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Abstract

Human parvovirus B19 (B19) is a small, non-enveloped virus and belongs to Parvoviridae family. B19 persists in many tissues such as thyroid tissue and even thyroid cancer. The main aim of this study was to determine the presence of B19, its association with increased inflammation in thyroid tissue, and thus its possible role in thyroid cancer progression. Studies have shown that virus replication in non-permissive tissue leads to overexpression of non-structural protein and results in upregulation of proinflammatory cytokines such as interleukin 6 and tumor necrosis factor alpha. A total of 36 paraffin-embedded thyroid specimens and serum were collected from patients and 12 samples were used as control. Various methods were employed, including polymerase chain reaction, real-time polymerase chain reaction, and enzyme-linked immunosorbent assay. The results have shown the presence of B19 DNA in 31 of 36 samples (86.11%). Almost in all samples, the levels of non-structural protein 1, nuclear factor kappa B, tumor necrosis factor alpha, and interleukin 6 were simultaneously high. The presence of parvovirus B19 has a significant positive correlation with nuclear factor kappa B, tumor necrosis factor alpha, and interleukin 6 levels. This study suggests that B19 infection may play an important role in tumorigenesis and thyroid cancer development via the inflammatory mechanisms.

Keywords

Parvovirus B19 thyroid cancer nuclear factor kappa B interleukin 6 tumor necrosis factor alpha non-structural protein 1

Introduction

One of the common endocrine malignancies is thyroid cancer which increased rapidly in recent decades.¹ Thyroid cancer exists in several histological forms, including follicular, papillary, medullary, and undifferentiated anaplastic thyroid carcinoma. Microenvironment changes in thyroid tissue such as inflammation and ionizing radiation possibly involve in tumor progression.¹ Human parvovirus B19 is a small, non-enveloped virus and belongs to Parvoviridae family. The B19 genome is a linear single-stranded DNA and encodes one non-structural protein 1 (NS1) and two viral capsid proteins (VP1 and VP2).^2,3 Erythroid progenitor cells are the main site of B19 replication, in the event that other cell types support the expression of B19 genome in the absence of detectable replication. B19 DNA has been reported in association with persistent infection in multiple tissues, including bone marrow, brain, colon, heart, kidneys, liver, lungs, lymphoid, skin, synovium, testis, thyroid, and tonsil.² NS1 plays a major role in the viral life cycle. It transactivates viral and cellular promoters such as HIV–long terminal repeat (HIV-LTR), interleukin 6 (IL-6), and tumor necrosis factor alpha (TNF-α) through the nuclear factor kappa B (NF-κB)-binding site in the IL-6 promoter. This suggests that the IL-6 upregulation may be important in the pathogenesis of B19 virus infection.^4–6 Many studies have shown that NF-κB has an important role in thyroid cancer since it controls the antiapoptotic and the proliferative signaling pathways of the thyroid cancer cells. Moreover, the demonstrated level of NF-κB is high in medullary, papillary, and follicular thyroid cancer cells whereas low in non-cancerous thyroid cells.⁷ The expression of B19 genes in different tissues such as thyroid cancer is associated with specific changes in inflammatory genes and effect on cellular microenvironment and may finally lead to tumor development.² However, little information about the B19 gene expression in thyroid cancer tissue is available.⁸ These changes in cellular gene expression may contribute to persistent B19 infection and expression as well as tumor progression. Reactive oxygen nitrogen species (RONS) can cause different DNA damages such as strand breaks, changes in DNA base, enhanced expression of proto-oncogenes, and tumor-suppressor genes damage. Also, it has been shown that RONS can induce malignant transformation in cultured cells. The development of human cancer depends on several factors such as DNA damage and inflammation.⁹

In this study, we determined the presence of B19 in thyroid cancer tissue; the expression level of NS1, VP1, TNF-α, IL-6, levels of NF-κB, RONS and its association with viral infection; and finally the association of this virus with thyroid cancer development.

Materials and methods

Samples

Thirty-six paraffin-embedded thyroid specimens and serum were collected from patients between March 2014 to December 2015 in Shahrekord and Tehran. All the cases were re-examined by pathologists to confirm the diagnosis. Tumor samples were classified histologically based on the World Health Organization (WHO) criteria. Patients’ charts were checked to ensure that no one was immunocompromised. The carcinoma samples included 14 medullary, 19 papillary, and 3 follicular thyroid cancer cells. Also, 12 normal thyroid tissues obtained from the areas surrounding the surgically removed adenomas were used. The age of patients in the study ranged from 27 to 76 years.

