Prenatal and postnatal antiviral therapies for the prevention and treatment of congenital cytomegalovirus infections.

PURPOSE OF REVIEW: Congenital cytomegalovirus infection (cCMV) is the leading infectious cause of sensorineural hearing loss and lifelong neurodevelopmental disabilities. Studies suggest antiviral therapy can prevent fetal infection after maternal primary infection, as well as halt the progression of hearing loss and neurodevelopmental disabilities in newborns with symptomatic cCMV. With growing worldwide momentum on early detection and diagnosis of cCMV, this review describes the exciting recent advances in antiviral therapies in CMV infected pregnant mothers and babies, as well as emerging evidence on anti-CMV vaccines. RECENT FINDINGS: New opportunities for prenatal and neonatal interventions have driven a rising interest in screening and identification of asymptomatic CMV infection. Routine screening of pregnant women to identify primary infection in first trimester is now advocated in Western Europe but has yet to be examined from a public health perspective in other regions. Evidence is emerging for maternal valaciclovir therapy to prevent fetal infection after a maternal primary CMV infection in the first trimester of pregnancy. For those infants who are born with symptomatic cCMV, a 6-month course of valganciclovir, started within the first 4 weeks of life, and possibly up to 13 weeks of life, is the current recommended therapy. However, there is unclear evidence for the benefit of treatment for asymptomatic cCMV and cCMV with isolated hearing loss. Research to identify more effective antivirals and an effective CMV vaccine continues. SUMMARY: More research is needed to determine the region-specific applicability of the new European recommendations for routine CMV screening in pregnancy. Areas of uncertainty in postnatal management include timing of initiation, duration of treatment and identifying pediatric subgroups that benefit from modification of the standard treatment recommendations.

Congenital Cytomegalovirus-Associated Sensorineural Hearing Loss in Children: Identification Following Universal Newborn Hearing Screening, Effect of Antiviral Treatment, and Long-Term Hearing Outcomes.

OBJECTIVES: Congenital cytomegalovirus (cCMV) is the most common cause of nongenetic sensorineural hearing loss (SNHL) in children. We examined the longitudinal hearing outcomes of children with cCMV in relation to their newborn hearing screening findings, and their use of antiviral therapy. DESIGN: The study was based on a retrospective chart review using a database of pediatric patients (N = 445) seen at the University of Minnesota Lions clinic. Chart review identified infants with cCMV, and records were reviewed for information about universal newborn hearing screen (UNHS) results, the clinical course of SNHL, and the use of antiviral therapy. RESULTS: A total of 44 children were identified with cCMV. In this group, 33 (75%) had SNHL of varying degree and age at onset. Notably, 17 (39%) children passed UNHS bilaterally. Of those children, 6 (35%) ultimately acquired bilateral or unilateral SNHL, detected at a mean age of 20 months (median age, 12 months). Five out of 10 children (50%) that did not pass UNHS in one ear acquired late-onset hearing loss in the contralateral ear, identified at a mean age of 24 months (median age, 4 months). Eleven (25%) children passed UNHS bilaterally and continued to demonstrate normal hearing in both ears at their most recent follow-up visit at a mean age of 19 months (SD, 18 months). Of the 33 children with cCMV and SNHL, 18 (55%) received antiviral medication (ganciclovir and/or valganciclovir). While, on average, both treated and untreated ears experienced a progression of hearing loss over time, the group that received antiviral treatment experienced less overall hearing change compared with the untreated group (baseline-adjusted expected mean difference, -10.5 dB; 95% confidence interval, -28.1 to 7.2 dB). CONCLUSIONS: Among children with cCMV included in this study who passed UNHS in both ears, 35% demonstrated delayed-onset SNHL. Notably, of those children who referred unilaterally, 50% later demonstrated SNHL in the contralateral ear. These findings have implications for audiological monitoring, and potentially antiviral therapy, of children with cCMV. As implementation of universal cCMV screening moves forward, a key aspect of follow-up will be appropriate long-term audiologic monitoring.

Updated National and State-Specific Prevalence of Congenital Cytomegalovirus Infection, United States, 2018-2022.

