Functional Analysis of Trichodysplasia Spinulosa-Associated Polyomavirus-Encoded Large T Antigen.

Trichodysplasia spinulosa-associated polyomavirus (TSPyV or human polyomavirus 8) was identified from patients with trichodysplasia spinulosa, a rare skin disease affecting the faces of immunocompromised patients. Like other polyomaviruses, the TSPyV genome encodes a large T antigen (LT). However, the expression and functions of TSPyV LT in infected cells remain largely unknown. In the present study, we cloned a full-length TSPyV LT cDNA from cells transfected with the full-length of TSPyV LT DNA. Transfection study using green fluorescence protein-tagged LT expression plasmids showed that TSPyV LT was expressed in the nucleus of transfected cells. Analysis of deletion mutants identified a nuclear localization signal in TSPyV LT. Recombinant TSPyV LT exhibited an ATPase activity. TSPyV LT has a chitinase-like domain; however, no chitinase activity was detected. Immunoprecipitation assays revealed that TSPyV LT bound to retinoblastoma 1, but not to p53 in transfected cells. Expression of TSPyV LT in NIH3T3 cells induced colony formation in soft agar, suggesting its transformation activity. These data indicate that TSPyV LT may be associated with the pathogenesis of trichodysplasia spinulosa, which is a hyperplasia of keratinocytes in inner hair follicles.

Interleukin-6-dependent growth in a newly established plasmablastic lymphoma cell line and its therapeutic targets.

Plasmablastic lymphoma (PBL) is a rare, highly aggressive subtype of non-Hodgkin lymphoma with plasma-cell differentiation occurring typically in immune-suppressed patients such as those with AIDS. This study reports the establishment and characterization of a new cell line, PBL-1, derived from a patient with AIDS-associated PBL. Morphological assessment of PBL-1 indicated plasma-cell differentiation with a CD20(-) CD38(+) CD138(+) immunophenotype and IgH/c-myc translocation. The cell line harbours Epstein-Barr virus, but a 52.7-kbp length defect was identified in its genome, resulting in no expression of viral microRNAs encoded in the BamHI-A Rightward Transcript region. Importantly, supplementation of culture medium with >5 ng/mL of interleukin-6 (IL-6) was required for PBL-1 growth. Starvation of IL-6 or addition of tocilizumab, an inhibitory antibody for the IL-6 receptor, induced apoptosis of PBL-1. Transduction of IL-6 into PBL-1 by lentivirus vector induced autologous growth without IL-6 supplementation of culture medium. These data indicate the IL-6 dependency of PBL-1 for proliferation and survival. mTOR inhibitors induced cell death effectively, suggesting mTOR in the IL-6 signalling pathway is a potential therapeutic target for PBL. This established PBL cell line will be a useful tool to further understand the pathophysiology of PBL and aid the future development of PBL treatment.

Conserved sequences of BART and BHRF regions encoding viral microRNAs in Epstein-Barr virus-associated lymphoma.

OBJECTIVE: Epstein-Barr virus (EBV) encodes at least 25 pri-microRNAs (miRNAs) in two regions of its DNA genome, BART and BHRF. B95-8, an EBV reference strain, has a deletion in the BART region. However, no information is available on the deletions or mutations in the BART and BHRF regions in clinical samples of EBV-associated lymphoma. RESULTS: Nine DNA fragments encoding miR-BARTs and two coding miR-BHRF1s were amplified by PCR from DNA samples extracted from 16 cases of EBV-associated lymphoma. All the PCR products were sequenced directly. DNA fragments encoding miR-BARTs and miR-BHRF1-1 were successfully amplified from all samples. An adenine-to-guanine mutation in the DNA fragment encoding miR-BART2-3p was detected in four of the 16 cases, and a cytosine-to-thymidine mutation in the DNA fragment encoding miR-BART11-3p was detected in one of the 16 samples. These mutations were not associated with any histological categories of lymphoma. In conclusion, mutations were rarely observed in the DNA encoding viral miRNAs in cases of lymphoma. This suggests that the DNA sequences of EBV-encoded miR-BARTs and miR-BHRF1-1 are conserved in EBV-associated lymphoma.

Next-generation sequencing of miRNAs in clinical samples of Epstein-Barr virus-associated B-cell lymphomas.

