The prevalence of enteroviral RNA and protein in mitral valves of chronic rheumatic heart disease

Introduction: Acute Rheumatic Fever/ Rheumatic Heart Disease (ARF/RHD), a sequel of group A streptococcal (GAS) infection, even today constitutes a public health issue in developing countries including India. Differences in the prevalence of ARF/RHD in countries with a similar prevalence of GAS infections indicate the role of other cofactors in pathogenesis of RHD. Methodology: We investigated the prevalence of enterovirus (EV) in RHD by probing for both EV RNA and VP1 protein using Nonisotopic In Situ Hybridization (NISH) and Immunohistochemistry (IHC) respectively in 75 valvectomy specimens obtained from RHD cases. Results: Twenty-eight (37%) of the valves showed tissue inflammation with lymphocytic infiltration in a majority of the cases. Twenty-six and 27 (38% and 40%) of the 68 valves showed the presence of EV by IHC and NISH respectively, indicating a very good association between the two tests; however, only about 46 to 48% of them exhibited tissue inflammation. In eight cases (12%) the EV genome was detectable in absence of VP1 protein perhaps indicating a latent viral infection. Conclusions: Due to a high degree of endemicity of EV in India, we are tempted to speculate that EV may be responsible for the severity and rapid progression of RHD. The virus could either be working synergistically with GAS or could be an opportunist infecting damaged valves.


Introduction
Acute Rheumatic Fever/ Rheumatic Heart Disease (ARF/RHD) is an autoimmune, multiorgan inflammatory disease, secondary to group A streptococcal (GAS) infection in genetically susceptible individuals [1]. Although RHD has long receded in the developed world, the threat of the disease in developing countries including India is still high [1,2]. Conservative estimates indicate that, of the ~34 million RHD affected individuals and 10 million disabilityadjusted life years (DALYs) lost per year [3], 13.2 million live in India [4]. Likewise, of the 1.4 million premature deaths annually worldwide due to RHD [5], nearly 120,000 are estimated to have occurred in India [4]. The global burden of ARF and RHD in lowresource settings is 444/100,000 population, compared with 3.4/100,000 population in non-endemic countries [4,5].
Epidemiological evidence supporting the association between GAS pharyngitis and ARF is strong [5], and RHD development or progression has been prevented with primary and secondary prophylaxis against GAS [3,[6][7][8][9]. However, a preceding GAS pharyngitis is not an absolute necessity for ARF and likewise, ARF and RHD do not necessarily accompany each other [6][7][8]10]. Further, there is a striking difference in prevalence of RHD in countries across the world despite similar prevalence of GAS pharyngitis. The low 'hit rate' of RHD following GAS pharyngitis (0.05% of >600 million) [2,10,11] points towards the role of additional cofactors in the pathogenesis of RHD. Co-infections with other viral pathogens -e.g. Coxsackie B, is a strong possibility as seen in both ARF and RHD [12][13][14]. Considering that EV is endemic in India and that ARF and chronic RHD too are significant problems, we thought it would be pertinent to investigate the prevalence of EV RNA and VP1 protein in rheumatic mitral valves using Nonisotopic In Situ Hybridization (NISH) and Immunohistochemistry (IHC) respectively.

Ethics Statement
The study was approved by Institutional Medical Ethics Committee of Sri Jayadeva Institute of Cardiovascular Sciences and Research (SJICR) (vide no.SJIC/RES/23dt.24 th September,2010). Written informed consent for using the samples for research purposes was obtained from all the patients/guardians prior to surgery.

Study Group
Seventy-five patients with chronic RHD (36 males and 39 females; mean age 38.06 ± 11.5 years) scheduled to undergo mitral valve replacement surgery, between the years 2010 and 2017at Sri Jayadeva Institute of Cardiovascular Sciences and Research (SJICR), Bangalore, a tertiary cardiac care center comprised the study group.

