murine CMV (MCMV) suggest prolonged periods of viral co-replication, rather than strong competitive suppression. An established model was employed to investigate the different possible outcomes of multi-strain infection in other mouse strains. In this study, co-replication of up to four strains of MCMV in the spleen, liver and salivary glands was observed in both MCMV-susceptible and MCMV-resistant mice. In the absence of apparent competition, no other forms of competition were unmasked. In addition, no evidence of complementation between viral strains was observed. Importantly, co-replication of MCMV strains was apparent for up to 90 days in the salivary glands. These data indicated that competition was not the default outcome of multi-strain CMV infection. Prolonged, essentially neutral, co-replication may be the norm, allowing for multi-strain transmission and prolonged opportunities for recombination.

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.

Respiratory viral pathogens associated with lower respiratory tract disease among young children in the highlands of Papua New Guinea.

BACKGROUND: Acute lower respiratory tract infections (ALRI) commonly result in fatal outcomes in the young children of Papua New Guinea (PNG). However, comprehensive studies of the viral aetiology of ALRI have not been conducted in PNG for almost 30 years. OBJECTIVES: To determine the viruses associated with ALRI among children living in the PNG highlands using sensitive molecular detection techniques. STUDY DESIGN: Pernasal swabs were collected routinely between 1 week and 18 months of age and also during episodes of ALRI, as part of a neonatal pneumococcal conjugate vaccine trial. A tandem multiplex real-time PCR assay was used to test for a comprehensive range of respiratory viruses in samples collected from 221 young children. Picornavirus typing was supported by DNA sequence analysis. RESULTS: Recognized pathogenic respiratory viruses were detected in 198/273 (73%) samples collected from children with no evidence of ALRI and 69/80 (86%) samples collected during ALRI episodes. Human rhinoviruses (HRV) species A, B and C were detected in 152 (56%) samples from non-ALRI children and 50 (63%) samples collected during ALRI episodes. Partial structural region sequences for two new species C rhinoviruses were added to the GenBank database. ALRI was associated with detection of adenovirus species B (p<0.01) or C (p<0.05), influenza A (p<0.0001) or respiratory syncytial virus (p<0.0001). Multiple viruses were detected more often during ALRI episodes (49%) than when children displayed no symptoms of ALRI (18%) (p<0.0001). CONCLUSIONS: The burden of infection with respiratory viruses remains significant in young children living in the PNG highlands.

B7-mediated costimulation of CD4 T cells constrains cytomegalovirus persistence.

Cytomegalovirus (CMV) utilizes multiple strategies to modulate immunity and promote lifelong, persistent/latent infection, including suppressing T cell activation pathways. Here we examined the role of B7 costimulatory ligands in establishing immune détente from both the host and virus perspectives. Mice lacking both B7.1 and B7.2 showed reduced early expansion of CMV-specific CD4 T cells, consequently allowing for enhanced levels of persistent virus replication. In turn, a CMV mutant lacking expression of the m138 and m147.5 gene products, which restrict B7.1 and B7.2 expression in infected antigen-presenting cells, induced a more robust CD4 T cell response and showed decreased persistence. Together, these data reveal a requirement for B7-mediated signaling in regulating the CMV-specific CD4 T cell response and establishing host-virus equilibrium.

Immunoglobulin to zona pellucida 3 mediates ovarian damage and infertility after contraceptive vaccination in mice.

Antibodies reactive with the ovarian glycoprotein zona pellucida (ZP) have been linked with human female infertility. Anti-fertility vaccines that target ZP antigens have been utilized to restrict pest animal populations and their efficacy is associated with ovary-specific antibody induction. However, the necessity for zona pellucida-specific antibody in mediating infertility has not been examined in vivo. A recombinant mouse cytomegalovirus vaccine encoding murine zona pellucida 3 that induces rapid and complete infertility in BALB/c mice has been produced. The onset of infertility is temporally related to the presence of antibody sequestered into ovarian follicles and binding to the ZP of infected mice and the loss of mature follicles. When this vaccine was inoculated into immunoglobulin-deficient BALB/c mice with a null mutation in the immunoglobulin mu chain gene Igh-6, fertility was unaffected. Passive transfer of serum containing ZP3 antibodies also elicited transient infertility. Electron microscopy of ovarian tissue collected from ZP3-immunized immunocompetent mice demonstrated significant focal thinning of the zona pellucida (ZP) with reduced length and concentration of transzonal processes and many oocytes displayed evidence of injury. None of these changes were found in vaccinated immunoglobulin-deficient mice. These data confirm that ZP3-reactive antibody is necessary and sufficient to induce autoimmune-mediated follicular depletion and fertility suppression following the inoculation of this vaccine, and suggest that this is due to impaired zona pellucida formation. These findings have relevance in understanding the etiology of autoimmune ovarian disease in woman where anti-ZP antibodies are likely to have a causal role in infertility.

