**Valerija Groma 1,\*, Mihails Tarasovs 1,2, Sandra Skuja 1, Sofija Semenistaja 1, Zaiga Nora-Krukle 3, Simons Svirskis <sup>3</sup> and Modra Murovska <sup>3</sup>**


Received: 1 July 2020; Accepted: 19 August 2020; Published: 20 August 2020

**Abstract:** A direct association between joint inflammation and the progression of osteoarthritis (OA) has been proposed, and synovitis is considered a powerful driver of the disease. Among infections implicated in the development of joint disease, human herpesvirus 7 (HHV-7) infection remains poorly characterized. Therefore, we assessed synovitis in OA patients; determined the occurrence and distribution of the HHV-7 antigen within the synovial membrane of OA-affected subjects; and correlated plasma levels of the pro-inflammatory cytokines tumor necrosis factor (TNF), interleukin-6 (IL-6), and TNF expressed locally within lesioned synovial tissues with HHV-7 observations, suggesting differences in persistent latent and active infection. Synovial HHV-7, CD4, CD68, and TNF antigens were detected immunohistochemically. The plasma levels of TNF and IL-6 were measured by an enzyme-linked immunosorbent assay. Our findings confirm the presence of persistent HHV-7 infection in 81.5% and reactivation in 20.5% of patients. In 35.2% of patients, virus-specific DNA was extracted from synovial membrane tissue samples. We evidenced the absence of histopathologically detectable synovitis and low-grade changes in the majority of OA patients enrolled in the study, in both HHV-7 PCR+ and HHV-7 PCR- groups. The number of synovial CD4-positive cells in the HHV-7 polymerase chain reaction (PCR)+ group was significantly higher than that in the HHV-7 PCR- group. CD4- and CD68-positive cells were differently distributed in both HHV-7 PCR+ and HHV-7 PCR- groups, as well as in latent and active HHV-7 infection. The number of TNF+ and HHV-7+ lymphocytes, as well as HHV-7+ vascular endothelial cells, was strongly correlated. Vascular endothelial cells, especially in the case of infection reactivation, appeared vulnerable. The balance between virus latency and reactivation is a long-term relationship between the host and infectious agent, and the immune system appears to be involved in displaying overreaction when a shift in the established equilibrium develops.

**Keywords:** osteoarthritis; synovium; cytokines; HHV-7; PCR; ELISA; immunohistochemistry

#### **1. Introduction**

Joint diseases are recognized as common, widespread disabling pathologies all over the globe [1]. Among chronic rheumatic diseases having a substantial impact on population health, osteoarthritis (OA) is the one destined to increase and become the most prevalent [2]. It is estimated that among persons with OA, about 80% have some degree of movement limitation and 25% are unable to perform daily activities [3]. Previous studies have accentuated the role of OA as the major cause of hip and knee replacement surgeries [4]. Osteoarthritis has long been viewed as a degenerative disease of cartilage, but accumulating evidence indicates that inflammation has a crucial role in its pathogenesis [5,6]. A direct association between joint inflammation and the progression of OA has been proposed [7,8], and synovitis has been considered a powerful driver of the OA process [9].

The synovial membrane comprises a tissue enclosing the synovial cavity around the opposing surfaces of articular cartilage. It contains a superficial layer, called the intima, composed of two types of synoviocytes—macrophages or type A cells and fibroblast-like or type B cells—and underneath is layer of subintima which houses blood vessels and nerves. Synoviocytes manufacture, secrete, absorb, and adjust the contents of the joint cavity by producing and remodeling the extracellular matrix molecules (ECMs), both collagenous and ground substance/adhesion molecules, thus controlling local homeostasis [10,11]. Intimal cells are essential producers of cytokines, including the pro-inflammatory factors tumor necrosis factor (TNF), interleukin-1β (IL-1β), and interleukin-6 (IL-6) [12–14]. Cytokines diffusing through the synovial fluid into the articular cartilage may further activate chondrocytes and synoviocytes, thus sustaining inflammation [15].

Arthritogenic viruses implicated in the development of joint pathologies [16], including those manifesting with synovial damage, have been explored [16–19]. A viral etiology is evident for approximately 1% of all cases of acute arthritis [20]. Human herpesviruses are ubiquitous pathogens establishing a persistent infection in the host for life, but their contribution to articular damage and the etiopathogenesis of OA remains obscure [21–24].

The results of several studies have confirmed the presence of human herpesvirus 6 (HHV-6) and 7 (HHV-7) DNA and viral antigens by polymerase chain reaction (PCR) techniques, in situ hybridization, immunohistochemistry, and electron microscopy in blood plasma, peripheral blood mononuclear cells, the brain, and skin [25–30]. Furthermore, our earlier study reported on the presence of human HHV-6 and HHV-7 infection markers in synovial fluid and synovial tissues of rheumatoid arthritis (RA)-affected patients [31]. HHV-6A, HHV-6B, and HHV-7 are genetically related to human cytomegalovirus (HCMV) constituting the β-herpesvirus subfamily [22,32]. There is evidence suggesting that Epstein–Barr virus (EBV) and HCMV infection contribute to the pathogenesis of RA [18]. Furthermore, the presence of DNA from varicella zoster (VZV), herpes simplex virus (HSV), EBV, and HHV-6 has been confirmed in the synovial fluid and peripheral blood mononuclear cells of patients with RA, OA, and axial spondyloarthritis. In RA and spondyloarthritis, the authors found that the PCR results were concordant with the inflammatory activity of the disease [33,34]. The frequency and extent of synovial inflammation in OA linked to the assessment of inflammatory cytokine-producing cells evidenced in the presence of HHV-7 infection has not yet been elucidated, including the latency and reactivation conditions.

#### **2. Results**

#### *2.1. Nested Polymerase Chain Reaction*

Qualitative nested PCR (nPCR) testing was performed on 54 patients. The presence of persistent HHV-7 infection (the presence of the HHV-7 genomic sequence in DNA extracted from whole peripheral blood (WPB)) was detected in 44/54 (81.5%) OA patients. Out of 44 OA patients, the HHV-7 sequence in WPB DNA samples was detected in 27 females and 17 males. In 19/54 (35.2%) patients, virus-specific DNA was also present in DNA extracted from synovial membrane tissue samples (Figure 1a). All samples from patients with HHV-7 genomic sequences in whole blood DNA were

analyzed for viral infection reactivation (viral genomic sequences in cell-free blood plasma DNA). HHV-7 reactivation was found in 9/44 (20.5%) patients. Interestingly, in seven out of nine patients with an active viral infection, the HHV-7 specific genomic sequence was also found in both WPB and synovial membrane DNA (Figure 1b).

**Figure 1.** Distribution of patients enrolled in the study according to nested PCR data. (**a**) Venn diagram circles are scaled according to the number of samples and depict the total number, the number of human herpesvirus 7 (HHV-7)-positive whole peripheral blood (WPB), and the synovial membrane tissue samples. (**b**) Extract from the HHV-7 PCR+ group depicts the number of positive samples detected in the case of persistent and active viral infection (blood plasma and synovial membrane samples).
