A novel digital PCR assay for detection and comprehensive characterization of Molluscum contagiosum virus genotypes MOCV1, MOCV2, and MOCV3 and recombinant lineages.

Molluscum contagiosum virus (MOCV) is an important human pathogen causing a high disease burden worldwide. It is the last exclusively human-infecting poxvirus still circulating in its natural reservoir-a valuable model of poxviral evolution. Unfortunately, MOCV remains neglected, and little is known about its evolutionary history and circulating genomic variants, especially in non-privileged countries. The design weaknesses of available MOCV detection/genotyping assays surfaced with recent accumulation of abundant sequence information: all existing MOCV assays fail at accurate genotyping and capturing sub-genotype level diversity. Because complete MOCV genome characterization is an expensive and labor-intensive task, it makes sense to prioritize samples for whole-genome sequencing by diversity triage screening. To meet this demand, we developed a novel assay for accurate MOCV detection and genotyping, and comprehensive sub-genotype qualification to the level of phylogenetic groups (PGs). The assay included a novel set of oligonucleotide primers and probes, and it was implemented using digital polymerase chain reaction (dPCR). It offers sensitive, specific, and accurate detection, genotyping (MOCV1-MOCV3), and PG qualification (PG1-6) of MOCV DNA from clinical samples. The novel dPCR assay is suitable for MOCV diversity triage screening and prioritization of samples for complete MOCV genome characterization.

Differential diagnosis of exanthematous viruses during the 2022 Mpox outbreak in Minas Gerais, Brazil.

The monkeypox virus (MPXV) outbreak, primarily endemic to Africa, has spread globally, with Brazil reporting the second-highest number of cases. The emergence of MPXV in non-endemic areas has raised concerns, particularly due to the co-circulation of other exanthematous viruses such as varicella-zoster virus (VZV) and molluscum contagiosum virus (MOCV). To perform an accurate differential diagnosis of MPXV during the ongoing outbreak in Minas Gerais, Brazil, a 5PLEX qPCR assay targeting orthopoxviruses (OPV), VZV, and MOCV was used to retrospectively analyze all clinical samples that tested negative for MPXV in the initial screening conducted at Funed. In summary, our study analyzed 1,175 clinical samples received from patients suspected of MPXV infection and found a positivity rate of 33.8% (397 samples) for MPXV using the non-variola qPCR assay. Testing the 778 MPXV-negative clinical samples using the 5PLEX qPCR assay revealed that 174 clinical samples (22.36%) tested positive for VZV. MOCV DNA was detected in 13 and other OPV in 3 clinical samples. The sequencing of randomly selected amplified clinical samples confirmed the initial molecular diagnosis. Analysis of patient profiles revealed a significant difference in the median age between groups testing positive for MPXV and VZV and a male predominance in MPXV cases. The geographic distribution of positive cases was concentrated in the most populous mesoregions of Minas Gerais state. This study highlights the challenges posed by emerging infectious diseases. It emphasizes the importance of epidemiological surveillance and accurate diagnosis in enabling timely responses for public health policies and appropriate medical care. IMPORTANCE: Brazil ranks second in the number of cases during the global monkeypox epidemic. The study, conducted in Minas Gerais, the second most populous state in Brazil with over 20 million inhabitants, utilized differential diagnostics, revealing a significant number of positive cases for other exanthematous viruses and emphasizing the need for accurate diagnoses. During the study, we were able to assess the co-circulation of other viruses alongside monkeypox, including varicella-zoster virus, molluscum contagiosum virus, and other orthopoxviruses. The significance of the research is underscored by the concentration of positive cases in populous areas, highlighting the challenges posed by emerging infectious diseases. This demographic context further amplifies the importance of the research in guiding public health policies and medical interventions, given the substantial population at risk. The study not only addresses a global concern but also holds critical implications for a state with such a large population and geographic expanse within Brazil. Overall, the study emphasizes the pivotal role of surveillance and precise diagnosis in guiding effective public health responses and ensuring appropriate medical interventions.

Physiotherapy Management in the Case of Primary Peritoneal Serous Carcinoma With Lower Segment Cesarean Section: A Case Report.