CONTEXT: Congenital cytomegalovirus (cCMV) infection is the most common infectious cause of birth defects and the leading non-genetic cause of sensorineural hearing loss in the United States. Prior national cCMV infection prevalence estimates were based on one multi-site screening study conducted between 2007 and 2012 and were not adjusted for sociodemographic characteristics, such as maternal race and ethnicity or age. OBJECTIVE: This study sought to estimate national and state-specific prevalence of cCMV infection in the United States, adjusted for maternal race and ethnicity and maternal age group, by pooling estimates from published studies. DESIGN: We searched PubMed for U.S. cCMV newborn screening studies conducted between 2003 and 2023. From included studies, we abstracted maternal race and ethnicity- and age group-stratified cCMV prevalence to estimate strata-specific pooled prevalence. We obtained strata-specific weights from live birth data. MAIN OUTCOME MEASURE: Estimated adjusted national and state-specific prevalence estimates from 2018 to 2022. RESULTS: Four studies (conducted 2004-2005, 2008, 2007-2012, and 2016-2021) were included for data abstraction. Overall, infants born to non-Hispanic Black (9.3 [8.2-10.5] per 1000) or non-Hispanic American Indian and Alaska Native (8.5 [2.1-33.2] per 1000) mothers had the highest cCMV prevalence. The estimated race and ethnicity-adjusted prevalence was 4.6-4.7 per 1000 live births nationally and ranged from 3.9 to 6.5 per 1000 across states from 2018 to 2022. Southern states and Alaska consistently had the highest cCMV prevalence. The estimated maternal age group-adjusted prevalence was 4.3-4.4 per 1000 live births nationally and ranged from 3.8 to 5.1 per 1000 across states from 2018 to 2022. CONCLUSIONS: States with larger proportions of racial and ethnic minorities had higher estimated prevalence of cCMV infection compared to states with larger proportions of White persons. These estimates may be useful for informing cCMV surveillance at the jurisdiction level and developing tailored, culturally relevant education and prevention strategies for persons at higher risk.

What are the key pediatric public policy priorities as the COVID-19 pandemic persists?

IMPACT: The COVID-19 pandemic is not over, and its impact is just beginning to be felt on children. COVID-19 vaccines protect both the pregnant patient and newborns, and breastfeeding provides a key component of passive protective immunity. "Long COVID" has contributed to the current crisis in pediatric mental health, and vaccines confer protection against this long-term complication of COVID-19 disease. Vaccine misinformation is not only impacting compliance with maternal and pediatric COVID-19 immunization efforts, but also other routine childhood vaccinations. As a public health priority, we must improve our response to vaccine misinformation and find novel strategies to improve vaccine compliance.

Gestational Antibodies to C. pneumoniae, H. pylori and CMV in Women with Preeclampsia and in Matched Controls.

OBJECTIVES: Some research has suggested a possible role for past infection in the development of preeclampsia. The objective of this study was to explore the role of Helicobacter pylori, cytomegalovirus, and Chlamydophila pneumoniae in the development of preeclampsia in a prospective pregnancy sample. METHODS: We conducted a nested case-control study in The Archive for Child Health (ARCH), a pregnancy cohort of 867 unselected women enrolled at the first prenatal visit with archived blood and urine in pregnancy. We matched 21 cases of preeclampsia to 52 unaffected controls on maternal age (±4 years), race, parity, and gestational age at blood draw. Using conditional logistic regression, we examined the association between preeclampsia status and immunoglobulins G (IgG) tested by indirect ELISA to each of the three microorganisms, adjusting for potential confounders. RESULTS: No significant difference was found between cases and controls. The unadjusted odds ratio was 1.5 (95%CI: 0.2-9.1), 0.6 (95%CI: 0.2-1.9), and 1.9 (95%CI: 0.6-5.6) for H. pylori, cytomegalovirus and C. pneumoniae respectively. After controlling for confounders analysis found increased odds of H. pylori IgG (AOR: 1.9; 95% CI: 0.2-15.3) and C. pneumoniae IgG (AOR: 2.3; 95% CI: 0.6-9.2) for preeclampsia, albeit being not significant. Conversely, cytomegalovirus IgG had lower odds for preeclampsia (AOR: 0.4; 95% CI: 0.1-1.7). CONCLUSIONS: Past infection with H. pylori, and C. pneumoniae in early pregnancy showed a higher risk of preeclampsia, but the findings failed to achieve statistical significance. Cytomegalovirus was not associated with preeclampsia in these data. These preliminary findings encourage future research in populations with high prevalence of these infections.