Promoter control over foreign antigen expression in a murine cytomegalovirus vaccine vector.

Previous studies have reported on the development of a recombinant murine cytomegalovirus (rMCMV) containing the mouse zona pellucida 3 (mZP3) gene for use as a virally vectored immunocontraceptive (VVIC). This study aimed to alter promoter control over foreign antigen expression and cellular localisation of the antigen expressed in order to overcome virus attenuation previously encountered. Early studies reported on the mZP3 gene expressed by a strong constitutive human cytomegalovirus immediate-early 1 promoter (pHCMV IE1). This virus was able to induce >90% infertility in BALB/c mice despite being heavily attenuated in vivo. In this study the mZP3 was placed under the control of the MCMV early 1 (pMCMV E1) promoter and the inducible tetracycline promoter (Tet-On). In both instances the recombinant virus was able to induce infertility in directly infected mice. However, the viruses remained attenuated. This study demonstrated the capacity to manipulate the nature of the immune response by altering promoter control over foreign antigen expression and cellular localisation of the expressed antigen. We were able to demonstrate that by using the MCMV E1 promoter it was still possible to sterilize female BALB/c mice with an MCMV vector expressing mZP3. The use of the MCMV E1 promoter provides an added level of safety to any MCMV based VVIC approach as it only allows for transgene expression in MCMV permissive cells.

Overcoming innate host resistance to vaccination: employing a genetically distinct strain of murine cytomegalovirus avoids vector-mediated resistance to virally vectored immunocontraception.

The laboratory strain of murine cytomegalovirus (MCMV), K181, has been successfully engineered as a vaccine expressing murine zona pellucida 3 (mZP3) for viral vectored immunocontraception (VVIC) in mice. However, certain laboratory strains of mice are resistant to infection with K181 and therefore demonstrate resistance to VVIC. Cmv1 is the best characterised innate resistance mechanism to MCMV and was first described in C57BL/6 mice. Resistance in C57BL/6 mice is due to early and strong activation of natural killer (NK) cells by an MCMV gene product, m157, that binds directly to the NK cell activating receptor Ly49H. In this study a wild strain of MCMV, G4, which expresses a variant m157 incapable of activating Ly49H, was engineered to express murine zona pellucida 3 (mZP3) and assessed for its ability to sterilise female C57BL/6 mice. When infected with K181-mZP3 female C57BL/6 mice remained fully fertile. In contrast, female C57BL/6 mice were sterilised by a single intraperitoneal inoculation of G4-mZP3. Infertility was induced by G4-mZP3 in three strains of mice that express Ly49H, on two different histocompatibility-2 (H-2) backgrounds. Finally, enhanced immunocontraception was observed in mice expressing H-2(k) mediated resistance to MCMV when infected with G4-mZP3 compared to K181-mZP3. These data indicate that when using viral vaccine vectors, variant vector strains may be used to circumvent powerful innate immune responses against the vector and promote effective vaccination. This study highlights the importance of vaccine vector genetics in vaccination strategies.

An economical tandem multiplex real-time PCR technique for the detection of a comprehensive range of respiratory pathogens.

This study used real-time PCR assays to screen small sample volumes for a comprehensive range of 35 respiratory pathogens. Initial thermocycling was limited to 20 cycles to avoid competition for reagents, followed by a secondary real-time multiplex PCR. Supplementary semi-nested human metapneumovirus and picornavirus PCR assays were required to complete the acute respiratory pathogen profile. Potential pathogens were detected in 85 (70%) of pernasal aspirates collected from 121 children with acute respiratory symptoms. Multiple pathogens were detected in 29 (24%) of those samples. The tandem multiplex real-time PCR was an efficient method for the rapid detection of multiple pathogens.

Memory inflation during chronic viral infection is maintained by continuous production of short-lived, functional T cells.

During persistent murine cytomegalovirus (MCMV) infection, the T cell response is maintained at extremely high intensity for the life of the host. These cells closely resemble human CMV-specific cells, which compose a major component of the peripheral T cell compartment in most people. Despite a phenotype that suggests extensive antigen-driven differentiation, MCMV-specific T cells remain functional and respond vigorously to viral challenge. We hypothesized that a low rate of antigen-driven proliferation would account for the maintenance of this population. Instead, we found that most of these cells divided only sporadically in chronically infected hosts and had a short half-life in circulation. The overall population was supported, at least in part, by memory T cells primed early in infection, as well as by recruitment of naive T cells at late times. Thus, these data show that memory inflation is maintained by a continuous replacement of short-lived, functional cells during chronic MCMV infection.

