Human cytomegalovirus infection impairs neural differentiation via repressing sterol regulatory element binding protein 2-mediated cholesterol biosynthesis.

Congenital human cytomegalovirus (HCMV) infection is a major cause of abnormalities and disorders in the central nervous system (CNS) and/or the peripheral nervous system (PNS). However, the complete pathogenesis of neural differentiation disorders caused by HCMV infection remains to be fully elucidated. Stem cells from human exfoliated deciduous teeth (SHEDs) are mesenchymal stem cells (MSCs) with a high proliferation and neurogenic differentiation capacity. Since SHEDs originate from the neural crest of the early embryonic ectoderm, SHEDs were hypothesized to serve as a promising cell line for investigating the pathogenesis of neural differentiation disorders in the PNS caused by congenital HCMV infection. In this work, SHEDs were demonstrated to be fully permissive to HCMV infection and the virus was able to complete its life cycle in SHEDs. Under neurogenic inductive conditions, HCMV infection of SHEDs caused an abnormal neural morphology. The expression of stem/neural cell markers was also disturbed by HCMV infection. The impairment of neural differentiation was mainly due to a reduction of intracellular cholesterol levels caused by HCMV infection. Sterol regulatory element binding protein-2 (SREBP2) is a critical transcription regulator that guides cholesterol synthesis. HCMV infection was shown to hinder the migration of SREBP2 into nucleus and resulted in perinuclear aggregations of SREBP2 during neural differentiation. Our findings provide new insights into the prevention and treatment of nervous system diseases caused by congenital HCMV infection.

Human Cytomegalovirus mRNA-1647 Vaccine Candidate Elicits Potent and Broad Neutralization and Higher Antibody-Dependent Cellular Cytotoxicity Responses Than the gB/MF59 Vaccine.

BACKGROUND: MF59-adjuvanted gB subunit (gB/MF59) vaccine demonstrated approximately 50% efficacy against human cytomegalovirus (HCMV) acquisition in multiple clinical trials, suggesting that efforts to improve this vaccine design might yield a vaccine suitable for licensure. METHODS: A messenger RNA (mRNA)-based vaccine candidate encoding HCMV gB and pentameric complex (PC), mRNA-1647, is currently in late-stage efficacy trials. However, its immunogenicity has not been compared to the partially effective gB/MF59 vaccine. We assessed neutralizing and Fc-mediated immunoglobulin G (IgG) effector antibody responses induced by mRNA-1647 in both HCMV-seropositive and -seronegative vaccinees from a first-in-human clinical trial through 1 year following third vaccination using a systems serology approach. Furthermore, we compared peak anti-gB antibody responses in seronegative mRNA-1647 vaccinees to that of seronegative gB/MF59 vaccine recipients. RESULTS: mRNA-1647 vaccination elicited and boosted HCMV-specific IgG responses in seronegative and seropositive vaccinees, respectively, including neutralizing and Fc-mediated effector antibody responses. gB-specific IgG responses were lower than PC-specific IgG responses. gB-specific IgG and antibody-dependent cellular phagocytosis responses were lower than those elicited by gB/MF59. However, mRNA-1647 elicited higher neutralization and antibody-dependent cellular cytotoxicity (ADCC) responses. CONCLUSIONS: Overall, mRNA-1647 vaccination induced polyfunctional and durable HCMV-specific antibody responses, with lower gB-specific IgG responses but higher neutralization and ADCC responses compared to the gB/MF59 vaccine. CLINICAL TRIALS REGISTRATION: NCT03382405 (mRNA-1647) and NCT00133497 (gB/MF59).

The role of viruses in oral mucosal lesions.

The mucosa of the oral cavity is exposed to a large number of different microorganisms such as archaea, bacteria, fungi, parasites, and viruses. Among those, viruses cause specific infections, which can easily be transmitted from one person to another. The infectious route may not only include patients and their relatives but also the dental professional team. Thus, a wide knowledge regarding specific viral infections is crucial for the daily routine. Signs and symptoms of oral viral infections can be completely absent or develop into a pronounced clinical picture, so that early detection and information determine the further course of the infection and its influence on other inflammatory diseases, such as periodontitis, as well as the safety of family members and the social environment. As the clinical manifestation of viral infections may be highly variable leading to heterogenous mucosal lesions it is, in most cases, mandatory to differentiate them by specific microbiological tests in addition to clinical examination procedures. This article will give an overview of the role of viruses infecting the oral mucosa, and in addition, describe their clinical manifestation and management.