This case report presents the medical path of a 24-year-old female patient, who had undergone lower (uterine) segment cesarean section (LSCS) while facing complications of having several diagnoses at the same time, including primary peritoneal serous cancer, sexually transmitted disease (STD), and IgM-positive dengue. The prevention and treatment of STDs require an integrated approach due to the persistent problems they provide in the global healthcare system. In India, there is a high birth rate, which makes LSCS a common treatment. The combination of dengue fever, STDs, cancer care, and such issues related to women's health emphasizes the necessity of specialized interventions to reduce the risk of problems both during and after pregnancy. A sophisticated, multidisciplinary approach to postoperative care is required due to the confluence of these disorders, with physiotherapy and rehabilitation serving as a crucial treatment approach. The patient received breathing exercises along with core strengthening exercises. For relaxation, Benson's relaxation technique was used. Significant improvement was seen in the patient's muscle strength and quality of life post rehabilitation.

The Antiviral Effect of Berdazimer Sodium on Molluscum Contagiosum Virus Using a Novel In Vitro Methodology.

Molluscum contagiosum (MC) is characterized by skin lesions containing the highly contagious molluscum contagiosum poxvirus (MCV). MCV primarily infects children, with one US Food and Drug Administration (FDA)-approved drug-device treatment in use but no approved medications. Assessing antivirals is hindered by the inability of MCV to replicate in vitro. Here, we use vaccinia virus as a surrogate to provide evidence of the anti-poxvirus properties of berdazimer sodium, a new chemical entity, and the active substance in berdazimer gel, 10.3%, a nitric oxide-releasing topical in phase 3 development for the treatment of MC. We show that berdazimer sodium reduced poxvirus replication and, through a novel methodology, demonstrate that cells infected with drug-treated MCV virions have reduced early gene expression. Specifically, this is accomplished by studying the nuclear factor kappa-light-chain-enhancer of activated B cell (NF-kB)-blocking protein MC160 as an example of an early gene. The results provide a plausible unique antiviral mechanism of action supporting increased MCV resolution observed in patients treated with berdazimer gel, 10.3% and describe a novel methodology that overcomes limitations in investigating MCV response in vitro to a potential new MC topical medication.

Comprehensive analysis of 66 complete molluscum contagiosum virus (MOCV) genomes: characterization and functional annotation of 47 novel complete MOCV genomes, including the first genome of MOCV genotype 3, and a proposal for harmonized MOCV genotyping indexing.

Four molluscum contagiosum virus (MOCV) genotypes (MOCV1-4) and four subtype variants (MOCV1p, MOCV1va, MOCV1vb, and MOCV1vc) were partially characterized using restriction enzyme profiling in the early 1980s/1990s. However, complete genome sequences of only MOCV1 and MOCV2 are available. The evolutionary pathways of MOCV genotypes and subtype variants with unavailable sequences remain unclear, and also whether all MOCV genotypes/subtype variants can be reliably detected and appropriately categorized using available PCR-based protocols. We de novo fully characterized and functionally annotated 47 complete MOCV genomes, including two putative non-MOCV1/2 isolates, expanding the number of fully characterized MOCV genomes to 66. To ascertain the placement of any putative novel MOCV sequence into the restriction profiling typing scheme, we developed an original framework for extracting complete MOCV genome sequence-based restriction profiles and matching them with reference restriction profiles. We confirmed that two putative non-MOCV1/2 isolates represent the first complete genomes of MOCV3. Comprehensive phylogenomic, recombination, and restriction enzyme recognition site analysis of all 66 currently available MOCV genomes showed that they can be agglomerated into six phylogenetic subgroups (PG1-6), corresponding to the subtype variants from the pioneering studies. PG5 was a novel subtype variant of MOCV2, but no PGs corresponded to the subtype variants MOCV1vb or MOCV4. We showed that the phylogenetic subgroups may have diverged from the prototype MOCV genotype lineages following large-scale recombination events and hinted at partial sequence content of MOCV4 and direction of recombinant transfer in the events that spawned PG5 and the yet undetected subtype variant MOCV1vb.IMPORTANCEFour molluscum contagiosum virus (MOCV) genotypes (MOCV1-4) and four subtype variants were partially characterized using restriction enzyme profiling in the 1980s/1990s, but complete genome sequences of only MOCV1 and MOCV2 are available. The evolutionary pathways whereby genotypes/subtype variants with unavailable sequences emerged and whether all MOCVs can be detected using current diagnostic approaches remain unclear. We fully characterized 47 novel complete MOCV genomes, including the first complete MOCV3 genome, expanding the number of fully characterized genomes to 66. For reliably classifying the novel non-MOCV1/2 genomes, we developed and validated a framework for matching sequence-derived restriction maps with those defining MOCV subtypes in pioneering studies. Six phylogenetic subgroups (PG1-6) were identified, PG5 representing a novel MOCV2 subtype. The phylogenetic subgroups diverged from the prototype lineages following large-scale recombination events and hinted at partial sequence content of MOCV4 and direction of recombinant transfer in the events spawning PG5 and yet undetected MOCV1vb variant.