Immunization with recombinant murine cytomegalovirus expressing murine zona pellucida 3 causes permanent infertility in BALB/c mice due to follicle depletion and ovulation failure.

Zona pellucida (ZP) glycoproteins are promising candidate antigens for use in immunocontraceptive vaccines because of their crucial role in mammalian fertilization. A single intraperitoneal immunization with recombinant murine cytomegalovirus engineered to express murine ZP3 (rMCMV-mZP3) induces permanent infertility with no evident systemic illness in female BALB/c mice. To investigate the mechanisms underpinning reproductive failure elicited by rMCMV-mZP3, ovarian parameters and reproductive function were evaluated at time points spanning 10 days to 5 wk after virus inoculation. Fertility was substantially impaired by 14 days after inoculation with rMCMV-mZP3 and was fully ablated by 21 days. Pregnancies established after inoculation but before complete infertility showed no adverse effects on fetal viability assessed at Day 17.5 post coitum (pc). Infertile mice retained estrous cycling activity and remained receptive to mating; however, at Day 3.5 pc there were fewer developing embryos and corpora lutea, plasma progesterone content was reduced, and there was no evidence of excess unfertilized oocytes. Consistent with this, profound ovarian pathology was evident from 10 days after rMCMV-mZP3 inoculation, with a decline first in mature ovarian follicles and then in immature ovarian follicles and with diminished expression of genes regulating follicle development, including Nobox, Gdf9, and Gja1 (connexin43). Follicle loss was associated with mild focal oophoritis and with recruitment of inflammatory leukocytes, predominantly CD4(+) and CD8(+) T cells evident from 10 days after virus inoculation. These data indicate that vaccination with rMCMV-mZP3 causes permanent infertility in BALB/c mice principally due to induction of ovarian autoimmune pathology leading to progressive oocyte depletion and eventual ovulation failure.

Prior infection with murine cytomegalovirus (MCMV) limits the immunocontraceptive effects of an MCMV vector expressing the mouse zona-pellucida-3 protein.

We have developed a murine cytomegalovirus (MCMV)-vectored vaccine expressing the mouse zona-pellucida-3 gene (rMCMV-ZP3), which successfully induces infertility in experimentally inoculated laboratory or wild-derived mice. However, the future success of this vector as a fully disseminating vaccine in free-living mice may be compromised by pre-existing immunity since there is a high prevalence of naturally acquired MCMV infection in these mice. To evaluate the effect of prior immunity to MCMV on vaccine efficacy, we constructed two new biologically effective recombinant MCMV vectors expressing the mouse ZP3 protein from two MCMV strains (N1 and G4) derived from free-living mice. In wild mice, mixed MCMV infection is common and could be acquired either by simultaneous coinfection or sequential infection with different MCMV strains. Interestingly, while coinfection with both wild-type and rMCMV-ZP3 via the intraperitoneal route reduced the impact of the rMCMV-ZP3, prior infection with the same wild-type strain as that used to construct the rMCMV-ZP3 abrogated the immunocontraceptive effects of either N1-ZP3 or G4-ZP3. However, prior infection with G4 28 days before the introduction of N1-ZP3 had a reduced influence on the efficacy of the rMCMV-ZP3. Thus, the strain of virus and the timing of prior infection are factors that may influence the efficacy of the rMCMV-ZP3. Given that mixed infection of mice with MCMV is common, it is possible that prior immunity acquired by natural mucosal infection may have less a less inhibitory effect on the immunocontraceptive outcome.

Innate antiviral resistance influences the efficacy of a recombinant murine cytomegalovirus immunocontraceptive vaccine.

Recombinant betaherpesviruses are attractive vaccine candidates because of their persistence in the host. A recombinant murine cytomegalovirus expressing the mouse ovarian glycoprotein zona pellucida 3 induces long lasting sterility in female BALB/c mice. Using inbred mouse strains selected for their innate resistance or susceptibility to MCMV, we show that genetically determined innate resistance to MCMV can reduce immunocontraceptive success. The Cmv1 locus that controls natural killer cell mediated responses to MCMV was implicated in determining vaccine efficacy. However, the role of the H-2 haplotype was less clear. Interestingly, Mus domesticus from an outbred colony of wild-derived mice were readily sterilised by vaccination, consistent with observations that strong innate immunity to MCMV is not common in Australian wild mice.

