Journal of Infection (2010) 60, 360e370

www.elsevierhealth.com/journals/jinf

Parvovirus B19 infection associated withHashimoto’s thyroiditis in adults

Juanhong Wang a,b, Weiping Zhang a, Hongxiang Liu c, Di Wang a,Wenqing Wang a, Yuanfei Li a, Zhe Wang a, Lu Wang a, Wei Zhang d,Gaosheng Huang a,*

a State Key Laboratory of Cancer Biology, Department of Pathology, Xijing Hospital, Fourth Military Medical University, No.17, Changle West Road, Xi’an 710032, PR Chinab Department of Pathology, Xi’an Central Hospital, Xi’an 710003, PR Chinac Department of Histopathology, Addenbrooke’s Hospital, Cambridge University Hospitals NHS Foundation Trust,Cambridge CB2 2QQ, UKd Department of Pathology, Tangdu Hospital, Fourth Military Medical University, Xi’an 710038, PR China

Accepted 7 February 2010Available online 12 February 2010

KEYWORDSHashimoto’s thyroiditis;Parvovirus B19;Viral protein

Abbreviations: B19, parvovirus B19; nlaser-capture microdissection; HT, Haular thyroid carcinoma; TMC, thyroid mcommon antigen.

* Corresponding author. Tel.: þ86 0E-mail address: huanggs@fmmu.ed

0163-4453/$36 ª 2010 The British Infdoi:10.1016/j.jinf.2010.02.006

Summary Objective: Parvovirus B19 is a common human pathogen, which has been linked toautoimmune diseases recently. The aim of the study is to evaluate whether B19 is involved inadult Hashimoto’s thyroiditis (HT).Methods: Eighty-six thyroid tissues from the adult patients with a spectrum of thyroid disor-ders were examined for B19 DNA and capsid protein by nested PCR, in-situ hybridization andimmunohistochemistry. The presence of viral DNA in HT epithelium was studied by laser-capture microdissection and sequencing of PCR products. The expressions of nuclear factor-kB (NF-kB) and interleukin-6 were investigated by immunohistochemistry.Results: B19 DNA was significantly present in HT tissues by both PCR (29/32, 90.6%) and in-situhybridization (23/32, 71.9%, all p < 0.01) compared with normal thyroid tissue (7/16, 43.8%; 2/16, 12.5%). Laser-capture microdissection further confirmed this difference. B19 capsid pro-tein in HT group was significantly higher than that in all the control groups (p < 0.01), andthe expression of NF-kB and interleukin-6 in HT tissues was up-regulated. NF-kB was wellco-localized with B19 protein in thyroid epithelia by double-labeling immunofluorescenceand confocal microscopy.

PCR, nested polymerase chain reaction; ISH, in-situ hybridization; IHC, immunohistochemistry; LCM,shimoto’s thyroiditis; NTT, normal thyroid tissues; NTMG, non-toxic multinodular goiters; FTC, follic-edullary carcinoma; NF-kB, nuclear factor-kB; IL-6, interleukin-6; CK8, cytokeratin 8; LCA, leucocyte

29 8477 4946; fax: þ86 029 8325 5697.u.cn (G. Huang).

ection Society. Published by Elsevier Ltd. All rights reserved.

B19 infection and HT in adults 361

Conclusions: The presence of B19 nuclear acid and viral protein was significantly common in HTtissues and it suggested a possible role of B19 in adult HT.ª 2010 The British Infection Society. Published by Elsevier Ltd. All rights reserved.

Introduction

Hashimoto’s thyroiditis (HT) is an organ-specific autoim-mune disorder, which is the most frequent cause ofhypothyroidism and goiter.1,2 This condition is named afterDr. Hakaru Hashimoto who first described it in 1912. HT usu-ally affected middle-aged females. It is characterized asgradual destruction of thyroid follicles, infiltration of thethyroid with mononuclear cells and production of variousautoantibodies such as anti-thyroglobulin and anti-thyroperoxidase antibodies.1,2 HT is quite complex, likemost other autoimmune diseases, numerous etiologicaland pathogenic factors involving genetic, hormonal and en-vironmental constituents participate in the initiation ofHT.2 Environmental agents such as virus infection havebeen investigated as potential etiologies of HT,3,4 but theetiology and pathogenesis of HT remain undetermined.