Epstein-Barr virus (EBV) encodes 49 microRNAs (miRNAs) in the BART and BHRF1 regions of its genome. Although expression profiles of EBV-encoded miRNAs have been reported for EBV-positive cell lines and nasopharyngeal carcinoma, to date there is little information about total miRNA expression, including cellular and viral miRNAs, in the primary tumors of EBV-associated B-lymphoproliferative disorders. In this study, next-generation sequencing and quantitative real-time reverse transcription-PCR were used to determine the expression profiles of miRNAs in EBV-infected cell lines and EBV-associated B-cell lymphomas, including AIDS-related diffuse large B-cell lymphoma (DLBCL), pyothorax-associated lymphoma, methotrexate-associated lymphoproliferative disorder, EBV-positive DLBCL of the elderly, and Hodgkin lymphoma. Next-generation sequencing revealed that EBV-encoded miRNAs accounted for up to 34% of total annotated miRNAs in these cases. Expression of three miR-BHRF1s was significantly higher in AIDS-related DLBCL and pyothorax-associated lymphoma compared with methotrexate-associated lymphoproliferative disorder and EBV-positive DLBCL of the elderly, suggesting the association of miR-BHRF1s expression with latency III EBV infection. Heat map/clustering analysis of expression of all miRNAs, including cellular and EBV miRNAs, by next-generation sequencing demonstrated that each EBV tumor, except methotrexate-associated lymphoproliferative disorder, formed an isolated cluster. Principal component analysis based on the EBV-encoded miRNA expression showed that each EBV tumor formed a distinguished cluster, but AIDS-related DLBCL and pyothorax-associated lymphoma formed larger clusters than other tumors. These data suggest that expression of miRNAs, including EBV-encoded miRNAs, is associated with the tumor type and status of virus infection in these tumors.

Deep-Sequence Identification and Role in Virus Replication of a JC Virus Quasispecies in Patients with Progressive Multifocal Leukoencephalopathy.

JC virus (JCV) is a DNA virus causing progressive multifocal leukoencephalopathy (PML) in immunodeficient patients. In the present study, 22 genetic quasispecies with more than 1.5% variant frequency were detected in JCV genomes from six clinical samples of PML by next-generation sequencing. A mutation from A to C at nucleotide (nt) 3495 in JCV Mad1 resulting in a V-to-G amino acid substitution at amino acid (aa) position 392 of the large T antigen (TAg) was identified in all six cases of PML at 3% to 19% variant frequencies. Transfection of JCV Mad1 DNA possessing the V392G substitution in TAg into IMR-32 and human embryonic kidney 293 (HEK293) cells resulted in dramatically decreased production of JCV-encoded proteins. The virus DNA copy number was also reduced in supernatants of the mutant virus-transfected cells. Transfection of the IMR-32 and HEK293 cells with a virus genome containing a revertant mutation recovered viral production and protein expression. Cotransfection with equal amounts of wild-type genome and mutated JCV genome did not reduce the expression of viral proteins or viral replication, suggesting that the mutation did not have any dominant-negative function. Finally, immunohistochemistry demonstrated that TAg was expressed in all six pathological samples in which the quasispecies were detected. In conclusion, the V392G amino acid substitution in TAg identified frequently in PML lesions has a function in suppressing JCV replication, but the frequency of the mutation was restricted and its role in PML lesions was limited. IMPORTANCE: DNA viruses generally have lower mutation frequency than RNA viruses, and the detection of quasispecies in JCV has rarely been reported. In the present study, a next-generation sequencer identified a JCV quasispecies with an amino acid substitution in the T antigen in patients with PML. In vitro studies showed that the mutation strongly repressed the expression of JC viral proteins and reduced the viral replication. However, because the frequency of the mutation was low in each case, the total expression of virus proteins was sustained in vivo. Thus, JC virus replicates in PML lesions in the presence of a mutant virus which is able to repress virus replication.

Establishing and characterizing a new primary effusion lymphoma cell line harboring Kaposi's sarcoma-associated herpesvirus.