Impairment in neurocognitive function following experimental neonatal guinea pig cytomegalovirus infection.

BACKGROUND: Cytomegalovirus (CMV) is a leading infectious cause of neurologic deficits, both in the settings of congenital and perinatal infection, but few animal models exist to study neurodevelopmental outcomes. This study examined the impact of neonatal guinea pig CMV (GPCMV) infection on spatial learning and memory in a Morris water maze (MWM) model. METHODS: Newborn pups were challenged intraperitoneally (i.p.) with a pathogenic red fluorescent protein-tagged GPCMV, or sham inoculated. On days 15-19 post infection (p.i.), pups were tested in the MWM. Viral loads were measured in blood and tissue by quantitative PCR (qPCR), and brain samples collected at necropsy were examined by histology and immunohistochemistry. RESULTS: Viremia (DNAemia) was detected at day 3 p.i. in 7/8 challenged animals. End-organ dissemination was observed, by qPCR, in the lung, liver, and spleen. CD4-positive (CD4+) and CD8-positive (CD8+) T cell infiltrates were present in brains of challenged animals, particularly in periventricular and hippocampal regions. Reactive gliosis and microglial nodules were observed. Statistically significant spatial learning and memory deficits were observed by MWM, particularly for total maze distance traveled (p < 0.0001). CONCLUSION: Neonatal GPCMV infection in guinea pigs results in cognitive defects demonstrable by the MWM. This neonatal guinea pig challenge model can be exploited for studying antiviral interventions. IMPACT: CMV impairs neonatal neurocognition and memory in the setting of postnatal infection. The MWM can be used to examine memory and learning in a guinea pig model of neonatal CMV infection. CD4+ and CD8+ T cells infiltrate the brain following neonatal CMV challenge. This article demonstrates that the MWM can be used to evaluate memory and learning after neonatal GPCMV challenge. The guinea pig can be used to examine central nervous system pathology caused by neonatal CMV infection and this attribute may facilitate the study of vaccines and antivirals.

Impact of breast milk-acquired cytomegalovirus infection in premature infants: Pathogenesis, prevention, and clinical consequences?

Maternal-fetal transmission of cytomegalovirus (CMV) represents the most common infectious cause of long-term neurodevelopmental disability in children. Congenital CMV (cCMV) infection is associated with microcephaly, seizure disorders, cognitive disability, developmental delay, and sensorineural hearing loss (SNHL). Of these disabilities, SNHL is the most common, affecting approximately 10% of infants with cCMV. Although the sequelae of cCMV are well recognized, it is much less clear what long-term morbidities may occur in neonates that acquire post-natal CMV infection. Post-natal CMV (pCMV) infection is most commonly transmitted by breast-feeding, and in full-term infants is of little consequence. However, in preterm, very-low birthweight (VLBW) infants (<1500 g), pCMV can result in a severe sepsis-like syndrome, with wide-ranging end-organ disease manifestations. Although such short-term complications are well recognized among clinicians caring for premature infants, the long-term risks with respect to adverse neurodevelopmental outcomes remain controversial. In this review, we provide an overview of the clinical manifestations of breast milk-acquired pCMV infection. In particular, we summarize studies that have examined-sometimes with conflicting conclusions-the risks of long-term adverse neurodevelopmental outcome in VLBW infants that acquire pCMV from breast milk. We highlight proposed preventive strategies and antiviral interventions, and offer recommendations for high-priority areas for future basic science and clinical research.

Detection and treatment of cerebral toxoplasmosis in an aplastic pediatric post-allogeneic hematopoietic cell transplant patient: a case report.