Molluscum Contagiosum in HIV Patient Treated with 20% Topical Glycolic Acid After Resistance with Topical Tretinoin.

Molluscum contagiosum (MC) is a benign papular skin infection caused by Molluscum contagiosum virus (MCV). Over the past 30 years, the incidence of MK has continued to increased association with sexually transmitted infections and human immunodeficiency virus (HIV) infection. The incidence of MC in HIV patients is quite high at 5-8%. Until now there is no standard therapy used for the treatment of MC in patients with HIV. In HIV patients, anti retro viral therapy (ARV) is the main therapy with several other additional therapies such as cantaridin, chemical peeling agents such as glycolic acid (20-70%) and trichloroacetic acid (20-100%), cryosurgery, electrosurgery, incision, lactic acid, laser surgery, podophyllin, retinoic acid, and urea. There have been no studies regarding the administration of topical 20% glycolic acid in MC patients. We report a case of MC in an HIV patient who was treated with 20% topical glycolic acid after failing treatment with topical tretinoin. The diagnosis was made clinically, cytologically, and histopathologically, a white mass was found on compression of the lesion and Henderson-Paterson bodies. The lesions on the face, arms, and legs were given glycolic acid lotion 20% which was applied once a day at night. The lesions started to show responses to the treatment at week 6th as some of the MC papules became hyperpigmented macules. The side effects of therapy that appeared were itching and hyperpigmentation. Topical 20% glycolic acid can be used for MC therapy with minimal side effects, easy to apply and safe.

The MC160 protein of the molluscum contagiosum virus dampens cGAS/STING-induced interferon-ß activation.

Molluscum contagiosum virus (MCV) is a poxvirus that causes benign, persistent skin lesions. MCV encodes a variety of immune evasion molecules to dampen host immune responses. Two of these proteins are the MC159 and MC160 proteins. Both MC159 and MC160 contain two tandem death effector domains and share homology to the cellular FLIPs, FADD, and procaspase-8. MC159 and MC160 dampen several innate immune responses such as NF-κB activation and mitochondrial antiviral signaling (MAVS)-mediated induction of type 1 interferon (IFN). The type 1 IFN response is also activated by the cytosolic DNA sensors cyclic GMP-AMP synthase (cGAS) and stimulator of interferon genes (STING). Both cGAS and STING play a vital role in sensing a poxvirus infection. In this study, we demonstrate that there are nuanced differences between both MC160 and MC159 in terms of how the viral proteins modulate the cGAS/STING and MAVS pathways. Specifically, MC160 expression, but not MC159 expression, dampens cGAS/STING-mediated induction of IFN in HEK 293 T cells. Further, MC160 expression prevented the K63-ubiquitination of both STING and TBK1, a kinase downstream of cGAS/STING. Ectopic expression of the MC160 protein, but not the MC159 protein, resulted in a measurable decrease in the TBK1 protein levels as detected via immunoblotting. Finally, using a panel of MC160 truncation mutants, we report that the MC160 protein requires both DEDs to inhibit cGAS/STING-induced activation of IFN-ß. Our model indicates MC160 likely alters the TBK1 signaling complex to decrease IFN-ß activation at the molecular intersection of the cGAS/STING and MAVS signaling pathways.

A Novel Application of Zinc Oxide Nanoparticles for Inhibition of Molluscum contagiosum Virus Infection.