BACKGROUND: Primary effusion lymphoma is a rare distinct large B-cell neoplasm that is associated with Kaposi's sarcoma-associated herpesvirus (KSHV) infection. Over recent years, 9 KSHV-positive/Epstein-Barr virus (EBV)-negative PEL cell lines have been established. METHODS: Tumor cells were collected from the pleural effusion of a 49-year-old male with AIDS. Cells were grown in RPMI1640 culture medium supplemented with 10 % fetus bovine serum. Single cell cloning was performed successfully by a limiting dilution method in a 96-well plate. The cell line obtained was designated SPEL. RESULTS: SPEL cells showed gourd-shaped morphology with a polarized nucleus, expressing CD38, CD138, and Blimp-1, but not B cell markers such as CD19 and CD20. Polymerase chain reaction analysis revealed that SPEL cells were positive for KSHV but negative for EBV. Tetradecanoylphorbol acetate induced expression of KSHV lytic proteins and the production of KSHV particles in SPEL cells. Subcutaneous inoculation of SPEL cells into severe combined immunodeficiency mice resulted in the formation of solid tumors. Next-generation sequencing revealed the 138 kbp genome sequence of KSHV in SPEL cells. Suberic bishydroxamate, a histone deacetylase inhibitor, induced the expression of KSHV-encoded lytic proteins and cell death in SPEL cells. CONCLUSIONS: A new KSHV-positive and EBV-negative PEL cell line, SPEL was established. This cell line may contribute to furthering our understanding of the pathogenesis of PEL and KSHV infection.

Evaluation of Vero-cell-derived simian endogenous retrovirus infection in humans by detection of viral genome in clinicopathological samples and commercialized vaccines and by serology of Japanese general population.

BACKGROUND: Vero cells are used in laboratories for the isolation of viruses and the production of vaccines. Recently, the sequence of simian endogenous retrovirus (SERV) was identified in Vero cells (SERVagm-Vero), with homology to exogenously transmitted, pathogenic simian retroviruses (SRVs). Although SERVagm-Vero was shown to be noninfectious to human cells in vitro, SERV infection in humans is controversial. In this study, we evaluated the status of SERV infection in humans by detecting the viral genome in clinicopathological samples and commercialized vaccines, and with a serological survey of the Japanese general population. METHODS: Real-time polymerase chain reaction (PCR) and reverse transcription-PCR were used to detect the SERVagm-Vero genome. We also examined the seroprevalence of SERV in 1000 individuals in the Japanese population with an enzyme-linked immunoabsorbent assay (ELISA) using mixed SERVagm-Vero gag, pol, and env proteins as antigens. RESULTS: Real-time PCR failed to detect SERVagm-Vero genomic fragments in 783 human clinicopathological samples, and all 13 human cell lines tested were negative for the SERVagm-Vero genome. Thirteen commercialized vaccines, including five Vero-based vaccines, were also negative for the SERVagm-Vero genome on real-time PCR and reverse transcription-PCR. Eight (0.8%) were seropositive on ELISA, and western blotting showed that all eight sera contained anti-pol antibodies. All SERV-seropositive individuals were born before 1965, suggesting that SERV infection in Japan might not be associated with vaccine, because more than 90% of Japanese children born from 1964 to 2012 have received live poliovirus vaccines containing virus produced in Vero cells since the 1980s. CONCLUSION: We have confirmed that the vaccines we use today are free of SERVagm-Vero. Moreover, SERV infection in humans is very rare and unlikely to be associated with vaccines in the Japanese general population.

Fumagillin, a potent angiogenesis inhibitor, induces Kaposi sarcoma-associated herpesvirus replication in primary effusion lymphoma cells.

Kaposi sarcoma and primary effusion lymphoma cells are infected with Kaposi sarcoma-associated herpesvirus (KSHV), predominantly in the latent form, and KSHV replication is observed rarely. Angiogenesis plays a crucial role in the pathogenesis of both Kaposi sarcoma and primary effusion lymphoma. In this study, we found that fumagillin, a potent angiogenesis inhibitor, induced replication of KSHV in primary effusion lymphoma cell lines. The transcript and protein product of replication transcriptional activator (RTA) were induced by 1-10 µM fumagillin at 24 and 48 h, respectively. Western blot analysis demonstrated that 10 µM fumagillin induced not only RTA expression but also other KSHV-encoded lytic proteins. A real-time PCR array detecting KSHV gene expression demonstrated that the expression profiles of KSHV induced by fumagillin were similar to those induced by 12-O-tetradecanoylphorbol-13-acetate (TPA), but the amounts of each transcript were lower than those induced by TPA. Finally, real-time PCR demonstrated an increase in that viral DNA copy number per cell in fumagillin-stimulated primary effusion lymphoma cell lines, indicating replication of KSHV. In addition to TPA, 10 µM fumagillin resulted in growth inhibition of primary effusion lymphoma cell lines. These observations suggest that an angiogenesis inhibitor is an agent with potent effects on cell growth and KSHV reactivation in primary effusion lymphoma cells.