DNA extraction

Sections were cut from each block which were previously deparaffinized in 1 mL of xylene and then by 1 mL of ethanol 100%. DNA extraction was done by QIAamp DNA Mini Kit according to the manufacturer’s instructions (Qiagen, Germany).

Primer

Primers used in NS1 gene amplification are as follows:¹⁰

Forward primer: 5′-AATACACTGTGGTTTTATGGGCCG-3′

Reverse primer: 5′-CCATTGCTGGTTATAACCACAGGT-3′

Polymerase chain reaction

A volume of 1 µg of DNA was amplified in a 2× Taq Master mix, 1 µM forward and reverse primers (metabion, Germany), and water upto 25 µL. Amplification was carried out in a thermocycler (EppendorfMastercycler^®, Hamburg, Germany) for 35 rounds: denaturation at 95°C for 1 min, annealing at 55°C for 45 s, extension at 70°C for 1 min, and final extension at 72°C for 10 min. Agarose gel electrophoresis (3%) of polymerase chain reaction (PCR) products was carried out using 1 mM Tris–Borate–ethylenediaminetetraacetic acid (EDTA) (TBE) buffer at 95 V for 1 h and then the DNA bands were stained with DNA green viewer dye (SinaClon, Iran).

IL-6 gene expression

Thyroid tissue RNA extraction was performed with RNeasy Mini Kit (Qiagen) according to the complementary DNA (cDNA) synthesis protocol. For reverse transcription, 1 µg of total RNA was reverse transcribed using the QuantiNova Reverse Transcription Kit (Qiagen). The primers and probes used for the measurement of IL-6 level are as follows:¹¹

Forward: 5′-GGTACATCCTCGACGGCATCT-3′

Reverse: 5′-GTGCCTCTTTGCTGCTTTCAC-3′

Probe: 5′-FAM-TGTTACTCTTGTTACATGTCTCCTTTCTCAGGGCT-TAMRA-3′

A reagent mixture of 75 mL was made up for each sample with 1× Master Mix, 900 nM of IL-6 forward primer, 300 nM of reverse primer, 100 nM of IL-6 probe, 1× 18S mix (primers and probe), and 50–100 ng of sample and made upto a final volume of 75 mL with water. Each sample was run in triplicate in a reaction volume of 20 mL for 50 cycles using standard real-time PCR cycling conditions.

TNF-α gene expression

Synthesis of cDNA: A volume of 1 µg of total RNA was reverse transcribed using the QuantiNova Reverse Transcription Kit (Qiagen). Total RNAs (1 µg) were reverse transcribed using random hexamers and Superscript II reverse transcriptase (Invitrogen, Thermo Fisher Scientific, USA). Real-time PCR was performed with 12.5 ng of cDNA and both the sense and antisense oligonucleotides in a final volume of 25 µL using the SYBR Green TaqMan Universal PCR Master Mix (Qiagen). Fluorescence was monitored and analyzed in a GeneAmp 7000 detection system instrument (Applied Biosystems, UK). Analysis of the 18S ribosomal RNA (rRNA) was carried out in parallel using the rRNA control TaqMan Assay Kit in order to normalize the gene expression. Results are expressed as 2(Ct^18SCtgene)(1−(1/2(C^tgeneCtRT−))) where Ct corresponds to the number of cycles needed to generate a fluorescent signal above a pre-defined threshold. Oligonucleotide primers were designed using the Primer Express software (Applied Biosystems). All the primers used were validated for PCR efficiency. The same reaction was done without Superscript II reverse transcriptase (RT−) to estimate the DNA contamination.

NS1 gene expression

Synthesis of cDNA: A volume of 1 µg of total RNA was reverse transcribed using the QuantiNova Reverse Transcription Kit (Qiagen). Quantitative reverse transcription PCR (qRT-PCR) was performed using pre-designed TaqMan Gene Expression Assays for NS1. Real-time PCR was performed with 14 ng cDNA and both the sense and antisense oligonucleotides in a final volume of 25 µL using the SYBR Green TaqMan Universal PCR Master Mix (Qiagen).