BACKGROUND: Cerebral toxoplasmosis infection presents with non-specific neurologic symptoms in immunocompromised patients. With lack of measurable adaptive immune responses and reluctance to sample affected brain tissue, expedient diagnosis to guide directed treatment is often delayed. CASE PRESENTATION: We describe the use of cerebrospinal fluid polymerase chain reaction and plasma cell-free DNA technologies to supplement neuroimaging in the diagnosis of cerebral toxoplasmosis in an immunocompromised pediatric patient following allogeneic hematopoietic cell transplantation for idiopathic severe aplastic anemia. Successful cerebral toxoplasmosis treatment included antibiotic therapy for 1 year following restoration of cellular immunity with an allogeneic stem cell boost. CONCLUSIONS: Plasma cell-free DNA technology provides a non-invasive method of rapid diagnosis, improving the likelihood of survival from often lethal opportunistic infection in a high risk, immunocompromised patient population.

Animal Models of Congenital Cytomegalovirus Transmission: Implications for Vaccine Development.

Although cytomegaloviruses (CMVs) are species-specific, the study of nonhuman CMVs in animal models can help to inform and direct research aimed at developing a human CMV (HCMV) vaccine. Because the driving force behind the development of HCMV vaccines is to prevent congenital infection, the animal model in question must be one in which vertical transmission of virus occurs to the fetus. Fortunately, two such animal models-the rhesus macaque CMV and guinea pig CMV-are characterized by congenital infection. Hence, each model can be evaluated in "proof-of-concept" studies of preconception vaccination aimed at blocking transplacental transmission. This review focuses on similarities and differences in the respective model systems, and it discusses key insights from each model germane to the study of HCMV vaccines.

Cytomegalovirus Viremia Associated With Increased Mortality in Cryptococcal Meningitis in Sub-Saharan Africa.

BACKGROUND: Cryptococcal meningitis and tuberculosis are both important causes of death in persons with advanced human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS). Cytomegalovirus (CMV) viremia may be associated with increased mortality in persons living with HIV who have tuberculosis. It is unknown whether concurrent CMV viremia is associated with mortality in other AIDS-related opportunistic infections. METHODS: We prospectively enrolled Ugandans living with HIV who had cryptococcal meningitis from 2010-2012. Subsequently, we analyzed stored baseline plasma samples from 111 subjects for CMV DNA. We compared 10-week survival rates among those with and without CMV viremia. RESULTS: Of 111 participants, 52% (58/111) had detectable CMV DNA (median plasma viral load 498 IU/mL, interquartile range [IQR] 259-2390). All samples tested were positive on immunoglobin G serology. The median CD4+ T cell count was 19 cells/µL (IQR 9-70) and did not differ by the presence of CMV viremia (P = .47). The 10-week mortality rates were 40% (23/58) in those with CMV viremia and 21% (11/53) in those without CMV viremia (hazard ratio 2.19, 95% confidence interval [CI] 1.07-4.49; P = .03), which remained significant after a multivariate adjustment for known risk factors of mortality (adjusted hazard ratio 3.25, 95% CI 1.49-7.10; P = .003). Serum and cerebrospinal fluid cytokine levels were generally similar and cryptococcal antigen-specific immune stimulation responses did not differ between groups. CONCLUSIONS: Half of persons with advanced AIDS and cryptococcal meningitis had detectable CMV viremia. CMV viremia was associated with an over 2-fold higher mortality rate. It remains unclear whether CMV viremia in severely immunocompromised persons with cryptococcal meningitis contributes directly to this mortality or may reflect an underlying immune dysfunction (ie, cause vs effect). CLINICAL TRIALS REGISTRATION: NCT01075152.

Chest palpitations in a teenager as an unusual presentation of Lyme disease: case report.