Molluscum contagiosum virus (MCV) is an infection caused by the molluscum contagiosum virus. Antiviral medications used to treat MCV infections have several problems, including drug-resistant and toxicity. As a result, improving safe, innovative, and effective antiviral drugs is critical. Therefore the current study aimed to investigate ZnO-NPs effects on M. contagisum infection and molluscum contagiosum virus replication, among the main exciting viruses that menace human health. The antiviral activity of zinc oxide nanoparticles (ZnO-NPs) against MCV infection was investigated in this work. FESEM and TEM electron microscopy were used to examine the nanoparticles. The cytotoxicity of the nanoparticles was assessed using the MTT assay, and anti-influenza effects were detected using RT-PCR and TCID50. An indirect immunofluorescence experiment was used to investigate the inhibitory effect of nanoparticles on viral antigen expression. In all tests, acyclovir was employed as a control. Compared to virus control, post-exposure of MCV with ZnO nanoparticles at the highest dose but is not toxic (100 g/mL) resulted in 0.2, 0.9, 1.9, and 2.8 log10 TCID50 reductions in infectious diseases virus titer (P=0.0001). This ZnO-nanoparticles level was accompanied by an inhibition percentage (17.8%, 27.3%, 53.3%, 62.5 %, and 75.9%), respectively, measured based on viral load compared with the virus control. Compared to the positive control, fluorescence emission intensity in virally infected cells that administrated ZnO nanoparticles was statically decreased. Our findings demonstrated that ZnO-NPs have antiviral effects against the MCV. This property indicates that ZnO-NP has a high potential for usage in topical formulations to treat facial and labial lesions.

Strategies and Patterns of Codon Bias in Molluscum Contagiosum Virus.

Trends associated with codon usage in molluscum contagiosum virus (MCV) and factors governing the evolution of codon usage have not been investigated so far. In this study, attempts were made to decipher the codon usage trends and discover the major evolutionary forces that influence the patterns of codon usage in MCV with special reference to sub-types 1 and 2, MCV-1 and MCV-2, respectively. Three hypotheses were tested: (1) codon usage patterns of MCV-1 and MCV-2 are identical; (2) SCUB (synonymous codon usage bias) patterns of MCV-1 and MCV-2 slightly deviate from that of human host to avoid affecting the fitness of host; and (3) translational selection predominantly shapes the SCUB of MCV-1 and MCV-2. Various codon usage indices viz. relative codon usage value, effective number of codons and codon adaptation index were calculated to infer the nature of codon usage. Correspondence analysis and correlation analysis were performed to assess the relative contribution of silent base contents and significance of codon usage indices in defining bias in codon usage. Among the tested hypotheses, only the second and third hypotheses were accepted.

The Molluscum Contagiosum Gene MC021L Partially Compensates for the Loss of Its Vaccinia Virus Homolog, F13L.

Orthopoxviruses produce two antigenically distinct infectious enveloped virions termed intracellular mature virions and extracellular virions (EV). EV have an additional membrane compared to intracellular mature virions due to a wrapping process at the trans-Golgi network and are required for cell-to-cell spread and pathogenesis. Specific to the EV membrane are a number of proteins highly conserved among orthopoxviruses, including F13, which is required for the efficient wrapping of intracellular mature virions to produce EV and which plays a role in EV entry. The distantly related molluscipoxvirus, molluscum contagiosum virus, is predicted to encode several vaccinia virus homologs of EV-specific proteins, including the homolog of F13L, MC021L. To study the function of MC021, we replaced the F13L open reading frame in vaccinia virus with an epitope-tagged version of MC021L. The resulting virus (vMC021L-HA) had a small-plaque phenotype compared to vF13L-HA but larger than vΔF13L. The localization of MC021-HA was markedly different from that of F13-HA in infected cells, but MC021-HA was still incorporated in the EV membrane. Similar to F13-HA, MC021-HA was capable of interacting with both A33 and B5. Although MC021-HA expression did not fully restore plaque size, vMC021L-HA produced amounts of EV similar to those produced by vF13L-HA, suggesting that MC021 retained some of the functionality of F13. Further analysis revealed that EV produced from vMC021L-HA exhibit a marked reduction in target cell binding and an increase in dissolution, both of which correlated with a small-plaque phenotype.IMPORTANCE The vaccinia virus extracellular virion protein F13 is required for the production and release of infectious extracellular virus, which in turn is essential for the subsequent spread and pathogenesis of orthopoxviruses. Molluscum contagiosum virus infects millions of people worldwide each year, but it is unknown whether EV are produced during infection for spread. Molluscum contagiosum virus contains a homolog of F13L termed MC021L. To study the potential function of this homolog during infection, we utilized vaccinia virus as a surrogate and showed that a vaccinia virus expressing MC021L-HA in place of F13L-HA exhibits a small-plaque phenotype but produces similar levels of EV. These results suggest that MC021-HA can compensate for the loss of F13-HA by facilitating wrapping to produce EV and further delineates the dual role of F13 during infection.

Selenium, Selenoproteins and Viral Infection.