Levels of NF-κB

Levels of NF-κB p65 were determined using Transcription Factor Assay Kit (Cambridge, MA, USA) according to the protocol.

VP1 gene expression

A volume of 1 µg of total RNA was reverse transcribed using the QuantiNova Reverse Transcription Kit (Qiagen). qRT-PCR was conducted using pre-designed TaqMan Gene Expression Assays for VP1. Real-time PCR was performed with 12 ng cDNA and both the sense and antisense oligonucleotides in a final volume of 25 µL using the SYBR Green TaqMan Universal PCR Master Mix (Qiagen).

Measurement of reactive oxygen species and reactive nitrogen species

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) were measured by OxiSelect^™ Intracellular ROS/RNS Assay Kit (Cell Biolabs, Inc., San Diego, CA, USA) according to the protocol.

Statistical methods

Normality test was done using Kolmogorov–Smirnov test for continuous variables. The two-independent sample t test and Mann–Whitney non-parametric test were performed for comparing the central tendency (e.g. mean for normal and median for non-normal gene expression) of gene expressions in the two groups. Correlation analysis was done by implementing the Spearman’s rank-order. Generalized linear model was used for assessing the association between gene expression and parvovirus B19 and Thyroid cancer. All the data were analyzed using IBM SPSS version 21.0 (SPSS, Chicago, IL, USA) and GraphPad Prism version 6 (La Jolla, CA, USA). The p values less than 0.05 were considered as statistical significance.

Results

Among the 48 participants who were included in this study, 36 subjects were thyroid cancer patients and 12 others were normal thyroid patients. In this study, the minimum and maximum age was 27 and 76 years, respectively. Of the patients, 14 (39%) were males. More information is presented in Supplementary material.

Detection of B19 DNA and serological status

B19 DNA was found in 31 of 36 patients (86.1%) and 3 of 12 controls (25%) by PCR method (Figure 1). Two patients were immunoglobulin M (IgM) positive. Serological status and information on DNA detection of patients and controls are listed in (Table 1).

Figure 1.

Detection of B19 DNA in samples—2, 11: negative controls; 3: positive control; and 4–10 and 12: positive samples.

Table 1.

Serological status of patients and controls.

	IgM	IgG	B19 DNA
Patients	2 (5.5%)	35 (97.2%)	31 (86.1%)
Controls	0 (0%)	10 (83.3%)	3 (25%)
All	2 (4.1%)	45 (93.7%)	34 (70.8%)

Ig: immunoglobulin.

Levels of NS1, TNF-α, IL-6, VP1, and NF-κB

We utilized real-time PCR to determine the expression of IL-6, TNF-α, NS1, and VP1 mRNAs and enzyme-linked immunosorbent assay (ELISA) to determine the expression of NF-κB. As described in Table 2, cytokines, IL-6, and NS1 gene expression levels were statistically significantly higher in patients than controls (odds ratio (OR) = 12.4, 95% confidence interval (CI) = 2.65–58.29, p < 0.001). Also, TNF-α and VP1 gene expression levels were statistically significantly higher in patients than controls (OR = 5.8, 95% CI = 1.41–23.84, p < 0.01), (OR = 1.38, 95% CI = 1.13–1.69, p = 0.04), respectively. The level of NF-κB was more in the tumor tissue than in normal tissue (OR = 2.96, 95% CI = 0.72–12.16, p = 0.12). More details are shown in the Table 2.

Table 2.

Association between expression of IL-6, NS1, TNF-α, VP1, and NF-κB and tumor tissue.

Expression level	Categories	Tumor tissue	Control	p value	Adjusted p value^a	OR (95% CI)
IL-6	Low	Reference	Reference	–	–
	High	29 (81%)	3 (25%)	<0.001	0.0025	12.4 (2.65–58.29)
NS1	Low	Reference	Reference	–	–
	High	29 (81%)	3 (25%)	<0.001	0.0025	12.4 (2.65–58.29)
TNF-α	Low	Reference	Reference	–	–
	High	29 (81%)	5 (42%)	<0.01	0.016	5.8 (11.41–23.84)
VP1	Low	Reference	Reference	–	–
	High	10 (27.7%)	1 (8%)	0.04	0.05	1.38 (1.13–1.69)
NF-κB	Low	Reference	Reference	–	–
	High	29 (81%)	7 (58%)	0.12	0.12	2.96 (0.72–12.16)

IL-6: interleukin 6; NS1: non-structural protein 1; TNF-α: tumor necrosis factor alpha; VP1: viral capsid protein 1; NF-κB: nuclear factor kappa B; OR: odds ratio; CI: confidence interval; FDR: false discovery rate.