Multicenter Evaluation of the DiaSorin Molecular Simplexa Congenital CMV Direct PCR Test on Neonatal Saliva and Urine Specimens.

Cytomegalovirus (CMV) is the most common virus associated with congenital infection worldwide and is a major cause of sensorineural hearing loss (SNHL) and developmental delay. Up to 90% of infants with congenital CMV (cCMV) infection are asymptomatic at birth, making the diagnosis challenging. Postnatal diagnosis involves testing newborn saliva and/or urine collected before 21 days of life to confirm cCMV infection. This multicenter study evaluated the performance of the Simplexa Congenital CMV Direct real-time PCR assay for the qualitative detection of CMV in newborn saliva (n = 2,023) and urine (n = 1,797) specimens. Compared to two PCR/bidirectional sequencing assays, the Simplexa Congenital CMV Direct assay demonstrated positive percent agreement (PPA) and negative percent agreement (NPA) of 98.6% and 99.9%, respectively, for saliva samples and a PPA of 97.8% and an NPA of 99.9% for urine specimens. Overall concordance was κ = 0.98 or near perfect compared to the composite reference methods with both sample types. By 95% probit analysis, the limit of detection (LoD) using the AD-169 reference strain was 350 ± 12 copies/mL in urine. The LoDs of saliva swabs in either 1 mL or 3 mL of transport medium were 274 ± 12 copies/mL and 300 ± 14 copies/mL, respectively. The Simplexa Congenital CMV Direct assay can be applied to both saliva and urine specimens collected from newborns less than 21 days of age to rapidly and reliably identify CMV infection.

Performance evaluation of fully automated cobas® 6800 CMV PCR for the detection and quantification of cytomegalovirus DNA in neonatal urine and saliva, and adult urine, saliva, and vaginal secretion.

Laboratory testing for cytomegalovirus (CMV) in bodily fluids is essential to manage congenital and prenatal CMV infection. The rapid and fully automated cobas® CMV PCR is approved only for the testing of plasma in transplant patients. To evaluate the performance of the cobas® CMV to detect and quantify CMV DNA in neonatal and adult female urine, saliva, and vaginal secretion, the limit of detection (LoD), limit of quantification (LoQ), imprecision, linearity, PCR efficiency, bias, analytical specificity, cross-reactivity, and cross-contamination of the cobas® CMV for urine, saliva, and vaginal secretion was determined. The performance of the assay was evaluated prospectively with two laboratory-developed PCR assays using neonatal and adult urine, saliva swabs, and vaginal swabs. The LoD and LoQ were 31 and 100 IU/mL, respectively, for urine, and 81 and 100 IU/mL, respectively, for vaginal secretion. The LoD and LoQ for saliva were the same (200 IU/mL). The cobas® CMV was precise (coefficient of variation ≤10%), linear (R2 ≥ 0.995), and efficient (1.07 and 1.09) between 100 and 250,000 IU/mL for the sample types. The bias and analytical specificity was <±0.30 log10 IU/mL and 100%, respectively. Cross-reactivity with non-CMV pathogens was not detected. Cross-contamination rate was 0.28%. The diagnostic accuracy, sensitivity, and specificity of the cobas® CMV for neonatal urine and saliva were ≥95.0%, ≥93.3%, and ≥90.4%, respectively. The overall percent agreement for adult urine, saliva, and vaginal secretion was 86.6%, 94.5%, and 89.4%, respectively. Taken together, the cobas® CMV demonstrated acceptable analytical and diagnostic performance, and is suitable for routine diagnostic laboratory investigation of CMV infection in neonates and adults.

Detecting human cytomegalovirus in urine, vagina and saliva: Impact of biological fluids and storage durations and temperatures on CMV DNA recovery.