Controls
Thirty age matched individuals (28 males and 2 females; mean age 34.77 ± 13.52 years) who succumbed to road traffic accidents (RTA) at the National Institute of Mental Health And Neurosciences (NIMHANS), Bangalore formed the control for mitral valves.

Specimens
Venous blood was collected from all the patients of the study group before surgery. Surgical specimens of mitral valves received in the Department of Pathology, SJICR, formed the study material. Normal mitral valves collected at autopsy, from victims of RTA in Department of Neuropathology, (NIMHANS) served as controls.
Positive control: Coxsackie B2 virus infected mouse tissue (kind gift from Dr. S. A Huber); digoxigenin labeled group specific oligonucleotide probe complementary to the conserved group common sequence in the 5' non coding region of the EV genome (5'to3' GAA ACA CGG ACA CCC AAA GTA GTCGGT TCC GCT GCR GAG TTR CCC RTT ACG ACA) [15][16][17]

Nonisotopic In Situ Hybridization
NISH was carried out as per the protocol described earlier [15]. The integrity of RNA in tissue sections was verified by NISH using digoxigenin labeled β actin RNA probe. With every batch of NISH, EV infected mouse tissue sections served as positive controls and no probe tissue sections served as negative controls.

Scoring NISH
A blinded, independent microscopic scoring of the stained slides was carried out by three senior pathologists. Sections were considered to express EV RNA only when the intracytoplasmic signals were consistently localized to the same region in serial sections upon repeat testing. Also, a slide was labeled as positive only when at least two of the three pathologists consensually agreed upon the scoring. All positives were confirmed by a repeat NISH test on a different day.
Hematoxylin and Eosin stained tissue sections of the valve were graded for cellularity and interstitial inflammatory cell infiltrate.
Quantitative reverse transcription PCR was attempted to validate NISH results, insufficient yield of RNA however compelled us to abandon this procedure.

Immunohistochemistry
The presence of EV in the tissues was subsequently validated by indirect immunohistochemistry using antibodies to VPI. EV infected mouse tissue constituted positive control (detailed under NISH) and tissue sections without addition of primary antibodies served as negative controls. Three pathologists (SRK, AM, RSJ) blinded to groups independently scored each slide as positive and negative. Intracytoplasmic staining consistently localized to the same region on serial sections and upon repeat testing was considered positive with at least two of the three pathologists consensually agreeing upon the scoring.

Statistical tests
Chi-square test was used to calculate the association between various parameters.

Prevalence of Enterovirus in mitral valves
The prevalence of the virus could be analyzed for expression of both EVRNA and VP1 protein in 68 of the 75 cases.

NISH
Positive signals were seen within cells morphologically resembling myofibroblasts and endothelial cells and smooth muscle cells of the newly formed vessels in the valve (Figure 2). Enteroviral RNA was detected in 27/68 valves (40%) (Table1).
Hence, out of the 68 cases, 19 (28%) were positive and 34 (50%) negative, by both NISH and IHC, thereby exhibiting a concordance between the tests. Eight cases (8/68, 12%) were positive by NISH, but negative by IHC that may represent latent / dormant viral infection (Table 1) and likewise seven cases (7/68, 10%) positive by IHC, were negative by NISH probably secondary to viral RNA degradation (Table 1), during the procedure.
While there was a significant association between the detection of the viral RNA and protein (p<0.0001; Table 1), there was no association between tissue inflammation and prevalence of the virus either by NISH or IHC (p>0.5; Tables 2 and 3). No viral

