A Case of Hypersensitivity to Mosquito Bites with Type III Latency of EB Virus Infection in NK Cells with Prolonged Survival Due to Induction of Specific CD8+ Cytotoxic T Cells.
Authors: Tsuchiya Shigeru , Du Wei , Kikuta Hideaki , Uchiyama Toru , Onuma Masaei , Rikiishi Takeshi , Sasahara Yoji , Ohuchi Miyako Yoshinari , Minegishi Masayoshi , Kumaki Satoru 
Published: April 2020

Abstract
Hypersensitivity to mosquito bites (HMB) is a rare disease classified into chronic active EB Virus (EBV) infection (CAEBV), characterized by clonal expansion of EBV-infected NK cells. The immunological background of the disease has not been elucidated yet. In this report we extensively studied EBV infected NK cells of a patient with HMB in view of several aspects, including latency, usage of promoter Cp/Wp and Qp of EBNAs, and induction of CD8+ cytotoxic T lymphocytes (CTL) to peptide antigens in order to know the relation between latency of EBV infected NK cells and induction of CTL.
Case report: A female patient aged 19 complained of skin symptoms after a mosquito bite. A blister developed at the bite site that subsequently transformed into ulcer. The patient also had general fever and liver dysfunction. Past medical history showed that she suffered from HMB when she was 7 years old. The titers of serum antibodies to EBV were as follows: viral capsid antigen (VCA) IgG, 1280X; IgA, 80X; IgM, 10X; EBNA, 20X; EA DR IgG, 160X and IgA, 10X. These data strongly indicated lytic type of EBV infection. Subset analysis of peripheral blood lymphocytes revealed CD3+ lymphocytes, 50.2%; CD4+, 34.2%; CD8+, 12.7%; CD19, 10.0% and CD56/16+, 22.6%. Real time polymerase chain analysis of her peripheral mononuclear cells showed markedly increased copy number of EBV genome (5 x 10^5 copies/μgDNA). Purification of each lymphocyte fraction by immunomagnetic beads and subsequent PCR analysis showed that EBV infected lymphocytes were NK cells. Latency and Cp and Qp of EBNA promoter usage analyses: In order to determine the latency of EBV infected NK cells RT-PCR was carried out. EBNA1, EBNA2, LMP1, LMP2A, and EBER1 were positive and BZLF was negative. These results indicated that EBV infection in NK cells is associated with type III latency. RT-PCR analyses confirmed that both promoters Cp/Wp and Qp were involved in this disease. Through bisulfite PCR analysis, eighty four % of Cp was found to be methylated, on the other hand, only 6% of Qp was methylated. These methylation patterns were similar to those of hemophagocytic syndrome with latency III, also known in a few patients with CAEBV. Detection and expansion of EBV specific CD8+ CTL: We used HLA-A24 restricted BRLF1, EBNA3A, EBNA3B and LMP2 tetramers for quantification of specific CTL and we found 0.48% of BRLF1 and 0.15% of EBNA3 peptide-specific CTL among CD8+ cells. These cells could be expanded to 6.6% for BRLF1 and 0.22% for EBNA3A peptide-specific CTL after stimulation with an autologous lymphoblastoid cell line (LCL) for 10 days and then with LCL and IL-2 for 7 days.
Conclusion: In this study we reported a long-term survivor of HMB with EBV infected NK cell proliferation. Promoter usage of EBNA genes and their methylation patterns might be important to determine latency and host immune responses against EBV infected cells.