Infection with parvovirus B19 (B19) is a global concernand the infection rate is similar in the United States, Europeand Asia,5 with approximately half of 15-year-old adoles-cents and over 60% of adults being seropositive.6 Both inchildren and adults, B19 infections is generally regardedas a cause or trigger of various forms of autoimmune di-seases,7 such as rheumatoid arthritis (RA), idiopathicthrombocytopenic purpura (ITP),8 systemic lupus erythe-matosus (SLE) and other autoimmune hematologic diseases.However, few literatures are available on B19 involvementin autoimmune thyroiditis. More recently, Lehmann HWet al. analyzed the presence of viral DNA and antibodiesagainst B19 proteins in serum samples from children withHT and concluded that acute parvovirus B19 infections areinvolved in the pathogenesis of HT in children.9 Mori Ket al. reported an adult patient with HT in whom B19 DNAhas been persistently detected in the thyroid.10 Neverthe-less, whether B19 infection is associated with Hashimoto’sthyroiditis in adults still remains unknown.

B19 is a small non-enveloped single stranded DNA viruswith a genome size of 5596 bp. The viral genome encodesthree major proteins: the nonstructural protein NS1 and twoviral capsid proteins VP1 and VP2.5 VP1 is the same as VP2except for an additional unique portion (VP1u) of 227 aminoacids at its amino terminal. NS1 is cytotoxic to host cells.5,11

VP1 and VP2 which form the icosahedral viral capsid are im-munogenic.5,12,13 We previously reported that B19 proteincan activate and up regulate the expression of nuclear fac-tor-kB (NF-kB).14 It is also reported by Moffatt S et al. thatB19 NS1 protein can activate proinflammatory cytokineinterleukin-6 (IL-6) gene expression through the NF-kB bind-ing site in the IL-6 promoter.15 Both NF-kB and IL-6 arethought to be pivotal in triggering the various inflammatoryand autoimmune disorders.16 In addition, a secreted phos-pholipase A2 (PLA2) motif has been identified in the VP1u re-gion of B1917 and the VP1u-associated PLA2 activity isnecessary for the induction of autoimmune reactions.18

In the light of the above, we wonder whether humanparvovirus B19 is involved in the pathogenesis of adult HT

due to its ability to infect human individuals and to induceautoimmunity in humans. In order to examine the associ-ation between B19 and adult HT, we have done a retrospec-tive study, which indicates a potential role of B19 in thepathogenesis of HT.

Materials and methods

Specimen collection

In this study, 70 paraffin-embedded thyroid tissue blocksfrom the patients with HT (32 samples), non-toxic multi-nodular goiter (NTMG, 19 samples), follicular thyroidcarcinoma (FTC, 10 samples) and thyroid medullary carci-noma (TMC, 9 samples) were retrieved from the pathologyarchives of Xijing Hospital from 2000 to 2004. Normalthyroid tissues (NTT, 16 samples) were obtained from theareas surrounding surgically removed adenomas. The age ofthe 32 HT patients ranged from 22 to 69 and all of themwere female except one. Statistical analysis showed thatthere was no significant difference between HT patientsand control groups (All p > 0.05) in age and gender. All theHT patients and control cases were of Chinese origin. Thediagnoses were confirmed histologically by two patholo-gists. Specimen collection and the study procedures wereapproved by the Xijing Hospital Ethics Committee. Table 1summarizes the diagnoses and clinical data from thepatients.

