**4. Materials and Methods**

#### *4.1. Patients' Characteristics*

Fifty-four patients that presented with advanced OA and underwent joint replacement surgery for the disease at the Riga East University Hospital Clinic "Gailezers" between March 2019 and January 2020 were enrolled in the study. The inclusion criterion was a primary or previous diagnosis of OA established in the given hospital. All OA subjects fulfilled relevant American College of Rheumatology (ACR) criteria for the disease-affected joints: Hip [69] and knee [70]. All subjects enrolled in the study (mean age 69 (range 35–85 years), standard deviation (SD) ± 12.28 years); 19 (35.2%) males and 35 (64.8%) females) had OA confirmed clinically and radiologically. They did not reveal any objective and subjective evidence of any other inflammatory disease apart from OA. The clinical data of patients included information on the duration, course, and clinical features of the disease at the time of presentation, whereas laboratory analyses employed to monitor OA progress included complete blood count, hemoglobin, and C reactive protein (CPR) data. PCR data were obtained from 48 out of 54 patients. These OA patients were subdivided into two study groups: HHV-7 PCR+ (*n* = 19) subjects (the first group) and HHV-7 PCR- (*n* = 29) subjects (the second group), accordingly. The average age of OA subjects of the first group was 68.95 years (SD ± 9.35), whereas that of the second group was

63.93 years (SD ± 12.9). The study was approved by the Ethical Committee of Riga Stradins University (Decisions No. 6-2/7/9 and No. 6-1/01/62) and was conducted according to the Declaration of Helsinki.

#### *4.2. Blood Sample Collection and Detection of TNF and IL-6 Levels*

Ethylenediamine tetraacetic acid anti-coagulated peripheral blood samples from OA patients were collected. Plasma samples were separated from peripheral blood by centrifugation. The levels of TNF and IL-6 (pg/mL) were measured by an enzyme-linked immunosorbent assay (ELISA) (TNF-α by Nordic Biosite, Copenhagen, Denmark), according to the manufacturer's guidelines, and analyzed in comparison with the presence or absence of HHV-7 infection markers. The optical density was measured by a microplate reader (Multiscan Ascent, Thermo Electron Corporation, Waltham, MA, USA) at a 450 nm wavelength using Ascent Software, and the results were calculated using Microsoft Excel.