FDR correction for multiple comparisons by Benjamini–Hochberg method.

None of the associations between IL-6, TNF-α, NS1, VP1, and NF-κB with tumor tissue types were statistically significant (p > 0.20). More details are summarized in the Table 3.

Table 3.

Association between expression of IL-6, NS1, TNF-α, VP1, and NF-κB and tumor tissue types.

Expression level	Categories	Tumor type (medullary carcinoma)	Tumor type (papillary carcinoma)	Tumor type (follicular carcinoma)	p value	Adjusted p value^a
IL-6	Low	Reference	Reference	Reference	–	–
	High	10 (71%)	16 (84%)	3 (300%)	0.24	0.30
NS1	Low	Reference	Reference	Reference	–	–
	High	9 (64%)	17 (89%)	3 (300%)	0.21	0.30
TNF-α	Low	Reference	Reference	Reference	–	–
	High	10 (71%)	15 (79%)	4 (400%)	0.22	0.30
VP1	Low	Reference	Reference	Reference	–	–
	High	5 (35.7%)	4 (21.1%)	1 (33.3%)	0.63	0.63
NF-κB	Low	Reference	Reference	Reference	–	–
	High	10 (71.4%)	16 (84.2%)	3 (300%)	0.20	0.30

IL-6: interleukin 6; NS1: non-structural protein 1; TNF-α: tumor necrosis factor alpha; VP1: viral capsid protein 1; NF-κB: nuclear factor kappa B; FDR: false discovery rate.

FDR correction for multiple comparisons by Benjamini–Hochberg method.

Presence of parvovirus B19 has a significant positive correlation with the levels of NS1, TNF-α, IL-6, and NF-κB (r = 0.81, p < 0.001), (r = 0.70, p < 0.001), (r = 0.79, p < 0.001), and (r = 0.51, p < 0.001), respectively. But, the positive correlation between VP1 gene expression and presence of parvovirus B19 was not statistically significant (r = 0.21, p = 0.13). The association between NS1 gene expression and the presence of parvovirus B19 was stronger than others. In other words, OR of high expression of NS1 in the subjects with the presence of parvovirus B19 was 134.3 (95% CI = 12.76–1414.22), which is comparatively higher than that in those with the absence of parvovirus B19 (Table 4).

Table 4.

Associations between expressions of NS1, TNF-α, IL-6, VP1, and NF-κB in presence of parvovirus B19.

Gene expression	Correlation coefficient	p value	OR (95% CI)
Presence of parvovirus B19
NS1	0.81	<0.001	134.3 (12.76–1414.22)
TNF-α	0.70	<0.001	37.8 (6.63–216.27)
IL-6	0.79	<0.001	124.8 (10.53–1016.85)
VP1	0.21	0.13	4.68 (0.53–41.07)
NF-κB	0.51	<0.001	18.60 (3.71–93.27)

NS1: non-structural protein 1; TNF-α: tumor necrosis factor alpha; IL-6: interleukin 6; VP1: viral capsid protein 1; NF-κB: nuclear factor kappa B; OR: odds ratio; CI: confidence interval.

There was significantly increased expression of IL-6, NS1, TNF-α, and VP1 in the thyroid cancer patients relative to the controls (p = 0.0015, fold changes of 4.76; p = 0.0012, fold changes of 4.82; p = 0.009, fold changes of 3.86; p = 0.041, fold changes of 2.95, respectively; Figure 2).

Figure 2.

Comparison of IL-6, NS1, TNF-α, and VP1 gene expression in the cancer tissue and controls.

ROS and RNS assay

The levels of RONS in thyroid cancer cells were statistically higher than the normal cells (p = 0.022 and p = 0.043, respectively). Presence of parvovirus B19 has a positive correlation with RONS level (r = 0.13, p = 0.38; r = 0.15, p = 0.33, respectively).