Nested PCR detection and DNA sequence analysis

DNA was extracted from the paraffin-embedded tissues.19

The integrity of the extracted DNA fragments was con-firmed by PCR amplification of a 110bp fragment of b-glob-ulin DNA by using the primer pair 50-ACACAACTGTGTTCACTAGC-30 and 50-CAACTTCATCCACGTTCACC-30.19

Nested PCR was used to amplify B19 DNA.20 The first-round PCR was carried out with outer primers 50-AGC ATGTGG AGT GAG GGG GC and 50-AAA GCA TCA GGA GCT ATACTT CC (nucleotides 3370e3389 and 3659e3637, respec-tively). The second round PCR was carried out with the in-ner primers 50-GCC AAC TCT GTA ACT TGT AC and 50-AAATAT CTC CAT GGG GTT GAG (nucleotides 3400e3419 and3572e3552, respectively), which amplified a region of theB19 VP1/VP2 capsid protein sequence. The reaction wasperformed under the following conditions: denaturationat 94 �C for 5 min; amplification for 30 cycles at 94 �C for30 s, 56 �C for 30 s, and 72 �C for 45 s; and extension at72 �C for 10 min. After the first-round PCR, 1 mL of thefirst-run PCR products was used as a template for the sec-ond round PCR, which yielded a 173 bp product. All thenested PCR reactions were performed in duplicate and inparallel with positive and negative controls. pGEM-1/B19plasmid carrying the whole genome of B19 (provided byProf. J. P. Clewley of Central Public Health Laboratory, Lon-don, UK) and DNA obtained from paraffin-embedded kidney

Table 1 Clinical, nested PCR, in-situ hybridization and immunohistochemistry data of thyroid diseases.

Cases Age, y Sex B19 nested PCR B19 in-situ hybridization Immunohistochemistry

B19 VP1/VP2 NF-kB p65 IL-6

Hashimoto’s Thyroiditis1 41 F � � � � þ2 22 F þ þ þ þ þ3 45 F þ þ þ þ þ4 62 F þ þ þ þ þ5 38 F þ þ þ þ þ6 36 F þ � � þ �7 52 F þ � � þ þ8 68 F þ þ þ þ þ9 58 F þ þ � � �10 48 F þ � � � �11 52 F þ � � � �12 58 F þ þ þ þ �13 44 F þ þ þ þ þ14 40 F þ þ þ þ þ15 65 F þ þ þ þ þ16 48 F þ þ þ þ þ17 41 F þ þ � � þ18 40 F þ � � � �19 53 F þ þ � þ �20 53 F þ þ � � �21 67 M þ � � þ �22 57 F þ þ � þ þ23 50 F þ þ þ þ þ24 54 F þ þ þ þ þ25 54 F þ þ þ � þ26 41 F � � � � þ27 35 F þ þ þ þ þ28 44 F þ þ þ þ þ29 69 F þ þ þ þ ND30 66 F � � � � �31 55 F þ þ þ � �32 40 F þ þ þ þ þ

Normal thyroid tissue1 36 F þ � � � ND2 42 F � � � þ þ3 67 F � � � � �4 59 F þ � � ND ND5 44 F þ þ � ND ND6 25 F � � � � �7 57 F þ þ þ þ ND8 49 F � � � � �9 60 M � � � � ND10 45 F þ � � � ND11 58 F þ � � � ND12 36 F � � � � �13 51 F � � � � ND14 40 F þ � � � ND15 32 F � � � � ND16 48 F � � � � ND

Non-toxic multinodular goiter1 28 F þ þ � ND ND2 40 F þ þ � ND ND3 53 F þ þ þ ND ND4 50 F þ þ � ND ND

362 J. Wang et al.

Table 1 (continued )

Cases Age, y Sex B19 nested PCR B19 in-situ hybridization Immunohistochemistry

B19 VP1/VP2 NF-kB p65 IL-6

5 35 F þ þ � ND ND6 51 M � � � ND ND7 44 M þ þ � ND ND8 62 M þ þ þ ND ND9 21 F � � � ND ND10 39 F þ þ � ND ND11 54 F þ þ � ND ND12 57 F þ � � ND ND13 54 F þ þ � ND ND14 41 F þ � � ND ND15 46 F þ þ � ND ND16 48 F þ þ � ND ND17 51 F þ � � ND ND18 32 F þ þ � ND ND19 63 F þ þ � ND ND

Follicular thyroid carcinoma1 37 F þ þ � � �2 72 F þ þ � þ �3 35 M þ � � � ND4 57 F þ � � � ND5 56 F þ þ � � þ6 33 F þ þ � � �7 64 F þ þ � � �8 57 F þ þ � � ND9 41 F þ þ � � �10 70 F þ � � � �

Medullary thyroid carcinoma1 33 F � � � � �2 68 F � � � � �3 68 F þ þ � þ �4 55 M þ � � þ �5 30 F þ � � � �6 39 F � � � � �7 58 M � � � � �8 46 F þ � � � �9 32 F þ � � � �F, female; M, male; �, negative; þ, positive; ND, not done.