Viral vectored immunocontraception: screening of multiple fertility antigens using murine cytomegalovirus as a vaccine vector.

Mouse cytomegalovirus (MCMV) has previously been used as a vaccine vector for viral vectored immunocontraception (VVIC). MCMV expressing murine zona pellucida 3 (mZP3) induces long term infertility in up to 100% of female BALB/c mice following a single inoculation. Whilst a large number of antigens have been investigated as potential immunocontraceptive vaccines, it has been difficult to compare these antigens as few studies have used identical approaches or even animal species. Here a range of protein and polyepitope antigens, all expressed by MCMV, were tested for the ability to sterilise female mice. The antigens tested were bone morphogenic protein 15 (BMP15), oviduct glycoprotein (OGP) and ubiquitin-tagged mZP3. In addition, four polyepitope constructs that contain rodent or mouse specific epitopes were tested. This study found that when expressed by an MCMV vector, only full-length mZP3 or ubiquitin-tagged mZP3 induced infertility in female mice. BMP15 and OGP had no effect. Of the four polyepitopes tested, one had a partial effect on fertility. These data indicate that while MCMV is an effective vector for VVIC, the antigen used needs to be tested empirically. The partial infertility seen in mice infected with one of the polyepitope vaccines is a promising finding suggesting that it may be possible to combine a species specific virus with a species specific antigen for use as a disseminating mouse control agent.

Genes of murine cytomegalovirus exist as a number of distinct genotypes.

Murine cytomegaloviruses encode a number of genes which modulate polymorphic host immune responses. We suggest that these viral genes should themselves therefore exhibit sequence polymorphism. Additionally, clinical isolates of human cytomegalovirus (HCMV) have been shown to vary extensively from the common laboratory strains. Almost all research conducted on murine cytomegalovirus (MCMV) has used the laboratory strains Smith and K181, which have been extensively passaged in vitro and in vivo since isolation. Using the heteroduplex mobility assay (HMA) to determine levels of sequence variation 11 MCMV genes were examined from 26 isolates of MCMV from wild mice, as well as both laboratory strains. Both the HMA and sequencing of selected genes demonstrated that whilst certain genes (M33, mck-2, m147.5, m152) were highly conserved, others (m04, m06, M44, m138, m144, m145 and m155) contained significant sequence variation. Several of these genes (m06, m144 and m155) exist in wild MCMV strains as one of several distinct genotypes.

Mixed infection with multiple strains of murine cytomegalovirus occurs following simultaneous or sequential infection of immunocompetent mice.

As with human cytomegalovirus (HCMV) infection of humans, murine CMV (MCMV) infection is widespread in its natural host, the house mouse Mus domesticus, and may consist of mixed infection with different CMV isolates. The incidence and mechanisms by which mixed infection occurs in free-living mice are unknown. This study used two approaches to determine whether mixed infection with MCMV could be established in laboratory mice. The first utilized two naturally occurring MCMV strains, N1 and G4, into which the lacZ gene was inserted by homologous recombination. The lacZ gene was used to track recombinant and parental viruses in simultaneously coinfected mice. In the second approach, a real-time quantitative PCR (qPCR) assay was used to detect viral immediate-early 1 (ie1) gene sequences in mice successively coinfected with G4 and then with the K181 MCMV strain. In both systems, mixed infection was detected in the salivary glands and lungs of experimentally infected mice. MCMV-specific antibody in sera and G4 IE1-specific cytotoxic lymphocyte responses in the spleens of twice-infected mice did not prevent reinfection. Finally, the prevalence of mixed infection in free-living mice trapped in four Australian locations was investigated using real-time qPCR to detect ie1 DNA sequences of N1, G4 and K181. Mixed infection with MCMVs containing the G4 and K181 ie1 sequences was detected in the salivary glands of 34.2 % of trapped mice. The observations that mixed infections are common in free-living M. domesticus and are acquired by immunocompetent mice through simultaneous or successive infections are important for vaccine development.

Multiplex nested PCR (MNP) assay for the detection of 15 high risk genotypes of human papillomavirus.