Sample collection, transport and storage conditions vary in the human cytomegalovirus (CMV) shedding literature. Currently, limited data exist on the impact of biological fluids and pre-analytical sample handling on the detection of CMV DNA. To evaluate CMV DNA recovery from urine, vaginal fluid and saliva stored in different conditions, adult urine, vaginal and saliva fluids and swabs, stored with or without selected nucleic acid preservation media at various durations and temperatures, was compared by polymerase chain reaction (PCR) quantitation of spiked samples and self-collected urine (n = 45) and vaginal swabs (n = 58) from CMV seropositive pregnant women. There was a time-dependent reduction in CMV DNA recovery from urine, urine diluted in phosphate-buffered saline, and saliva stored at 2-8°C, but not from urine preserved in cobas® PCR transport media (CPM) (urine/CPM). For vaginal fluid, a reduction in recovery was evident after 7 days storage at 2-8°C. CMV DNA recovery over 91 days was similar between -80°C and -20°C storage for urine and vaginal swabs preserved in CPM, and saliva swabs preserved in eNAT® PCR transport media. A statistically significant change in CMV DNA recovery after 25 months storage (median) at -80°C was not observed for self-collected urine/CPM and vaginal swab/CPM from pregnant women. Taken together, recovery of CMV DNA is dependent on fluid type and storage conditions. To improve the validity and reliability of detection at different storage durations and temperatures, the use of nucleic acid preserving transport media at the point of collection for urine, vaginal fluid and saliva may be essential.

Optimizing congenital cytomegalovirus detection by pool testing in saliva by a rapid molecular test.

Universal congenital cytomegalovirus (cCMV) screening in saliva is increasingly recommended. The aim of our study was to correlate the performance of a point-of-care rapid molecular test with CMV real time PCR (CMV RT-PCR) detection, using saliva pool-testing in newborns under a universal screening strategy. Saliva swabs were prospectively collected from newborns < 21 days old and tested by Alethia-LAMP-CMV assay in pools of 5 samples. In positive pools, subjects were tested individually and by saliva and urine CMV RT-PCR. A subset of negative pools were studied with both techniques and viral loads in whole blood were determined in positive patients. From 1,642 newborns included in 328 pools, 8 were confirmed by urine CMV RT-PCR, (cCMV prevalence 0,49%). The PPA and NNA of the pooled saliva Alethia-LAMP-CMV testing were 87,5% and 99,8% with a negative and positive predictive value of 99,9% and 77,7%, respectively. Two false positives were detected (0,12%). A subset of 17 negative pools (85 samples), studied by saliva CMV RT-PCR, showed 100% concordance.  Conclusion: CMV pool-testing using a rapid molecular test in saliva proved feasible when compared to PCR gold standards. This strategy could improve cost-effectiveness for cCMV universal neonatal screening, based on the low prevalence of the infection and could be a more affordable approach in less developed regions with reduced detection capacity. What is Known: • cCMV is the most frequent congenital infection and a leading nongenetic cause of sensorineural hearing loss and brain disease. • Universal screening could allow early detection of congenitally infected infants, improving clinical outcome. • Saliva PCR is the preferred and non-invasive test for newborn cCMV screening. What is New: • The feasibility of a universal cCMV screening by pool-testing in saliva using a rapid test in pools of 5 samples. • PPA and NPA were 87,5 and 99,8% compared to CMV PCR in urine. • This strategy could be relevant specially in LMIC where detection capacity is reduced and could improve cost-effectiveness. • cCMV prevalence in our center was 0,49%.

Association of Epstein-Barr virus, cytomegalovirus and lipocalin with periodontitis in type 2 diabetic subjects.