B19 infection and HT in adults 363

tissue of a hydropic fetus (16 weeks’ gestation) which hadbeen proved to have B19 infection by nested PCR, B19DNA ISH and IHC were used as positive controls in parallel.Reaction without template and DNA obtained from fetalkidney tissues of therapeutic abortion (13 weeks’ gestation)which had no clinical, histological or serological evidence ofB19 infection was used as negative controls. The amplifiedDNA with the expected size was purified with Qiaquick PCRpurification kit (Qiagene, Germany) and sequenced.

In-situ hybridization detection

The nPCR products of B19 DNA amplified by TaKaRaPyrobest DNA polymerase (TaKaRa Biotech) were purifiedand labeled to generate the B19 DNA probe by random-primed incorporation of digoxigenin-labeled dUTP by using

a Dig DNA labeling and detection kit (Roche, Germany). TheISH was then performed according to previously describedmethods.21 Briefly, paraffin sections of 4 mm thickness weredeparaffinized and hydrated, then placed in 0.2 M HCl for10 min and followed by digestion in 25 mg/ml proteinase K(Merck, Darmstadt, Germany) for 10 min at 37 �C. Aftertwo washes with PBS, the sections were dehydrated, air-dried, and incubated with pre-hybridization solution at42 �C for 30 min. The sections and the B19 probe (with a fi-nal concentration of 200 ng/mL) were simultaneously dena-tured at 95 �C for 10 min, chilled on ice and incubated at42 �C overnight. The slides were then washed sequentiallyfor 15 min twice with SSC containing 10% (w/v) SDS respec-tively, blocked with blocking buffer at 37 �C for 30 min, andincubated for 2 h with anti-digoxigenin-alkaline phospha-tase conjugate. After two 15-min washes with washing

364 J. Wang et al.

buffer, the sections were stained with NBT/BCIP (Roche,Mannheim, Germany) solution until satisfactory blue signalswere detected. The sections were then dehydrated andmounted with neutral balsam prior to cover slipping. Thesections of the kidney tissues were used as positive andnegative control tissues in each test run. Moreover, the du-plicated sections of HT were hybridized with pre-hybridization mixture or digoxigenin-labeled pBR328 DNAthat was linearized with Bam HI as negative controls.

Immunohistochemical staining

Paraffin sections of 4 mm thickness were deparaffinizedand treated with 3% (v/v) hydrogen peroxide to blockendogenous peroxidase activity. Heat-induced antigenretrieval was performed in 0.01 M sodium citrate (PH6.0) and 10% (v/v) bovine serum albumin (BSA; Sigma,USA) in PBS at room temperature for 10 min to block thenon-specific antibody-binding sites. Then the sectionswere incubated overnight at 4 �C with mouse monoclonalantibody against the B19 proteins VP1/VP2 (1:20 dilution;clone R92F6, Novocastra, UK), with the mouse mono-clonal antibody against the nuclear transcription factorsubset NF-kB p65 (1:400 dilution; clone sc-8008, SantaCruz, USA) and with the mouse monoclonal antibodyagainst the interleukin-6 (1:200 dilution; clone B-E8,Chemicon, CA, USA), respectively. A streptavidin-biotin-peroxidase complex kit (SABC kit, Zymed, USA) was usedto detect the protein conjugates.22 The sections were de-veloped with a diaminobenzidine substrate (Sigma, St.Louis, MO) and counterstained with hematoxylin. The pri-mary antibodies replaced with PBS and mouse IgG1 (AMS/Immunokontact) were used as negative controls. In eachbatch of the sections tested for B19 VP1/VP2-antigen,the kidney tissues were used as positive and negativecontrol tissues.