Discussion
Rheumatic Heart Disease is still a major public health concern in developing countries, and India has the dubious distinction of being declared the "RHD capital of the world" [4]. Acute Rheumatic Fever and RHD are known to be post GAS sequel. Various factors influence ASO reactivity in RHD, which could vary from negative to high positive and reinfection with GAS augments sustained or continuous rise in antibody titers [2][3]18].We found only 4% of RHD cases to be ASO reactive; one of the reasons for this could be ascribed to Penicillin prophylaxis.
There remain several unanswered questions in the etiopathogenesis of RHD. Indeed, the classical sequence of GAS pharyngitis, followed by ARF, progressing to RHD does not appear in the majority of RHD: in only ~66% of the patients with ARF a history of pharyngitis could be elicited and similarly, only ~60% of patients with ARF progressed to RHD [6][7]. It is still not clear why only a small fraction of GAS pharyngitis patients worldwide, develop RHD [2,5]. Amongst the various factors contributing to the etiopathogenesis of RHD, co-existing viral infections also feature to play a role [14].Of the many viruses implicated in the etiopathogenesis of RHD, the contribution of Enteroviruses in particular viruses of the Coxsackie group, is thought to be significant [19]. While an Indian study carried out in the 90s recorded Coxsackie B virus antibodies in about 91% of patients with ARF but considered it incidental [13], an Egyptian study found a low association between Coxsackie B viral infection and ARF and chronic RHD [20]. However, direct demonstration of the virus or its proteins in the lesions of RHD reveal the true prevalence in any geographical region [14,[21][22][23][24][25]. Replicating forms of the virus have also been shown in cardiac tissues, strengthening the association of the virus with RHD [15]. The latter Chinese study, found that while 24% of the mitral valves harbored viral RNA, almost twice this number expressed viral protein. In the present study, ~40% of the RHD valves showed presence of EV both by NISH and IHC. Though some studies in the past have reported cross reactivity of 5-D8/1 clone of antibodies with heat shock proteins and to uninfected human cardiomyocytes [26,27], we and others didn't observe any positive signals in normal control valves [15]. Valves which showed the presence of the viral genome without expression of viral protein were interpreted to be dormant infections; particularly since viruses are known to remain latent for long periods in the cardiac tissues following systemic spread, and become reactivated into an acute fulminating infection with rapidly progressive myocardial disease Table 1. Association between NISH and IHC for prevalence of EV in RHD.

NISH (n) Positive
Negative Total  Positive  19  7  26  Negative  8  34  42  Total  27  41  68 There was a significant association between the prevalence of the virus in the tissues by IHC and NISH (p < 0.0001). Table 2. Association between Inflammation and IHC prevalence of EV in the cardiac valves of chronic RHD.

Inflammation (n) Positive
Negative Total  Positive  12  14  26  Negative  15  27  42  Total  27  41  68 There was no association between inflammation and presence of EV in the tissues (p = 0.45). Table 3. Association between Inflammation and NISH prevalence of EV in the cardiac valves of chronic RHD.

Inflammation (n) Positive
Negative Total  Positive  13  14  27  Negative  15  33  48  Total  28  47  75 There was no association between inflammation and presence of EV in the tissues by NISH (p=0.21). and death [28,29]. Although we did not use EV antigenome as a probe to confirm the findings obtained by NISH, expression of the viral protein VP1 was considered as proof of viral replication in the tissues. Moreover, detecting viral capsid protein is more sensitive than detecting RNA, which is prone to fragmentation and degradation in FFPE tissues [15]. In the present study, only 46% to 48% of EV infected valves exhibited a tissue inflammatory response, representing active viral infection. The alternative possibility of EV playing an opportunistic role by establishing itself in a damaged RHD milieu cannot be ruled out. However, the chances of the virus playing an active role are higher considering the renewed thinking on the etiopathogenesis of the disease [26]. Molecular mimicry between M protein of GAS and cardiac myosin [27] and between Coxsackie (CX) viral proteins and cardiac actin [28] has been known for some time now. Additionally, the elegant demonstration of molecular complementarity between GAS and CX; cardiac actin and myosin; and laminin and collagen IV has added a plausible dimension to the synergism of GAS and CX in the pathogenesis of RHD [26].

Conclusions
Considering the fact that India is endemic to both the microbes: GAS and EV, the results of the present study lend credence to the possibility that the two organisms could collaborate in the causation of RHD. The synergism between the two microbes may perhaps explain the severity and rapid progression of the disease in this part of the world. Alternatively, the possibility of EV being an opportunist colonizing the damaged valves cannot be entirely ruled out [30].