OBJECTIVE: The current study aims to evaluate and compare the lipocalin, adiponectin and periodontal viruses in the generalized periodontitis patients with and without diabetes mellitus. MATERIALS AND METHODS: Seventy subjects were grouped into 35 systemically healthy (GP) and 35 patients with diabetes mellitus (GP+DM). The periodontal parameters, demographic and diabetic variables were evaluated in both the groups. The subgingival tissue samples were procured from the diseased sites and were analysed for the detection of EBV, CMV, HSV and protein markers by real-time polymerase chain reaction (RT-PCR) and lipocalin and adiponectin were identified by enzyme-linked immunosorbent assay (ELISA). RESULTS: The demographic variables such as age and BMI did not differ between the groups. PI and CAL were found to be significantly higher in GP+DM (p < 0.05). EBV (82.9%), CMV (71.4%) and protein marker: lipocalin were also found to be statistically highly significant in GP+DM and adiponectin was found to be higher in GP group and reduced in GP+DM group (p < 0.0001). CONCLUSION: The increased prevalence of EBV and CMV and lipocalin with reduced levels of adiponectin in patients with diabetes and periodontitis which may show aggravation of the diabetic status of the periodontitis patients thereby reinforcing a strong Periodontitis-DM continuum.

Universal newborn screening for congenital cytomegalovirus infection: feasibility and relevance in a French type-III maternity cohort.

OBJECTIVE: Evaluation of relevance and feasibility of universal newborn congenital cytomegalovirus infection (cCMVI) screening in saliva. DESIGN: Retrospective, population-based cohort study. SETTING: Clamart, France, 2016-2020. POPULATION: All neonates born consecutively in our level III maternity unit. METHODS: CMV PCR in saliva for all neonates at birth, and, if positive, CMV PCR in urine to confirm or exclude cCMVI. Prospective and retrospective characterisation of maternal infections. ROC curve analysis to assess saliva PCR performances. Acceptability of screening among staff members evaluated by a survey. MAIN OUTCOME MEASURES: Number of cCMVI neonates; number of expected and unexpected cCMVI. RESULTS: Among 15 341 tested neonates, 63 had cCMVI (birth prevalence of 0.4%, 95% CI 0.3-0.5). In 50% of cases, maternal infection was a non-primary infection (NPI) during pregnancy. cCMVI was expected or suspected (maternal primary infection [PI], antenatal or neonatal signs) in 24/63 neonates (38%), and unexpected in 39/63 neonates (62%). The best CMV saliva threshold to predict cCMVI was 356 (2.55 log) copies/ml [95% CI 2.52 log-3.18 log], with an area under the ROC curve of 0.97. Over 90% of the 72 surveyed staff members reported that the screening was easy and quick. No parent refused the screening. CONCLUSIONS: Universal screening for cCMVI with CMV PCR on saliva samples is feasible and highly acceptable to parents and healthcare providers. Over half (62%) of the cases had no prenatal/neonatal signs of cCMVI or a maternal history of CMV infection during pregnancy and would probably not have been diagnosed without universal screening. TWEETABLE ABSTRACT: In 62% of congenital cytomegalovirus infection cases, only universal neonatal screening in saliva can detect infection.

Artificial stem cells mediated inflammation-tropic delivery of antiviral drugs for pneumonia treatment.

BACKGROUND: Cytomegalovirus (CMV) pneumonia is a major cause of morbidity and mortality in immunodeficiency individuals, including transplant recipients and Acquired Immune Deficiency Syndrome patients. Antiviral drugs ganciclovir (GCV) and phosphonoformate (PFA) are first-line agents for pneumonia caused by herpesvirus infection. However, the therapy suffers from various limitations such as low efficiency, drug resistance, toxicity, and lack of specificity. METHODS: The antiviral drugs GCV and PFA were loaded into the pH-responsive nanoparticles fabricated by poly(lactic-co-glycolic acid) (PLGA) and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), and further coated with cell membranes derived from bone marrow mesenchymal stem cells to form artificial stem cells, namely MPDGP. We evaluated the viral suppression effects of MPDGP in vitro and in vivo. RESULTS: MPDGP showed significant inflammation tropism and efficient suppression of viral replication and virus infection-associated inflammation in the CMV-induced pneumonia model. The synergistic effects of the combination of viral DNA elongation inhibitor GCV and viral DNA polymerase inhibitor PFA on suppressing the inflammation efficiently. CONCLUSION: The present study develops a novel therapeutic intervention using artificial stem cells to deliver antiviral drugs at inflammatory sites, which shows great potential for the targeted treatment of pneumonia. To our best knowledge, we are the first to fabricate this kind of artificial stem cell to deliver antiviral drugs for pneumonia treatment.

