Santa Rasa ASSOCIATION OF PERSISTENT VIRAL INFECTIONS WITH MYALGIC ENCEPHALOMYELITIS/ CHRONIC FATIGUE SYNDROME Doctoral Thesis for obtaining the degree of a Doctor of Medicine Speciality – Microbiology and Virology Scientific supervisor: Dr. med., Assoc. Prof. Modra Murovska The Doctoral Thesis are developed with the support of the ESF project “Support to implementation of doctoral study programmes and obtaining the scientific degree in RSU” Agreement No. 2009/0147/1DP/1.1.2.1.2/09/IPIA/VIAA/009 Riga, 2017 ANNOTATION Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a multifactorial disease with unexplained aetiology. Human herpesvirus (HHV)-6, HHV-7, human parvovirus B19 (B19V) and xenotropic murine leukemia virus-related virus (XMRV) are thought to be possible trigger factors in ME/CFS development. The aim of this study was to determine the involvement of HHV-6, HHV-7, B19V and XMRV in etiopathogenesis of ME/CFS. Various polymerase chain reaction (PCR) methods to detect presence of viral genomic sequences were used, viral load and expression of virus-specific genes. Presence of HHV-6A and HHV-6B by PCR and restriction analysis was distinguished with following visualization of amplification products electrophoretically. Immunoenzymatic methods were used to estimate presence of virus-specific antibodies, reaction patterns of these antibodies, as well as the level of cytokines in blood plasma, while the HHV-6 antigen expression was detected by indirect immunofluorescence. The analysis of patients with ME/CFS and apparently healthy individuals revealed absence of XMRV proviral gag and env gene sequences in DNA from patients and apparently healthy individuals. These data are in concordance with the results obtained in many laboratories worldwide. HHV-6 specific antibodies in 92.1% of ME/CFS patients’ and 76.7% of apparently healthy individuals’ plasma samples were found. Markers of persistent HHV-6 infection in a latent phase had 42% of patients and 28.7% of healthy individuals, though in an active phase – 11% of ME/CFS cases and none of healthy individuals. HHV-6B is prevalent in Latvian ME/CFS patients. HHV-7 specific antibodies had 84.6% of patients and 93.8% of analysed controls. Markers of persistent HHV-7 infection in latent and active phase had 58% vs 34% of ME/CFS patients and 67.3% vs 8% of apparently healthy individuals. B19V specific antibodies in 78% of patients with ME/CFS and 67.4% of healthy individuals were detected. Presence of latent/persistent B19V infection markers had 12% of patients and 1.9% of controls but 17% of patients and 1.9% of healthy individuals had an active infection. According to the antibody pattern, 36% of ME/CFS patients had recent B19V infection and 43% – sustained infection. In patients with a persistent viral infection in an active phase median HHV-6 load (1927 vs 279 copies/106 cells), median HHV-7 load (238.6 vs 196.7 copies/106 cells) and median B19V load (251.8 vs 37.2 copies/106 cells) was higher than in patients with a persistent viral infection in a latent phase. 2 Analysing HHV-6, HHV-7 and B19V co-infection, latent infection/co-infection was observed to 51.5% of patients and 76.7% of apparently healthy individuals, whereas active – 45% of ME/CFS patients and 8.7% of healthy individuals. HHV-6 load in patients with persistent infection/co-infection in a latent and active phase was 262 and 653.2 copies/106 cells, respectively, whereas HHV-7 load was 166.5 and 248.5 copies/106 cells, respectively. In case of latent/persistent B19V co-infection, the viral load was 96.8, in case of active co-infection – 250.8 copies/106 cells. ME/CFS patients with a persistent infection in an active phase had higher level of pro-inflammatory (IL-6, TNF-α and IL-12) and anti-inflammatory (IL-10) cytokines than with a persistent infection in a latent phase, however without any statistical difference in part of cases. No difference was found in the level of IL-6 among patient groups without infection, with latent infection/co-infection, active single, double and triple co-infection, in turn a significant difference was revealed in the levels of TNF-α, IL-12 and IL-10 among these five groups. Furthermore, the level of TNF-α, IL-12 and IL-10 is significantly higher in patients with severe compared with moderate course of ME/CFS. All patients had unexplained chronic fatigue lasting for more than 6 months. Impaired memory, decreased concentration and sleep disturbances were most frequently observed symptoms in patients with ME/CFS. Patients with B19V genomic sequence and NS1 specific antibodies significantly often had lymphadenopathy and multi-joint pain. Moreover, onset of symptoms corresponded to B19V infection appearance time. The obtained data allow to conclude that XMRV infection is not associated with ME/CFS. Significantly more frequent findings of persistent HHV-6, HHV-7 and B19V infection/co-infection in an active phase with a higher viral load and elevated levels of pro- and anti-inflammatory cytokines among patients with ME/CFS than apparently healthy individuals indicate the importance of these infections/co-infections in ME/CFS development. Moreover, they are accompanied by a more severe ME/CFS clinical course. 