BACKGROUND: The incidence of Lyme disease (LD) in North America has increased substantially in the past two decades. Concomitant with the increased incidence of infection has been an enhancement in the recognition of LD complications. Here, we report a case of Lyme carditis complicated by heart block in a pediatric patient admitted to our children's hospital. What is unique about this case is that the complaint of chest palpitations is an infrequent presentation of LD, and what it adds to the scientific literature is an improved understanding of LD in the pediatric population. CASE PRESENTATION: The patient was a 16-year-old male who presented with the main concerns of acute onset of palpitations and chest pain. An important clinical finding was Erythema migrans (EM) on physical exam. The primary diagnoses were LD with associated Lyme carditis, based on the finding of 1st degree atrioventricular heart block (AVB) and positive IgM and IgG antibodies to Borrelia burgdorferi. Interventions included echocardiography, electrocardiography (EKG), and intravenous antibiotics. The hospital course was further remarkable for transition to 2nd degree heart block and transient episodes of complete heart block. A normal sinus rhythm and PR interval were restored after antibiotic therapy and the primary outcome was that of an uneventful recovery. CONCLUSIONS: Lyme carditis occurs in < 5% of LD cases, but the "take-away" lesson of this case is that carditis can be the presenting manifestation of B. burgdorferi infection in pediatric patients. Any patient with suspected Lyme carditis manifesting cardiac symptoms such as syncope, chest pain, or EKG changes should be admitted for parenteral antibiotic therapy and cardiac monitoring. The most common manifestation of Lyme carditis is AVB. AVB may manifest as first-degree block, or may present as high-grade second or third-degree block. Other manifestations of Lyme carditis may include myopericarditis, left ventricular dysfunction, and cardiomegaly. Resolution of carditis is typically achieved through antibiotic administration, although pacemaker placement should be considered if the PR interval fails to normalize or if higher degrees of heart block, with accompanying symptoms, are encountered. With the rising incidence of LD, providers must maintain a high level of suspicion in order to promptly diagnose and treat Lyme carditis.

Advancing Our Understanding of Protective Maternal Immunity as a Guide for Development of Vaccines To Reduce Congenital Cytomegalovirus Infections.

Human cytomegalovirus (HCMV) is the most common congenitally transmitted pathogen worldwide, impacting an estimated 1 million newborns annually. Congenital HCMV (cCMV) infection is a major global contributor to long-term neurologic deficits, including deafness, microcephaly, and neurodevelopmental delay, as well as to fetal loss and occasional infant mortality. Accordingly, design of a maternal vaccine to prevent cCMV continues to be a top public health priority. Nevertheless, we remain without a licensed vaccine. Maternal immunity provides partial protection, as the risk of vertical HCMV transmission from chronically infected mothers is reduced compared to settings in which the mother is newly infected during pregnancy. Therefore, an understanding of the maternal immune correlates of protection against cCMV is critical to informing design of an efficacious maternal vaccine. Although vaccine development is being assiduously pursued by a large number of pharmaceutical manufacturers, biotechnology organizations, and academic researchers, some pessimism has been expressed regarding the issue of whether a vaccine to protect against cCMV is possible. This pessimism is based on observations that natural immunity is not completely protective against maternal reinfection and congenital transmission. However, we assert that optimism regarding vaccine development is indeed justified, on the basis of accruing evidence of immune correlates of protection-readily achievable by vaccination-that are associated with reduced transmission of HCMV to the fetus in seronegative women. In light of the substantial burden on society conferred by cCMV infection, even a modest reduction in the occurrence of this fetal disease is an important public health goal and justifies aggressive clinical evaluation of vaccines currently in the pipeline.

Case report of congenital asplenia presenting with Haemophilus influenzae type a (Hia) sepsis: an emerging pediatric infection in Minnesota.