Herpesviruses in Periodontitis: An Umbrella Review.

BACKGROUND: Despite numerous studies indicating a high prevalence of herpesviruses in both apical and marginal periodontitis samples, their exact role in the pathogenesis of a periodontal disease is still unclear. OBJECTIVE: This umbrella review aimed to summarize data on herpesviruses detection in marginal periodontitis (MP) and apical periodontitis of endodontic origin (APEO) samples. METHODS: The study protocol has been drafted a priori and registered to the International Prospective Register of Systematic Reviews (PROSPERO) (CRD42020215922). The literature search was conducted using the following electronic databases: Clarivate Analytics' Web of Science, Scopus, PubMed and Cochrane Database of Systematic Reviews, from inception to October 2020, with no language restrictions. Systematic reviews with or without meta-analysis that evaluated the association between the occurrence of herpesviruses and different forms of periodontal diseases were included. Other types of studies, including narrative reviews, were excluded. Two reviewers independently performed a literature search, data extraction, and quality assessment of included studies. Any disagreements or doubts were resolved by a third reviewer. The quality of the reviews was assessed using the AMSTAR 2 tool (A measurement tool to assess systematic reviews). RESULTS: Six systematic reviews were included in the current review. One was graded as high quality, another one was graded as moderate quality, whereas the other four were graded as critically low-quality reviews. The presence of herpesviruses in subgingival samples was associated with an increased risk of MP, supported by the corresponding meta-analyses. Although the association was strong (OR > 3.0), the confidence intervals were wide, heterogeneity was significant, and studies were of small sample size. In addition, publication bias was detected. Contrary, data from systematic reviews that assessed APEO and herpesviruses did not show any significant associations. CONCLUSIONS: Low-quality studies with high uncertainty suggest a strong association between herpesviruses and MP, but not with APEO.

Evaluation of the effects of 0.05% sodium hypochlorite and 0.12% chlorhexidine gluconate twice daily rinse on periodontal parameters and gingival crevicular fluid HSV1 and CMV levels in patients with chronic periodontitis: a multicentric study.

Background: Mechanical debridement of periodontal pockets remains the mainstay of therapy in all forms of periodontitis. There is 47% greater reduction in plaque amount when sodium hypochlorite (NaOCl) is used as an adjunct when compared with water rinsing. The aim of this study was to evaluate the effects of 0.05% NaOCl and 0.12% chlorhexidine gluconate twice daily rinse on periodontal parameters and gingival crevicular fluid (GCF) HSV1 and CMV levels in chronic periodontitis. Methods: Patients assigned to group A were prescribed 0.05% NaOCl mouthwash for twice daily rinse. Patients in group B were prescribed 0.12% chlorhexidine gluconate mouthwash to be used twice daily. Evaluation of periodontal parameters was done at baseline and after six months following therapy. GCF HSV1 and CMV levels were evaluated using a polymerase chain reaction. Results: A statistically significant difference was noted in the improvement in periodontal parameters between both groups, when evaluated six months following therapy with greater reduction in group A vis-a-vis group B. Conclusion: NaOCl when prescribed as a twice daily mouthwash can be recommended as a part of the home care regime in patients with chronic periodontitis. It is more cost-effective, easily available and can be beneficial to the troops in difficult terrains and extremes of climates, where oral healthcare facilities are not easily accessible.

IgA binds to the AD-2 epitope of glycoprotein B and neutralizes human cytomegalovirus.