Seroprevalence of trichodysplasia spinulosa-associated polyomavirus in Japan.

BACKGROUND: Trichodysplasia spinulosa-associated polyomavirus (TSV) was identified in, and shown to be the probable cause of, trichodysplasia spinulosa, a rare skin disease. To date, serological analyses have revealed that TSV infection is common among adults in the general population of Europe and Australia. However, there have been no reports of TSV in Asia. OBJECTIVE: To study the prevalence of TSV infection in Japan. STUDY DESIGN: TSV-VP1 expressed in a recombinant baculovirus expression system in an insect cell line, Tn5, self-assembled into virus-like particles. Overall, 1000 serum samples were examined by enzyme-linked immunosorbent assays using virus-like particles of TSV as antigen. Participants ranged in age from 0 to 94 years. RESULTS: Overall, 629 of 1000 serum samples (62.9%) were positive for anti-TSV antibodies. The seropositive rate increased with age and the seroprevalence of TSV significantly increased from 17.1% (25/146) in children aged from 0 to 4 years to 78.7% (472/600) in adults aged over 20 years (odds ratio = 0.056, 95% confidence interval = 0.035-0.900, P = 0.000, Chi-squared test). TSV seropositivity was not different between sera obtained in 1980 and 2012, and was not associated with sex. Competitive assay demonstrated that TSV antibodies did not cross-react with BK virus or Merkel cell polyomavirus. CONCLUSIONS: These results provide evidence that TSV circulates widely in the Japanese population, with primary exposure occurring mainly at early childhood, similar to that previously reported in other countries.

Case of Merkel cell carcinoma with squamous cell carcinoma possibly arising in chronic radiodermatitis of the hand.

We report a case of Merkel cell carcinoma (MCC) on the dorsal aspect of the right middle finger associated with multiple squamous cell carcinomas (SCC) possibly arising in chronic radiation dermatitis of the hand of an 80-year-old surgeon. In spite of resection of the primary lesion and right axillary lymph nodes, he died of the tumor 5 months after the first visit. Cutaneous and lymph node lesions of MCC were negative for Merkel cell polyoma virus (MCPyV) by immunostaining using monoclonal antibody (CM2B4) and anti-large T antigen of MCPyV polyclonal antibody, and real-time polymerase chain reaction. Several differences in clinicopathological findings have been found between MCPyV-positive cases and negative ones. Several authors have reported that MCPyV-negative cases have a worse prognosis than MCPyV-positive ones. Furthermore, in cases of MCC associated with SCC, most tumors have been reported to be MCPyV-negative. We should pay more attention to the relationship between the carcinogenesis of MCC and ionizing irradiation.

Development of a new real-time PCR system for simultaneous detection of bacteria and fungi in pathological samples.

A novel system for simultaneous detection of pathogenic bacteria and fungi in pathological samples was developed using a real-time polymerase chain reaction (PCR) system. This system, designated the "multi-microbial real-time PCR", has the potential to simultaneously detect 68 bacterial and 9 fungal species in a 96-well plate format. All probe-primer sets were designed to produce amplicons smaller than 210 bp using formalin-fixed paraffin-embedded samples as input. The specificity and sensitivity of each probe-primer set were tested against DNA extracted from pure cultures of specific pathogens. The multi-microbial real-time PCR system revealed profiles of microorganism infection in lung samples collected at autopsy from 10 patients with acquired immunodeficiency syndrome. Staphylococcus aureus was the most common microbe detected (n=8), but with low copy numbers. High copy numbers of Pseudomonas aeruginosa were detected in the lung samples with abscess (n=6). Enterococcus faecium (n=6), Elizabethkingia meningoseptica (n=4), and Candida albicans (n=4) were also frequently detected. In addition, a latent infection of Mycobacterium tuberculosis was detected in one case of pneumonia. In conclusion, this multi-microbial real-time PCR system can be useful for detecting bacteria and fungi in pathological specimens from patients with uncertain diagnoses.