Reactive oxygen species (ROS) are frequently produced during viral infections. Generation of these ROS can be both beneficial and detrimental for many cellular functions. When overwhelming the antioxidant defense system, the excess of ROS induces oxidative stress. Viral infections lead to diseases characterized by a broad spectrum of clinical symptoms, with oxidative stress being one of their hallmarks. In many cases, ROS can, in turn, enhance viral replication leading to an amplification loop. Another important parameter for viral replication and pathogenicity is the nutritional status of the host. Viral infection simultaneously increases the demand for micronutrients and causes their loss, which leads to a deficiency that can be compensated by micronutrient supplementation. Among the nutrients implicated in viral infection, selenium (Se) has an important role in antioxidant defense, redox signaling and redox homeostasis. Most of biological activities of selenium is performed through its incorporation as a rare amino acid selenocysteine in the essential family of selenoproteins. Selenium deficiency, which is the main regulator of selenoprotein expression, has been associated with the pathogenicity of several viruses. In addition, several selenoprotein members, including glutathione peroxidases (GPX), thioredoxin reductases (TXNRD) seemed important in different models of viral replication. Finally, the formal identification of viral selenoproteins in the genome of molluscum contagiosum and fowlpox viruses demonstrated the importance of selenoproteins in viral cycle.

Molluscum contagiosum: an update and review of new perspectives in etiology, diagnosis, and treatment.

Molluscum contagiosum (MC) is a self-limited infectious dermatosis, frequent in pediatric population, sexually active adults, and immunocompromised individuals. It is caused by molluscum contagiosum virus (MCV) which is a virus of the Poxviridae family. MCV is transmitted mainly by direct contact with infected skin, which can be sexual, non-sexual, or autoinoculation. Clinically, MC presents as firm rounded papules, pink or skin-colored, with a shiny and umbilicated surface. The duration of the lesions is variable, but in most cases, they are self-limited in a period of 6-9 months. The skin lesions may vary in size, shape, and location, which is more frequent in immunosuppressed patients, and could present complications such as eczema and bacterial superinfection. The diagnosis is based on clinical findings. A useful clinical tool is dermoscopy. If the diagnostic doubt persists, confocal microscopy or skin biopsy could be performed. The need for active treatment for MC is controversial; however, there is a consensus that it should be indicated in cases of extensive disease, associated with complications or aesthetic complaints. There are several treatment modalities which include mechanical, chemical, immunomodulatory, and antivirals. The objective of this article is to review the current evidence in etiology, clinical manifestations, diagnosis, and management alternatives of MC.

Clinical, Histopathological, and Virological Evaluation of 203 Patients With a Clinical Diagnosis of Molluscum Contagiosum.

Molluscum contagiosum (MC) manifests as small, umbilicated papules caused by the molluscum contagiosum virus (MCV). The extent of clinical misdiagnosis of MC is unknown. Combined clinical, histopathological, and virological evaluation of 203 consecutive patients with clinical diagnosis of MC treated at a university hospital during a 5-year period showed the correct clinical diagnosis in 188 of 203 (92.6%) patients. All 15 clinically misdiagnosed MC lesions were histopathologically and virologically confirmed as either common or anogenital warts caused by different human papillomaviruses. The MCV1/MCV2 subtypes ratio was 1.54:1, and the distribution of MCV subtypes differed across patients' age and anatomical location of lesions.

New Insights into the Evolutionary and Genomic Landscape of Molluscum Contagiosum Virus (MCV) based on Nine MCV1 and Six MCV2 Complete Genome Sequences.

Molluscum contagiosum virus (MCV) is the sole member of the Molluscipoxvirus genus and the causative agent of molluscum contagiosum (MC), a common skin disease. Although it is an important and frequent human pathogen, its genetic landscape and evolutionary history remain largely unknown. In this study, ten novel complete MCV genome sequences of the two most common MCV genotypes were determined (five MCV1 and five MCV2 sequences) and analyzed together with all MCV complete genomes previously deposited in freely accessible sequence repositories (four MCV1 and a single MCV2). In comparison to MCV1, a higher degree of nucleotide sequence conservation was observed among MCV2 genomes. Large-scale recombination events were identified in two newly assembled MCV1 genomes and one MCV2 genome. One recombination event was located in a newly identified recombinant region of the viral genome, and all previously described recombinant regions were re-identified in at least one novel MCV genome. MCV genes comprising the identified recombinant segments have been previously associated with viral interference with host T-cell and NK-cell immune responses. In conclusion, the two most common MCV genotypes emerged along divergent evolutionary pathways from a common ancestor, and the differences in the heterogeneity of MCV1 and MCV2 populations may be attributed to the strictness of the constraints imposed by the host immune response.