Human cytomegalovirus (HCMV) is a ubiquitous pathogen that is potentially pathogenic in immunosuppressed individuals and pregnant females during primary infection. The HCMV envelope glycoprotein B (gB) facilitates viral entry into all cell types and induces a potent immune response. AD-2 epitope is a highly conserved linear neutralizing epitope of gB and a critical target for antibodies; however, only 50% of sero-positive individuals make IgG antibodies to this site and IgA responses have not been fully investigated. This study aimed to compare IgG and IgA responses against gB and the AD-2 epitope in naturally exposed individuals and those receiving a recombinant gB/MF59 adjuvant vaccine. Thus, vaccination of sero-positive individuals improved pre-existing gB-specific IgA and IgG levels and induced de novo gB-specific IgA and IgG responses in sero-negative recipients. Pre-existing AD-2 IgG and IgA responses were boosted with vaccination, but de novo AD-2 responses were not detected. Naturally exposed individuals had dominant IgG responses towards gB and AD-2 compared with weaker and variable IgA responses, although a significant IgA binding response to AD-2 was observed within human breastmilk samples. All antibodies binding AD-2 contained kappa light chains, whereas balanced kappa/lambda light chain usage was found for those binding to gB. V region-matched AD-2-specific recombinant IgG and IgA bound both to gB and to AD-2 and neutralized HCMV infection in vitro. Overall, these results indicate that although human IgG responses dominate, IgA class antibodies against AD-2 are a significant component of human milk, which may function to protect neonates from HCMV.

Human Cytomegalovirus Glycoprotein B Nucleoside-Modified mRNA Vaccine Elicits Antibody Responses with Greater Durability and Breadth than MF59-Adjuvanted gB Protein Immunization.

A vaccine to prevent maternal acquisition of human cytomegalovirus (HCMV) during pregnancy is a primary strategy to reduce the incidence of congenital disease. The MF59-adjuvanted glycoprotein B (gB) protein subunit vaccine (gB/MF59) is the most efficacious vaccine tested to date for this indication. We previously identified that gB/MF59 vaccination elicited poor neutralizing antibody responses and an immunodominant response against gB antigenic domain 3 (AD-3). Thus, we sought to test novel gB vaccines to improve functional antibody responses and reduce AD-3 immunodominance. Groups of juvenile New Zealand White rabbits were administered 3 sequential doses of the full-length gB protein with an MF59-like squalene-based adjuvant, the gB ectodomain protein (lacking AD-3) with squalene adjuvant, or lipid nanoparticle (LNP)-encapsulated nucleoside-modified mRNA encoding full-length gB. All vaccines were highly immunogenic with similar kinetics and comparable peak gB-binding and functional antibody responses. The AD-3-immunodominant IgG response following human gB/MF59 vaccination was closely mimicked in rabbits. Though gB ectodomain subunit vaccination eliminated targeting of epitopes in AD-3, it did not improve vaccine-elicited neutralizing or nonneutralizing antibody functions. gB nucleoside-modified mRNA-LNP-immunized rabbits exhibited an enhanced durability of vaccine-elicited antibody responses. Furthermore, the gB mRNA-LNP vaccine enhanced the breadth of IgG binding responses against discrete gB peptides. Finally, low-magnitude gB-specific T cell activity was observed in the full-length gB protein and mRNA-LNP groups, though not in ectodomain-vaccinated rabbits. Altogether, these data suggest that the use of gB nucleoside-modified mRNA-LNP vaccines is a viable strategy for improving on the partial efficacy of gB/MF59 vaccination and should be further evaluated in preclinical models.IMPORTANCE Human cytomegalovirus (HCMV) is the most common infectious cause of infant birth defects, resulting in permanent neurological disability for one newborn child every hour in the United States. After more than a half century of research and development, we remain without a clinically licensed vaccine or immunotherapeutic to reduce the burden of HCMV-associated disease. In this study, we sought to improve upon the glycoprotein B protein vaccine (gB/MF59), the most efficacious HCMV vaccine evaluated in a clinical trial, via targeted modifications to either the protein structure or vaccine formulation. Utilization of a novel vaccine platform, nucleoside-modified mRNA formulated in lipid nanoparticles, increased the durability and breadth of vaccine-elicited antibody responses. We propose that an mRNA-based gB vaccine may ultimately prove more efficacious than the gB/MF59 vaccine and should be further evaluated for its ability to elicit antiviral immune factors that can prevent HCMV-associated disease.

Epidemiology of cytomegalovirus Infection among mothers and infants in Colombia.