Evidence for human herpesvirus-6B infection of regulatory T-cells in acute systemic lymphadenitis in an immunocompetent adult with the drug reaction with eosinophilia and systemic symptoms syndrome: a case report.

We describe a fatal case of drug reaction with eosinophilia and systemic symptoms (DRESS) syndrome with human herpesvirus-6B (HHV-6B)-associated lymphadenitis and virus-associated hemophagocytic syndrome triggered by an over-the-counter medication to treat respiratory and influenza-like symptoms. Histologically, the structure of the lymph node was disrupted with infiltration of large lymphocytes carrying intranuclear acidophilic inclusion bodies. Immunohistochemistry and real-time PCR analysis revealed that these large lymphocytes were positive for HHV-6B. Numerous HHV-6 particles were detected in the inclusion body of the lymphocytes by electron microscopy. Interestingly, immunohistochemistry revealed that HHV-6B-infected cells in the lymph node were CD3(+), CD4(+), CD25(+), and FoxP3(+) T cells, indicating a phenotypic resemblance to regulatory T-cells. This case provides direct evidence of HHV-6 infection in CD25(+)/FoxP3(+) T cells in a case of acute lymphadenitis of DRESS syndrome, suggesting a significant role of HHV-6 infection of regulatory T-cells in the pathogenesis of DRESS syndrome.

Detection of trichodysplasia spinulosa-associated polyomavirus in a fatal case of myocarditis in a seven-month-old girl.

Trichodysplasia spinulosa-associated polyomavirus (TSV) was identified in a seven-month-old girl with myocarditis. The number of TSV genomes detected was higher in the heart than in the other organs. The full-length TSV genome was cloned from the heart. This suggests a possible role of TSV infection in the pathogenesis of myocarditis in infants.

The first identification and retrospective study of Severe Fever with Thrombocytopenia Syndrome in Japan.

BACKGROUND: Severe fever with thrombocytopenia syndrome (SFTS) is caused by SFTS virus (SFTSV), a novel bunyavirus reported to be endemic in central and northeastern China. This article describes the first identified patient with SFTS and a retrospective study on SFTS in Japan. METHODS: Virologic and pathologic examinations were performed on the patient's samples. Laboratory diagnosis of SFTS was made by isolation/genome amplification and/or the detection of anti-SFTSV immunoglobulin G antibody in sera. Physicians were alerted to the initial diagnosis and asked whether they had previously treated patients with symptoms similar to those of SFTS. RESULTS: A female patient who died in 2012 received a diagnosis of SFTS. Ten additional patients with SFTS were then retrospectively identified. All patients were aged ≥50 years and lived in western Japan. Six cases were fatal. The ratio of males to females was 8:3. SFTSV was isolated from 8 patients. Phylogenetic analyses indicated that all of the Japanese SFTSV isolates formed a genotype independent to those from China. Most patients showed symptoms due to hemorrhage, possibly because of disseminated intravascular coagulation and/or hemophagocytosis. CONCLUSIONS: SFTS has been endemic to Japan, and SFTSV has been circulating naturally within the country.

Seroprevalence of Kaposi's sarcoma-associated herpesvirus among men who have sex with men in Japan.

Kaposi's sarcoma-associated herpesvirus (KSHV), the etiologic agent of Kaposi's sarcoma, causes malignancies frequently in patients with acquired immunodeficiency syndrome. In the United States and Europe, KSHV infection is common among men who have sex with men. However, the seroprevalence of KSHV among men who have sex with men in Japan is unknown. In the present study, the seroprevalence of KSHV was investigated among 230 men who have sex with men and 400 age- and area of residence-matched men (controls) using a mixed-antigen (KSHV-encoded K8.1, open reading frame 59, 65, and 73 proteins) enzyme-linked immunosorbent assay and an immunofluorescence assay. Among the Japanese men who have sex with men, serological assays revealed that 27 (11.7%) were seropositive for KSHV; 20 (5%) of the men in the control group were also KSHV seropositive. The seroprevalence of KSHV among men who have sex with men was significantly higher than in the control group (odds ratio = 2.52, 95% confidence intervals = 1.38-4.62, P = 0.0019, Chi-square test). Infection with the human immunodeficiency virus, Treponema pallidum, or hepatitis B and C virus did not correlate with KSHV infection. Furthermore, the association of KSHV seropositivity with specific sexual activities was not statistically significant. In conclusion, a higher KSHV seroprevalence was found among Japanese men who have sex with men than among the controls, suggesting that the circulation of KSHV infection is more efficient among men who have sex with men in Japan than among men who do not engage in such sexual activities.