BACKGROUND: Human papillomavirus (HPV) is now recognized as the causative agent in cervical cancer. The HPV genotypes that infect the genital region have been classified into high and low risk types according to their oncogenic potential. There is still uncertainty regarding rare HPV genotypes, however the types considered high risk in this study are: HPV-16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68 and 70. OBJECTIVES: We have set out to develop a multiplex nested PCR (MNP) assay with primers directed at the early region of the HPV genome to detect 15 high risk HPV (HRHPV) genotypes. Since it is known that the late region of HPV is lost on integration into the host cell genome, the primers are directed at the early region of the HPV genome so as to ensure the detection of integrated virus, in the absence of the episomal form of the virus. STUDY DESIGN: Primers were designed to detect specifically the high risk HPV in the MNP assay. The MNP assay was compared to a generic mucosal HPV nested PCR and another nested HRHPV PCR assay. DNA sequencing was carried out on the samples tested and matched with the PCR results. RESULTS: The MNP assay demonstrated that it was able to detect all 15 HRHPV types and was positive for more CIN1, CIN2 and CIN3 cases than the other nested HRHPV PCR. Further to this, the PCR product sizes differ for most of the HRHPV types detected in this system, so it is possible to type most of these HRHPV by the molecular size of the PCR products. CONCLUSION: The MNP assay detects 15 currently recognized HRHPV and could be very useful, in conjunction with the Pap smear, as a screening assay or to help manage Pap smears of uncertain cytology.

Human papillomavirus genotypes and their association with cervical neoplasia in a cohort of Western Australian women.

Human papillomavirus (HPV) is known to be the cause of almost all cervical cancers. The genotypes have been classified into high and low risk types according to their oncogenic potential. However, data for many of the genotypes are limited and some (HPV-26, 53, and 66) have no agreed status. A study was undertaken to determine the HPV genotype distribution in women of Western Australia and the association with cervical neoplasia. Liquid based cervical samples from a cohort of 282 Western Australian women were tested for HPV DNA by PCR followed by DNA sequencing to determine HPV genotypes. HPV-53 and HPV-16 were the most common genotypes found in this population. In addition 86 archived liquid based cervical samples from women with cervical intraepithelial neoplasia grades 1-3 (CIN 1-3) were tested for HPV DNA. Also 32 archived paraffin biopsy samples from women with squamous cell carcinoma were also tested. HPV-16 was the most common genotype found in these samples. Of the cohort of Western Australian women tested, 27% were found to contain HPV and approximately half of these contained known high-risk HPV genotypes, but only 30% of these were types 16 or 18. The data from this study indicate that HPV-53 is not oncogenic based on an R value and odds ratio (OR) of zero. The data also suggest that HPV-73 may be oncogenic, while HPV-66 is unlikely to be. Two high-risk HPV genotypes that are associated with the Asian region (HPV-52 and HPV-58) were found in Western Australian women suggesting a possible epidemiological link between women in these countries.

NK gene complex haplotype variability and host resistance alleles to murine cytomegalovirus in wild mouse populations.

The NK gene complex (NKC) on mouse chromosome 6 encodes receptors that are expressed on NK cells, such as Ly49H, and is involved in regulating NK cell control of virus infections, such as murine cytomegalovirus (MCMV). In the present study, we investigated the level of allelic heterogeneity in NKC loci in populations of outbred wild mice. This work revealed extensive levels of heterogeneity within two wild mouse populations. Analysis of MCMV replication in a population of specific pathogen-free outbred wild mice revealed that low viral titres, which are normally associated with the Cmv1(r) allele of the Cmv1 host resistance locus, were not prevalent in the mice tested. Hence, NKC-mediated resistance associated with Cmv1(r)/Ly49H-like effects was rare in this population. Overall, these data indicate that the NKC region is highly polymorphic and thus it is very likely that it confers on mice sufficient variability to cope with infection by a range of pathogens.

Use of a murine cytomegalovirus K181-derived bacterial artificial chromosome as a vaccine vector for immunocontraception.

Cytomegaloviruses (CMVs) are members of the Betaherpesvirinae subfamily of the Herpesviridae, and their properties of latency, large DNA size, gene redundancy, and ability to be cloned as bacterial artificial chromosomes (BACs) suggest their utility as vaccine vectors. While the K181 strain of murine CMV (MCMV) is widely used to study MCMV biology, a BAC clone of this virus had not previously been produced. We report here the construction of a BAC clone of the K181(Perth) strain of MCMV. The in vivo and in vitro growth characteristics of virus derived from the K181 BAC were similar to those of wild-type K181. The utility of the K181 BAC as a method for the rapid production of vaccine vectors was assessed. A vaccine strain of BAC virus, expressing the self-fertility antigen, murine zona pellucida 3, was produced rapidly using standard bacterial genetics techniques and rendered female BALB/c mice infertile with a single intraperitoneal inoculation. In addition, attenuated vaccine strains lacking the open reading frames m07 to m12 exhibited no reduction in efficacy compared to the full-length vaccine strain. In conclusion, we describe the production of a K181-based BAC virus which behaved essentially as wild-type K181 and allowed the rapid production of effective viral vaccine vectors.