Double staining of immunohistochemistry andin-situ hybridization

Thyroid epithelial cells and lymphocytes were marked bya mouse monoclonal anti-CK8 antibody (1:50 dilution, Dako,Denmark) and a mouse monoclonal anti-LCA antibody(1:100 dilution, Dako, Denmark), respectively. The sectionswere then hybridized with B19 specific DNA probe.

Double-labeling immunofluorescence assay

Paraffin-embedded sections went through deparaffiniza-tion, antigen retrieval, endogenous peroxidase quenchingand blocking. Then the sections were first incubated withthe mixture of the rabbit polyclonal anti-NF-kB p65 anti-body (1:50 dilution; Santa Cruz, CA, USA) and the mousemonoclonal anti-VP1/VP2-antigen antibody (1:10 dilution)overnight at 4 �C in a humid chamber. Next, they wereincubated by fluorescent antibody mixture of Texas-red-conjugated goat anti-rabbit and FITC-conjugated goat anti-mouse antibodies (both in 1:50 dilution and both fromDako, CA, USA) in PBS for 2 h. Finally, they were washedand mounted in aqueous mounting media and were

analyzed by Olympus FV1000 confocal microscope (Olym-pus; Tokyo, Japan).

Laser-capture microdissection, nested PCR andsequencing

Three representative specimens of HT and three normalthyroid specimens were subjected to LCM by the LeicaMicrosystems Wetzlar GmbH (Germany) according to themanufacturer’s protocols. First, the sections were labeledby IHC. The slides were dehydrated, cleared by xylene, andair-dried for 1 h. Next, LCM was performed under directmicroscopic visualization of the immunolabeled areas.Each section was overlaid by thermoplastic membraneand cells were captured by focal melting of the membraneby laser activation. Each of the captured samples con-tained 50e100 cells. The PicoPure DNA-Extraction kit(Qiagene, Germany) was used to isolate DNA, and thenPCR amplification, analysis and sequencing were per-formed. The individual sequence was used in a BLASTsearch against Genbank B19 sequences (National Center forBiotechnology Information).

Statistical analysis

Statistical analysis was performed by using Statistical Pro-gram for Social Sciences (SPSS) software (version 10.0, SPSSInc, Chicago, USA). The differences of gender and agedistributions among the groups were compared by PearsonChi-square (c2) test and unpaired student’s t test respec-tively. Data from the experimental results were analyzedby Pearson Chi-square (c2) test or Fisher’s exact test. Atwo-sided p value of less than 0.05 was consideredsignificant.

Results

Histopathology

Histologically, all the specimens of HT included in this studywere characterized as diffuse lymphocytic infiltration withwell-developed germinal centers in stroma. The enlargedthyroid epithelial cells with granular and pink cytoplasm,called Hurthle cells were clearly found in each case. All thenormal thyroid tissues showed normal morphology. Thesamples from the patients with non-toxic multinodulargoiter were characterized as many nodules, coexistingwith atrophic and hyperplastic thyroid follicles, as well asold hemorrhage and fibrosis in stroma. Thyroid nodule,which contained groups of cells invading the broad collag-enous capsule and/or adjacent tissues, was the criteria forFTC. Spindle or round cell tumor with amyloid stroma wasthe diagnostic feature of TMC.

Nested PCR assay for parvovirus B19 DNA

PCR products of 173 bp in size as predicted in the second roundPCR were amplified in 29 (90.6%) of 32 HTspecimens, 7 (43.8%)of 16 normal controls, 17 (89.5%) of 19 NTMG specimens, 10 of10 FTC and 5 of 9 TMC (Fig. 1). Sequencing of the randomly se-lected nPCR products confirmed that these amplicons

Figure 1 Nested PCR amplification of parvovirus B19 DNAsequences in Hashimoto’s thyroiditis. Representative agarosegel electrophoresis of nested PCR products. Lane M: 100 bpDNA Ladder. Lane P: Positive control. Lane N: Negative control.Lane 1, 2, 3, 4, 6 and 7: Positive samples. The amplicons areapproximately 173 bp in size. Lane 5: Negative sample.

B19 infection and HT in adults 365

originated from B19 DNA (GenBank accession no. GI9632996).Statistically, the positive rate of B19 DNA detected by nPCRin HT group was significantly higher than that of NTT(p Z 0.001) and that of TMC (p Z 0.031), respectively, butnot different than NTMG and FTC (both p Z 1.000).