A comparison of the effect of molluscum contagiosum virus MC159 and MC160 proteins on vaccinia virus virulence in intranasal and intradermal infection routes.

Molluscum contagiosum virus (MCV) causes persistent, benign skin neoplasm in children and adults. MCV is refractive to growth in standard tissue culture and there is no relevant animal model of infection. Here we investigated whether another poxvirus (vaccinia virus; VACV) could be used to examine MCV immunoevasion protein properties in vivo. The MCV MC159L or MC160L genes, which encode NF-κB antagonists, were inserted into an attenuated VACV lacking an NF-κB antagonist (vΔA49), creating vMC159 and vMC160. vMC160 slightly increased vΔA49 virulence in the intranasal and intradermal routes of inoculation. vMC159 infection was less virulent than vΔA49 in both inoculation routes. vMC159-infected ear pinnae did not form lesions, but virus replication still occurred. Thus, the lack of lesions was not due to abortive virus replication. This system provides a new approach to examine MCV immunoevasion proteins within the context of a complete and complex immune system.

Molecular Screening and Single Nucleotide Polymorphism Typing of Molluscum Contagiosum Virus (MCV) from Genital Specimens, between 2012 and 2015

Background: The present study is the first comprehensive report of the Molluscum contagiosum virus (MCV) in Iran based on the molecular technique for differentiation and typing of the MCV1 and MCV2. Methods: Patients were diagnosed as having tumor-like genital warts less than 5 mm in diameter, and HIV seronegative samples were chosen for this cross-sectional study. TaqMan real-time PCR was used to identify MCV following clinical examination. Typing of the MCV-positive specimens was performed in the SNP A27451G region of MC021L gene. Results: Of 1470 samples, 114 (7.75%) samples were positive for the MCV. From MCV-positive samples, 71.05% sequences were found to be related to the MCV1 and 28.95% to the MCV2. Conclusion: This assay constitutes a reliable method for identification and typing of the MCV genomic variants that could be valuable for reviewing the pathogenesis, molecular epidemiology, and the natural history of MCV-related situations.

POXVIRUS DISEASE OF SQUIRRELS (POXVIRIDAE, CHORDOPOXVIRINAE, SQPV - SQUIRREL POXVIRUS).

A new taxon of the subfamily Chordopoxvirinae that may represent a new genus of smallpox viruses is considered in this review. The distribution of gray squirrels (Sciurus carolinensis) throughout the UK during the 20th century and the decrease in the population of red squirrels (Sciurus vulgaris) is one of the most well-documented cases of ecological change of local fauna by the introduced species. The tendency to expand the distribution of the smallpox virus from Great Britain to the Western part of Europe has been noted. The genetic peculiarities of the genome of the poxvirus of squirrels, which determine its biological properties, as well as evolutionary relationships with other poxviruses, are separately described. Determination of the size of the genome by restriction analysis, sequencing of the whole genome, determination of the content of G/C nucleotide pairs, and functional mapping of the majority of genes made it possible to construct a phylogenetic tree. Phylogenetic analysis shows that this is a new representative of the subfamily Chordоpoxvirinae located between the viruses of the molluscum contagiosum and parapoxviruses. Serological and molecular biological methods are used to reveal and identify the causative agent of smallpox. The use of electron microscopy is limited in grey squirrels, due to the absence of organ damage and reproduction of the virus. Identification of the DNA of the causative agent of poxvirus of squirrels based on the use of different types of polymerase chain reaction (nested and in real time) overcomes all these limitations.

ELISA for Molluscum Contagiosum Virus.