BACKGROUND: In the pre-vaccine era, invasive disease with Haemophilus influenzae, type b (Hib) commonly presented with osteoarticular involvement. Haemophilus influenzae, type a (Hia) sepsis is a rare but emerging problem in recent years. Here, we report a case of sepsis with concomitant osteoarthritis due to Hia that was the presenting infectious disease manifestation of isolated asplenia in a young child. This unique observation adds to our understanding of sepsis and asplenia in children. CASE PRESENTATION: A five-year-old girl developed acute Hia bacteremia and sepsis. The patient developed arthritis shortly after onset of septic shock. Arthrocentesis was culture-negative, but given the difficulty differentiating between septic and reactive arthritis, prolonged antibiotic administration was provided for presumed osteoarticular infection, and the patient had an uneventful recovery. The finding of Howell-Jolly bodies on blood smear at the time of presentation prompted an evaluation that revealed isolated congenital asplenia. Evaluation for known genetic causes of asplenia was unrevealing. Investigation by the Minnesota Department of Health revealed an emergence of Hia infections over the past 5 years, particularly in children with an American Indian background. CONCLUSIONS: Hia is an important pathogen in the differential diagnosis of invasive bacterial infections in children and shares overlap in clinical presentation and pathogenesis with Hib. Invasive Hia disease can be a presenting manifestation of asplenia in children. Hia is an emerging pathogen in American Indian children.

Cytomegalovirus-induced pathology in human temporal bones with congenital and acquired infection.

OBJECTIVE: Publications on histopathology of human temporal bones with cytomegalovirus (CMV) infection are limited. We aim to determine histopathology of the inner ears and the middle ears in human temporal bones with congenital and acquired CMV infections. METHODS: Temporal bones from 2 infants with congenital and 2 adults with acquired CMV infection were evaluated by light microscopy. RESULTS: Two infants with congenital CMV infection showed striking pathological changes in the inner ear. There was a hypervascularization of the stria vascularis in the cochlea of the first infant, but no obvious loss of outer and inner hair cells was seen in the organ of Corti. However, cytomegalic cells and a loss of outer hair cells were found in the cochlea of the second infant. The vestibular organs of both infants showed cytomegalic cells, mostly located on dark cells. There was a loss of type I and type II hair cells in the macula of the saccule and utricle. Loss of hair cells and degeneration of nerve fibers was also seen in the semicircular canals. Both infants with congenital infection showed abundant inflammatory cells and fibrous structures in the middle ear cavity. No evidence of cytomegalic cells and hair cell loss was found in the cochlea or vestibular labyrinth in acquired CMV infection. CONCLUSIONS: In two infants with congenital CMV infection, the cochlea, vestibule, and middle ear were highly affected. Temporal bones of adult donors with acquired viral infection showed histological findings similar to donors of the same age without ear disease.

Limits and patterns of cytomegalovirus genomic diversity in humans.

Human cytomegalovirus (HCMV) exhibits surprisingly high genomic diversity during natural infection although little is known about the limits or patterns of HCMV diversity among humans. To address this deficiency, we analyzed genomic diversity among congenitally infected infants. We show that there is an upper limit to HCMV genomic diversity in these patient samples, with ∼ 25% of the genome being devoid of polymorphisms. These low diversity regions were distributed across 26 loci that were preferentially located in DNA-processing genes. Furthermore, by developing, to our knowledge, the first genome-wide mutation and recombination rate maps for HCMV, we show that genomic diversity is positively correlated with these two rates. In contrast, median levels of viral genomic diversity did not vary between putatively single or mixed strain infections. We also provide evidence that HCMV populations isolated from vascular compartments of hosts from different continents are genetically similar and that polymorphisms in glycoproteins and regulatory proteins are enriched in these viral populations. This analysis provides the most highly detailed map of HCMV genomic diversity in human hosts to date and informs our understanding of the distribution of HCMV genomic diversity within human hosts.

Targeted Mutagenesis of Guinea Pig Cytomegalovirus Using CRISPR/Cas9-Mediated Gene Editing.