In-situ hybridization assay for parvovirus B19 DNAin tissue sections

ISH for B19 DNA was performed in tissue sections of HT andcontrol specimens to confirm the local infection of the virusand its cellular distribution. The positive ISH signals werelocated in the nuclei of epithelial cells in different thyroiddisorders and they were also seen in infiltrating lympho-cytes in HT (Fig. 2A). In HT, the thyroid follicle epithelialcells showed strong nuclear staining (Fig. 2B), but no stain-ing or rather weak intensity was observed in the specimensof normal thyroid (Fig. 2C). The negative controls showedno staining, but the positive B19 control sample showedstrong staining distributed in the tissue section. The viralDNA was detected in 23 (71.9%) of 32 HT, 2 (12.5%) of 16normal, 14 (73.7%) of 19 NTMG, 1 of 9 TMC and 7 of 10FTC specimens. Statistically, the positive rate of B19 DNAdetected by ISH in HT group was significantly higher thanthat of NTT (p Z 0.0001) and that of TMC (p Z 0.002), re-spectively, but not different than NTMG (p Z 0.889) andFTC (p Z 1.000).

Immunohistochemistry assay for parvovirus B19VP1/VP2-antigen

The in-situ detection of viral protein showed that most ofthe positive IHC signals in 18 (56.3%) of 32 HT specimenswere from cytoplasm of the follicle epithelial cells (Fig. 2Dand E), and some of them were from nuclei of isolated orscattered infiltrating lymphocytes (Fig. 2F). The positivestaining in 1 (6.3%) of 16 NTT and 2 (10.5%) of 19 NTMGwas observed only in a few epithelial cells. In 15 of 16 nor-mal thyroid samples, normal epithelium demonstrated noexpression of VP1/VP2-antigen (Fig. 2G). The positive con-trol sample of B19 showed clear positive staining distrib-uted in the tissue section while the negative controlsshowed no evidence of VP1/VP2-antigen staining(Fig. 2H). 10 FTC and 9 TMC samples showed no expressionof viral protein. Statistically, the positive rate of B19 VP1/VP2-antigen in HT group was significantly higher than thatof all control groups (p < 0.01).

Double staining of immunohistochemistry and in-situ hybridization

Double-labeling method that combined immunohistochem-istry with in-situ hybridization showed that the B19-infected cells were both thyroid epithelial cells (Fig. 3A)and infiltrating lymphocytes (Fig. 3B).

Immunohistochemistry assay for NK-kB and IL-6

The positive signal of NF-kB in both cytoplasm and nuclear(Fig. 4A) was detected in 21 of 32 HT specimens by IHC. Theexpression of NF-kB greatly increased when compared withthat of control groups (Table 2, p < 0.05) and the increasedexpression correlated with the expression of B19 VP1/VP2-antigen (Fig. 4B, p < 0.001). The positive signal of IL-6 incytoplasm (Fig. 4C) was detected in 20 of 31 HT specimensby IHC and the expression increased greatly when com-pared with control groups (Table 2, p < 0.05), with one ex-ception - the normal control group due to a few samplestested in NTT.

Double-labeling immunofluorescence assay forparvovirus B19 antigen and NF-kB

We found plasma/nuclei expression of NF-kB (red, Fig. 4D)and robust plasma/nuclei presence of VP1/VP2 (green,Fig. 4E) in HT tissues using double-labeling immunohisto-chemistry. Superimposition of the images demonstratedthat the majority of cells showed co-localization of bothproteins in the plasma/nuclei of thyroid epithelia cells (yel-low, Fig. 4F).