Molluscum contagiosum virus (MCV) is a common skin pathogen of children and young adults. Infection with MCV causes benign skin tumors in children and young adults and is mostly self-limiting. In contrast to orthopoxviruses, MCV infections tend to take a subacute clinical course but may persist for up to 12 months. Current numbers for MCV seroprevalence in different geographical areas are based on a variety of historical serological methods from complement fixation assays to MCV ELISAs based on purified MCV virions and MC133 antigen expressed in a Semliki Forest Virus expression system. A standardized ELISA for the assessment of MCV seroprevalence would be useful to determine global MCV seroprevalence. The methods described show that polypeptides derived from MCV open reading frames MC084 (residues V123 to R230 and V33 to G117), mc133 (residues M1 to N370), and glutathione S-transferase (GST)-H3L (residues I142 to W251) expressed in E. coli RIL+ as GST fusion proteins can be used to assess antibody binding in a GST capture ELISA. We show how the ELISA can be used to screen a panel of patient sera previously characterized with the mc084 V123-R230 ELISA. © 2017 by John Wiley & Sons, Inc.

Poxviruses Utilize Multiple Strategies to Inhibit Apoptosis.

Cells have multiple means to induce apoptosis in response to viral infection. Poxviruses must prevent activation of cellular apoptosis to ensure successful replication. These viruses devote a substantial portion of their genome to immune evasion. Many of these immune evasion products expressed during infection antagonize cellular apoptotic pathways. Poxvirus products target multiple points in both the extrinsic and intrinsic apoptotic pathways, thereby mitigating apoptosis during infection. Interestingly, recent evidence indicates that poxviruses also hijack cellular means of eliminating apoptotic bodies as a means to spread cell to cell through a process called apoptotic mimicry. Poxviruses are the causative agent of many human and veterinary diseases. Further, there is substantial interest in developing these viruses as vectors for a variety of uses including vaccine delivery and as oncolytic viruses to treat certain human cancers. Therefore, an understanding of the molecular mechanisms through which poxviruses regulate the cellular apoptotic pathways remains a top research priority. In this review, we consider anti-apoptotic strategies of poxviruses focusing on three relevant poxvirus genera: Orthopoxvirus, Molluscipoxvirus, and Leporipoxvirus. All three genera express multiple products to inhibit both extrinsic and intrinsic apoptotic pathways with many of these products required for virulence.

Molluscum Contagiosum Virus Protein MC005 Inhibits NF-κB Activation by Targeting NEMO-Regulated IκB Kinase Activation.

Molluscum contagiosum virus (MCV), the only known extant human-adapted poxvirus, causes a long-duration infection characterized by skin lesions that typically display an absence of inflammation despite containing high titers of live virus. Despite this curious presentation, MCV is very poorly characterized in terms of host-pathogen interactions. The absence of inflammation around MCV lesions suggests the presence of potent inhibitors of human antiviral immunity and inflammation. However, only a small number of MCV immunomodulatory genes have been characterized in detail. It is likely that many more remain to be discovered, given the density of such sequences in other poxvirus genomes. NF-κB activation occurs in response to both virus-induced pattern recognition receptor (PRR) signaling and cellular activation by virus-induced proinflammatory cytokines like tumor necrosis factor and interleukin-1. Activated NF-κB drives cytokine and interferon gene expression, leading to inflammation and virus clearance. We report that MC005, which has no orthologs in other poxvirus genomes, is a novel inhibitor of PRR- and cytokine-stimulated NF-κB activation. MC005 inhibited NF-κB proximal to the IκB kinase (IKK) complex, and unbiased affinity purification revealed that MC005 interacts with the IKK subunit NEMO (NF-κB essential modulator). MC005 binding to NEMO prevents the conformational priming of the IKK complex that occurs when NEMO binds to ubiquitin chains during pathway activation. These data reveal a novel mechanism of poxvirus inhibition of human innate immunity, validate current dynamic models of NEMO-dependent IKK complex activation, and further clarify how the human-adapted poxvirus MCV can so effectively evade antiviral immunity and suppress inflammation to persist in human skin lesions.IMPORTANCE Poxviruses adapt to specific hosts over time, evolving and tailoring elegantly precise inhibitors of the rate-limiting steps within the signaling pathways that control innate immunity and inflammation. These inhibitors reveal new features of the antiviral response, clarify existing models of signaling regulation while offering potent new tools for approaching therapeutic intervention in autoimmunity and inflammatory disease. Molluscum contagiosum virus (MCV) is the only known extant poxvirus specifically adapted to human infection and appears adept at evading normal human antiviral responses, yet it remains poorly characterized. We report the identification of MCV protein MC005 as an inhibitor of the pathways leading to the activation of NF-κB, an essential regulator of innate immunity. Further, identification of the mechanism of inhibition of NF-κB by MC005 confirms current models of the complex way in which NF-κB is regulated and greatly expands our understanding of how MCV so effectively evades human immunity.