UNLABELLED: The cytomegaloviruses (CMVs) are among the most genetically complex mammalian viruses, with viral genomes that often exceed 230 kbp. Manipulation of cytomegalovirus genomes is largely performed using infectious bacterial artificial chromosomes (BACs), which necessitates the maintenance of the viral genome in Escherichia coli and successful reconstitution of virus from permissive cells after transfection of the BAC. Here we describe an alternative strategy for the mutagenesis of guinea pig cytomegalovirus that utilizes clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated genome editing to introduce targeted mutations to the viral genome. Transient transfection and drug selection were used to restrict lytic replication of guinea pig cytomegalovirus to cells that express Cas9 and virus-specific guide RNA. The result was highly efficient editing of the viral genome that introduced targeted insertion or deletion mutations to nonessential viral genes. Cotransfection of multiple virus-specific guide RNAs or a homology repair template was used for targeted, markerless deletions of viral sequence or to introduce exogenous sequence by homology-driven repair. As CRISPR/Cas9 mutagenesis occurs directly in infected cells, this methodology avoids selective pressures that may occur during propagation of the viral genome in bacteria and may facilitate genetic manipulation of low-passage or clinical CMV isolates. IMPORTANCE: The cytomegalovirus genome is complex, and viral adaptations to cell culture have complicated the study of infection in vivo Recombineering of viral bacterial artificial chromosomes enabled the study of recombinant cytomegaloviruses. Here we report the development of an alternative approach using CRISPR/Cas9-based mutagenesis in guinea pig cytomegalovirus, a small-animal model of congenital cytomegalovirus disease. CRISPR/Cas9 mutagenesis can introduce the same types of mutations to the viral genome as bacterial artificial chromosome recombineering but does so directly in virus-infected cells. CRISPR/Cas9 mutagenesis is not dependent on a bacterial intermediate, and defined viral mutants can be recovered after a limited number of viral genome replications, minimizing the risk of spontaneous mutation.

Repair of a Mutation Disrupting the Guinea Pig Cytomegalovirus Pentameric Complex Acquired during Fibroblast Passage Restores Pathogenesis in Immune-Suppressed Guinea Pigs and in the Context of Congenital Infection.

UNLABELLED: Guinea pig cytomegalovirus (GPCMV) provides a valuable model for congenital cytomegalovirus transmission. Salivary gland (SG)-passaged stocks of GPCMV are pathogenic, while tissue culture (TC) passage in fibroblasts results in attenuation. Nonpathogenic TC-derived virus N13R10 (cloned as a bacterial artificial chromosome [BAC]) has a 4-bp deletion that disrupts GP129, which encodes a subunit of the GPCMV pentameric complex (PC) believed to govern viral entry into select cell types, and GP130, an overlapping open reading frame (ORF) of unknown function. To determine if this deletion contributes to attenuation of N13R10, markerless gene transfer in Escherichia coli was used to construct virus r129, a variant of N13R10 in which the 4-bp deletion is repaired. Virions from r129 were found to contain GP129 as well as two other PC subunit proteins, GP131 and GP133, whereas these three PC subunits were absent from N13R10 virions. Replication of r129 in fibroblasts appeared unaltered compared to that of N13R10. However, following experimental challenge of immunocompromised guinea pigs, r129 induced significant weight loss, longer duration of viremia, and dramatically higher (up to 1.5 × 10(6)-fold) viral loads in blood and end organs compared to N13R10. In pregnant guinea pigs, challenge with doses of r129 virus of ≥5 × 10(6) PFU resulted in levels of maternal viremia, congenital transmission, pup viral loads, intrauterine growth restriction, and pup mortality comparable to that induced by pathogenic SG virus, although higher doses of r129 were required. These results suggest that the GP129-GP130 mutation is a significant contributor to attenuation of N13R10, likely by abrogating expression of a functional PC. IMPORTANCE: Tissue culture adaptation of cytomegaloviruses rapidly selects for mutations, deletions, and rearrangements in the genome, particularly for viruses passaged in fibroblast cells. Some of these mutations are focused in the region of the genome encoding components of the pentameric complex (PC), in particular homologs of human cytomegalovirus (HCMV) proteins UL128, UL130, and UL131A. These mutations can attenuate the course of infection when the virus is reintroduced into animals for vaccine and pathogenesis studies. This study demonstrates that a deletion that arose during the process of tissue culture passage can be repaired, with subsequent restoration of pathogenicity, using BAC-based mutagenesis. Restoration of pathogenicity by repair of a frameshift mutation in GPCMV gene GP129 using this approach provides a valuable genetic platform for future studies using the guinea pig model of congenital CMV infection.