We assessed maternal and infant cytomegalovirus (CMV) infection in Colombia. Maternal serum was tested for CMV immunoglobulin G antibodies at a median of 10 (interquartile range: 8-12) weeks gestation (n = 1501). CMV DNA polymerase chain reaction was performed on infant urine to diagnose congenital (≤21 days of life) and postnatal (>21 days) infection. Maternal CMV seroprevalence was 98.1% (95% confidence interval [CI]: 97.5%-98.8%). Congenital CMV prevalence was 8.4 (95% CI: 3.9%-18.3%; 6/711) per 1000 live births. Among 472 infants without confirmed congenital CMV infection subsequently tested at age 6 months, 258 (54.7%, 95% CI: 50.2%-59.1%) had postnatal infection.

The human oral virome: Shedding light on the dark matter.

Mediators of the initiation, development, and recurrence of periodontitis include the oral microbiome embedded in subgingival plaque and the host immune response to a dysbiosis within this dynamic and complex microbial community. Although mediators have been studied extensively, researchers in the field have been unable to fully ascribe certain clinical presentations of periodontitis to their nature. Emergence of high-throughput sequencing technologies has resulted in better characterization of the microbial oral dysbiosis that extends beyond the extensively studied putative bacterial periodontopathogens to a shift in the oral virome composition during disease conditions. Although the biological dark matter inserted by retroviruses was once believed to be nonfunctional, research has revealed that it encodes historical viral-eukaryotic interactions and influences host development. The objective of this review is to evaluate the proposed association of herpesviruses to the etiology and pathogenesis of periodontal disease and survey the highly abundant prokaryotic viruses to delineate their potential roles in biofilm dynamics, as well as their interactions with putative bacterial periodontopathogens and eukaryotic cells. The findings suggest that potential novel periodontal therapies targeting or utilizing the oral virome can alleviate certain clinical presentations of periodontitis. Perhaps it is time to embrace the viral dark matter within the periodontal environment to fully comprehend the pathogenesis and systemic implications of periodontitis.

Intraoral human herpes viruses detectable by PCR in majority of patients.

OBJECTIVES: To identify factors which influence the intraoral prevalence of human herpes viruses (HHVs) using mucosal swabs, saliva samples and qPCR analysis. METHODOLOGY: In this cross-sectional observational study, matched saliva and oral swabs were collected from a total of 115 subjects: 70 immunocompetent subjects with no mucosal abnormalities, 22 with mucosal abnormalities and 23 therapeutically immunocompromised individuals. Extracted DNA was analysed by multiplex qPCR for detection and quantification of HHVs 1-6. RESULTS: At least one human herpes virus was detected in 77.1% of immunocompetent individuals with no mucosal abnormalities, with EBV the most commonly detected at 61.4%. HHV-6 was detected in 17.1%, HSV-1 in 4.3% and CMV in 1.1%. Detection was higher in saliva than in oral swabs. There was no detection of HSV-2 or VZV. Neither presence of oral mucosal abnormality nor therapeutic immunocompromise was related to increased detection of human herpes virus. CONCLUSION: Commensal detection rates of EBV are high, and caution in clinical correlation of positive detection is warranted. Commensal CMV rates are low, and detection is likely to be clinically relevant. This study presents a comprehensive commensal detection rate of HHVs 1-6 by qPCR in saliva and swabs.

HCMV glycoprotein B subunit vaccine efficacy mediated by nonneutralizing antibody effector functions.