Frequent detection of Merkel cell polyomavirus DNA in sera of HIV-1-positive patients.

BACKGROUND: Merkel cell polyomavirus (MCPyV), human polyomavirus-6 (HPyV6), and human polyomavirus-7 (HPyV7) were identified as viruses shed from the skin. Serological analysis revealed that these viruses are common among the general population. However, there is little information about the presence of MCPyV, HPyV6, and HPyV7 in the sera and tissues of immunocompromised individuals. The aims of this study are to know if immune status affects the presence of MCPyV, HPyV6, and HPyV7 in the serum, and to reveal the presence of these viruses in diseased tissues of unknown etiology. METHODS: Sera from HIV-1-positive and -negative patients were examined by real-time PCR and nested PCR detecting MCPyV, HPyV6 and HPyV7. In addition, diseased tissue samples of unknown etiology were examined. RESULTS: Nine out of 23 serum samples (39.1%) from HIV-1-positive patients who had not received anti-retroviral therapy were positive for MCPyV, which is significantly higher than HIV-1-negative patients (6/110, 5.5%, P<0.01, Chi-square test). MCPyV DNA was detected in tissue samples of Merkel cell carcinoma (22/30 [73%]), encephalitis (4/19 [21%]), pneumonia (3/17 [18%]), and myocarditis (8/14 [57%]). With the exception of Merkel cell carcinoma samples, MCPyV-positive tissues showed low copy numbers of MCPyV DNA by real-time PCR and no expression of the MCPyV large T antigen by immunohistochemistry. HPyV6 and HPyV7 were rarely detected in serum and tissue samples. CONCLUSIONS: These results suggest that MCPyV viremia is associated with host immunity, and that circulation of HPyV6 and HPyV7 in the serum is rare.

Pathogenic characterization of a cervical lymph node derived from a patient with Kawasaki disease.

Kawasaki disease (KD) is the most common cause of multisystem vasculitis in childhood. Although cervical lymphadenitis is one of the major symptoms in KD, lymph node biopsy is rarely performed, because KD is usually diagnosed by clinical symptoms. A cervical lymph node biopsy was taken from a girl aged 1 year and 8 months who had suspected lymphoma, but she was diagnosed with KD after the biopsy. The cervical lymph node specimen was analyzed with multivirus real-time PCR that can detect >160 viruses, and unbiased direct sequencing with a next-generation DNA sequencer to detect potential pathogens in the lymph node. Histologically, focal necrosis with inflammatory cell infiltration, including neutrophils and macrophages, was observed in the marginal zone of the cervical lymph node, which was compatible with the acute phase of KD. Multivirus real-time PCR detected a low copy number of torque teno virus in the sample. Comprehensive direct sequencing of the cervical lymph node biopsy sample sequenced more than 8 million and 3 million reads from DNA and RNA samples, respectively. Bacterial genomes were detected in 0.03% and 1.79% of all reads in DNA and RNA samples, respectively. Although many reads corresponded to genomes of bacterial environmental microorganisms, Streptococcus spp. genome was detected in both DNA (77 reads) and RNA (2,925 reads) samples. Further studies are required to reveal any association of microbial or viral infection with the pathogenesis of KD.

Metachronous Merkel cell carcinoma on both cheeks.

Merkel cell carcinoma (MCC), an aggressive skin cancer with neuroendocrine features, has been found to be associated with a new type of human polyomavirus called Merkel cell polyomavirus (MCV). Patients diagnosed with MCC have a significantly increased risk of a second primary cancer. We report here the first case of two primary MCCs arising on the face at different times, associated with MCV infection. The tumour on the patient's right cheek was surgically removed, followed by chemoradiation. After a 10-year tumour-free period, a new tumour developed on the patient's left cheek. Histological and immunohistochemical findings were consistent with MCC. The tumours had high MCV copy numbers and expressed large T antigen, which may play a major role in MCV-mediated carcinogenesis. This case highlights the close links between MCC and MCV.