3 ANOTĀCIJA Mialģiskais encefalomielīts/hroniskā noguruma sindroms (ME/CFS) ir neskaidras izcelsmes multifaktoriāla slimība. Cilvēka herpesvīruss (HHV)-6, HHV-7, cilvēka parvovīruss B19 (B19V) un ksenotropajam peļu leikozes vīrusam radniecīgais vīruss (XMRV) tiek uzskatīti par iespējamajiem ME/CFS palajdējfaktoriem. Pētījuma “Persistentu vīrusu infekciju saistība ar mialģisko encefalomielītu/hroniskā noguruma sindromu” mērķis bija noteikt HHV-6, HHV-7, B19V un XMRV iesaisti ME/CFS etiopatoģenēzē. Dažādas polimerāzes ķēdes reakcijas (PCR) tika izmantotas vīrusu genoma secību klātbūtnes, vīrusu slodzes un gēnu ekspresijas noteikšanai. PCR un restrikcijas analīzi lietoja HHV-6A un HHV6-B izšķiršanai ar sekojošu fragmentu garuma elektroforētisku noteikšanu. Vīrusspecifisko antivielu klātbūtni un antivielu spektru, kā arī citokīnu līmeni asins plazmā noteica ar imūnfermentatīvajām metodēm, bet HHV-6 antigēnu ekspresiju – ar netiešās imūnfluorescences metodi. Analizējot pacientus ar ME/CFS un praktiski veselus indivīdus, XMRV provīrusa gag un env gēnu secības netika atrastas ne pacientu, ne arī praktiski veselu indivīdu DNS paraugos. Šie dati atbilst daudziem pasaulē veikto pētījumu rezultātiem. HHV-6 specifiskās antivielas atrada 92,1% ME/CFS pacientu un 76,7% praktiski veselu indivīdu plazmā. Marķierus persistentai HHV-6 infekcijai latentā fāzē konstatēja 42% pacientu un 28,7% veselu indivīdu, bet aktīvā fāzē – 11% ME/CFS pacientu, bet nevienam no veselajiem indivīdiem. Latvijā pacientiem ar ME/CFS prevalēja HHV-6B. HHV-7 specifiskās antivielas noteiktas 84,6% ME/CFS pacientu un 93,8% analizēto kontroles grupas indivīdu. Marķierus persistentai HHV-7 infekcijai latentā un aktīvā fāzē atrada 58% vs 34% ME/CFS pacientu un 67,3% vs 8% praktiski veselu indivīdu. B19V specifiskās antivielas detektētas 78% pacientu ar ME/CFS un 67,4% veselo indivīdu. Latentas/persistentas B19V infekcijas marķierus konstatēja 12% pacientu un 1,9% kontroles indivīdu, bet aktīvas infekcijas marķierus – 17% pacientu un 1,9% veselo indivīdu. Saskaņā ar noteikto antivielu spektru, nesen pārciesta B19V infekcija atrasta 36% pacientu un sen pārciesta (ilgstoša) – 43% pacientu. Pacientiem ar persistentu infekciju aktīvā fāzē mediānas HHV-6 slodze (1927 vs 279 kopijas/106 šūnu), mediānas HHV-7 slodze (238,6 vs 196,7 kopijas/106 šūnu) un mediānas B19V slodze (251,8 vs 37,2 kopijas/106 šūnās) bija augstāka nekā latentā fāzē. Analizējot HHV-6, HHV-7 un B19V koinfekciju, latenta infekcija/koinfekcija bija noteikta 51,5% pacientu un 76,7% praktiski veselo indivīdu, bet aktīva – 45% ME/CFS 4 pacientu un 8,7% veselo indivīdu. Pacientiem ar persistentu infekciju/koinfekciju latentā un aktīvā fāzē HHV-6 slodze bija 262 un 653,2 kopijas/106 šūnu, attiecīgi, bet HHV-7 slodze – 166,5 un 248,5 kopijas/106 šūnu, attiecīgi. Latentas/persistentas B19V koinfekcijas gadījumā B19V slodze bija 96,8, bet aktīvas – 250,8 kopijas/106 šūnu. Iekaisuma (IL-6, TNF-α un IL-12) un pretiekaisuma (IL-10) citokīnu līmenis augstāks bija pacientiem ar persistentu infekciju aktīvā fāzē, nekā latentā fāzē, taču ne visos gadījumos rezultāts bija statistiski būtisks. Lai gan starp pacientu grupām bez infekcijas, ar latentu infekciju/koinfekciju, aktīvu atsevišķu, dubultu un trīskāršu koinfekciju netika konstatētas būtiskas IL-6 līmeņa atšķirības, starp šīm piecām grupām atrastas būtiskas TNF-α, IL-12 un IL-10 atšķirības, turklāt, TNF-α, IL-12 un IL-10 līmenis bija būtiski augstāks pacientiem ar smagu nekā vidēji smagu slimības gaitu. Vairāk kā sešus mēnešus ilgs hronisks nogurums izpaudās visiem ME/CFS pacientiem. Visbiežāk pacientiem konstatēja atmiņas traucējumus, koncentrēšanās grūtības un miega traucējumus. Pacientiem ar detektētu B19V genoma secību un NS1 antivielām biežāk novēroja limfadenopātiju un locītavu sāpes, turklāt, simptomu parādīšanās atbilda inficēšanās ar B19V laikam. Šī pētījuma rezultāti ļauj secināt, ka XMRV infekcija nav saistīta ar ME/CFS. Būtiski biežāka persistentas HHV-6, HHV-7 un B19V infekcijas/koinfekcijas atrade aktīvā fāzē ar augstāku vīrusa slodzi un paaugstinātu iekaisuma un pretiekaisuma citokīnu līmeni pacientiem ar ME/CFS salīdzinot ar praktiski veseliem indivīdiem, kas saistīti ar smagāku ME/CFS klīnisko gaitu, liecina par šo vīrusu infekcijas/koinfekcijas nozīmīgo lomu ME/CFS attīstībā un klīniskajā norisē.