Human cytomegalovirus (HCMV) is the most common congenital infection worldwide, frequently causing hearing loss and brain damage in afflicted infants. A vaccine to prevent maternal acquisition of HCMV during pregnancy is necessary to reduce the incidence of infant disease. The glycoprotein B (gB) + MF59 adjuvant subunit vaccine platform is the most successful HCMV vaccine tested to date, demonstrating ∼50% efficacy in preventing HCMV acquisition in multiple phase 2 trials. However, the mechanism of vaccine protection remains unknown. Plasma from 33 postpartum women gB/MF59 vaccinees at peak immunogenicity was tested for gB epitope specificity as well as neutralizing and nonneutralizing anti-HCMV effector functions and compared with an HCMV-seropositive cohort. gB/MF59 vaccination elicited IgG responses with gB-binding magnitude and avidity comparable to natural infection. Additionally, IgG subclass distribution was similar with predominant IgG1 and IgG3 responses induced by gB vaccination and HCMV infection. However, vaccine-elicited antibodies exhibited limited neutralization of the autologous virus, negligible neutralization of multiple heterologous strains, and limited binding responses against gB structural motifs targeted by neutralizing antibodies including AD-1, AD-2, and domain I. Vaccinees had high-magnitude IgG responses against AD-3 linear epitopes, demonstrating immunodominance against this nonneutralizing, cytosolic region. Finally, vaccine-elicited IgG robustly bound membrane-associated gB on the surface of transfected or HCMV-infected cells and mediated virion phagocytosis, although were poor mediators of NK cell activation. Altogether, these data suggest that nonneutralizing antibody functions, including virion phagocytosis, likely played a role in the observed 50% vaccine-mediated protection against HCMV acquisition.

Cytomegalovirus Genetic Diversity Following Primary Infection.

BACKGROUND: Infection with multiple cytomegalovirus (CMV) strains (mixed infection) was reported in a variety of hosts. As the virus genetic diversity in primary CMV infection and the changes over time remain incompletely defined, we examined CMV diversity and changes in diversity over time in healthy adolescent females who participated in a phase 2 CMV gB/MF59 vaccine trial. METHODS: CMV genetic diversity was determined by genotyping of 5 genes-gB (UL55), gH (UL75), gN (UL73), US28, and UL144-in urine, saliva, and plasma samples from 15 study subjects. RESULTS: At the time of primary infection, 5 of 12 (42%) urine samples had multiple virus strains, and 50% of vaccine recipients were infected with gB1 genotype (vaccine strain). Mixed infection was documented in all 15 subjects within 3 months after primary infection, and the majority had different CMV genotypes in different compartments. Changes in genotypes over time were observed in all subjects. CONCLUSIONS: Infection with multiple CMV genotypes was common during primary infection and further diversification occurred over time. Infection with gB1 genotype in vaccine recipients suggests a lack of strain-specific protection from the vaccine. As only 5 polymorphic genes were assessed, this study likely underestimated the true genetic diversity in primary CMV infection.

Performance of the Alethia CMV Assay for Detection of Cytomegalovirus by Use of Neonatal Saliva Swabs.

Congenital cytomegalovirus (cCMV) infection is a major cause of childhood hearing loss and neurodevelopmental delay. Identification of newborns with cCMV infection allows provision of beneficial interventions. However, most infants with cCMV infection have subclinical infection and go undiagnosed. Thus, expanded neonatal CMV testing is increasingly recommended. Saliva is an attractive sample type for CMV testing of newborns, because it is easier to collect than urine and more sensitive for CMV detection than dried blood spots. We evaluated the Alethia CMV assay, a rapid, easy-to-use loop-mediated isothermal amplification method for qualitative detection of CMV DNA in neonatal saliva samples. Saliva swabs were collected prospectively from newborns <21 days old and tested by the Alethia assay according to the manufacturer's instructions. Archived saliva swabs from newborns with cCMV infection were also tested retrospectively. A composite reference method (CRM; two validated PCR assays followed by bidirectional sequencing of amplicons) was performed on all samples as the reference standard comparator. Of 1,480 prospectively collected saliva swabs, 1,472 (99.5%) were negative by both the Alethia assay and CRM, 5 (0.34%) were positive by both the Alethia assay and CRM, and 3 (0.20%) were positive only by the Alethia assay. All 34 (100%) archived swabs from newborns with cCMV infection were positive by both the CRM and the Alethia assay. Overall, the Alethia assay showed 100% and 99.8% positive and negative agreement with the CRM, respectively. The Alethia CMV assay is an accurate method for identifying neonates with cCMV infection and, given its simplicity, appears suitable for CMV testing using neonatal saliva outside a reference laboratory, including remote and resource-limited settings.