Discussion

Several factors such as inflammatory mechanism may be involved in the development and progression of cancer. Infectious agents are one of the main causes of inflammatory mechanism. For example, B19 may lead to downregulation of thyroid hormone receptor alpha (THRα) and retinoid X receptor alpha (RXRA); this downregulation leads to alteration in expression of THR/RXRA reregulated genes which are involved in thyroid tumorigenesis.⁴ In this study, we observed high positive rate of B19 DNA (86.11%). Furthermore, the prevalence of B19 infection in different types of thyroid cancer is as follows: 17 papillary, 11 medullary, and 3 follicular samples. This study suggests that B19 infection may play a key role in tumorigenesis and thyroid cancer progression via an inflammatory mechanism. Wang et al. detected B19 infection in papillary thyroid cancer (PTC) tissue samples using nested PCR, in situ hybridization (ISH), and immunohistochemistry (IHC) in 95%–97%, 83.3%, and 63% of cases, respectively.²⁶ Page et al.¹² showed the presence of B19 in thyroid tissue by IHC and quantitative PCR (qPCR). Adamson et al. found B19 capsid protein in 88% of PTC cells by IHC. They showed an obvious difference in the localization of detected capsid protein between tumor tissue and normal tissue in the same patient. This phenomenon proposed a shift in the microenvironment that is ideal for B19 infection.¹³ B19 DNA was detected in 16 (27.1%), IgM in 3 (5.1%), and IgG in 36 (61%) patients by Soliman et al.¹⁴ Because of high proliferation activity of thyroid cancer cells, rate of B19 infection in these cells is higher than that in the normal thyroid tissue.¹⁵ We demonstrated that almost in all samples, the levels of NS1, NF-κB, TNF-α IL-6, and RONS were simultaneously high. Such an increase in the level of cytokines and RONS could be due to the inflammation and may/could lead to thyroid cancer progression. Cytokine levels are affected by several factors, including the viral load, inflammation, and disease phases.^16–18 NS1 transactivates cellular promoters such as IL-6 and TNF-α through the NF-κB-binding site in the IL-6 promoter. However, IL-6 and TNF-α are considered as inflammatory agents.^4–6 Al-Gharrawi et al. demonstrated that IL-6 could play a role in the immunological microenvironment of thyroid tumors and IL-6 expression could be increased by B19 infection.¹⁹ Adamson also indicated in another study that B19 infection increased in adenoma and thyroid tumor, and this rise could be correlated with IL-6 expression in thyroid tissue and may result in/contribute to increased inflammation.²⁰ Kerr et al. reported that 13 of the 51 patients (25%) with B19 virus infection had detectable levels of IL-6 by ELISA. They further showed that TNF-α and interferon gamma (IFN-γ) levels were raised during B19 infection.⁶ In similar investigation, Tominga et al. indicated that IL-6 leads to reduced T3 secretion and inhibits thyroid-stimulating hormone (TSH)-induced peroxidase gene expression in cultures of human thyroid follicular cells (TFCs).²¹ High levels of IL-6 in pre-malignant cells can signify that this cytokine serves as a growth and survival factor that acts on these cells.²² Also, IL-6 is a protumorigenic cytokine and is initially suspected to activate the oncogenic transcription factor NF-κB.²³ However, several studies have demonstrated the role of NF-κB in solid tumors.⁷ VP1 was detected in 28% of tumor tissues and 8% of normal tissues. In a similar study, VP1 detection rate in tumor tissue and normal tissue was 83% and 6.25%, respectively.²⁴ These results support the hypothesis that B19 may play a direct or indirect role in the pathogenesis of thyroid cancer.

It is not an easy task to determine a correlation between cancer and a viral infection. Based on the results of B19 DNA and measurement of IL-6, TNF-α, NF-κB, NS1, VP1, and RONS levels in thyroid cancer, we arrived at the conclusion that there is a correlation between parvovirus B19 and this type of cancer. However, further work is required to investigate the exact role of B19 infection in thyroid cancer.

Footnotes

Acknowledgements

The authors are greatly thankful to the director and staff of hospitals in Tehran and Shahrekord, Iran.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

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