Laser-capture microdissection, nested PCR andsequencing

LCM was performed in 3 samples of HT that were positivefor VP1/VP2-antigen (Fig. 5A1 and A2) and in 3 samples ofNTT that was negative for both B19 genome and VP1/VP2-antigen (Fig. 5B1 and B2). Nested PCR analysis demon-strated the presence of DNA fragment of the VP1/VP2-antigen coding regions in all the 3 HT samples while nonein the normal controls (Fig. 5C). Sequencing analysisshowed that all the PCR amplicons were from human parvo-virus B19 genome. Moreover, the randomly distributedpoint mutations in the VP1/VP2 region of the B19 isolateswere also revealed. It is noteworthy that the B19 isolatesshowed sequence differences when compared with eachother. These sequence variations may be due to differentB19 genotypes as discussed previously.23 Therefore, cross-contaminations of the patient’s samples with B19 thatmay especially occur during nPCR methods could beexcluded.

Discussion

In the light of the evidence implicating B19 in some patientswith autoimmune diseases,7,8,24 we extended our study toHT, the most common organ-specific autoimmune diseasein the world. In our study, by nested PCR and ISH, we found

Figure 2 In-situ hybridization (ISH) and immunohistochemistry staining (IHC). (A) and (B) DNA ISH for B19 in the thyroid tissue ofa patient with HT. (A) The infiltrating lymphoid tissue and thyroid follicles show strong nuclear staining (ISH, original magnifica-tion � 100). (B) Thyroid follicles show positive nuclear staining (ISH, original magnification � 400). (C) The staining intensity israther weaker in epithelia of normal thyroid tissue (ISH, original magnification � 200). (D), (E) and (F) Staining for B19 proteinsby IHC in the thyroid tissue of the patients with HT. (D) Many thyroid glands between the lymphoid follicles show positive stainingfor B19 protein (IHC, original magnification � 40). (E) Positive IHC signals in many thyroid epithelial cells around a lymphoid follicle(IHC, original magnification � 100). (F) Positive IHC signals in both the numerous thyroid epithelial cells and scattered infiltratinglymphocytes (red arrow) (IHC, original magnification � 200). (G) Normal epithelium demonstrates no expression of VP1/VP2-antigen in sample of normal thyroid (IHC, original magnification � 200). (H) Negative control, the primary antibody was replacedwith mouse IgG1 (IHC, original magnification � 100). Diaminobenzidine was used as chromogen and hematoxylin was used forcounterstain.

366 J. Wang et al.

Figure 3 Double-labeling of in-situ hybridization (ISH) and immunohistochemistry (IHC). (A) Many thyroid epithelial cells are bothpositive for CK8 immunoassaying and B19 DNA detection by ISH in Hashimoto’s thyroiditis (HT) (ISH-IHC, originalmagnification � 200). (B) Numerous lymphocytes are both positive for LCA immunoassaying and B19 DNA detection in HT (ISH-IHC, original magnification � 400).

B19 infection and HT in adults 367

B19 DNA was in high proportion of diseased thyroid tissuesof HT, NTMG and FTC, whereas it was significantly lowerin NTT and TMC. Also, in this study, laser-capture microdis-section confirmed that B19 DNA was present in HT thyroidtissues but absent in normal thyroid tissues. By IHC, the

Figure 4 Immunohistochemical (IHC) expression of NF-kB and iNF-kB in samples of Hashimoto’s thyroiditis (HT). (A) Expression of(IHC, original magnification � 200). (B) The thyroid follicular epitheoriginal magnification � 200). (C) Expression of the interleukin-6 in(IHC, original magnification� 100). (D, E and F) Double-labeling immroid follicular epithelial cells (Texas-red, red. original magnification�green. original magnification � 600). (F) Superimposition of the imagproteins in the cytoplasm and nuclei of thyroid follicular epithelial c

viral capsid proteins were detected only in 6.3% of NTT,10.5% of NTMG and none of FTC and TMC samples, however,VP1/VP2-antigen was expressed in many of the HT samples(56.3%, p < 0.01). Moreover, our findings also showed thatboth B19 nucleic acid and proteins were mainly prevalent

nterleukin-6 and co-localization of B19 VP1/VP2-antigen andNF-kB p65 in thyroid follicular epithelial cells in samples of HTlial cells in samples of HT also expressed VP1/VP2-antigen (IHC,cytoplasm of thyroid follicular epithelial cells in samples of HTunofluorescence in samples of HT. (D) Expression of NF-kB in thy-

600). (E) Robust cytoplasm presence of VP1/VP2-antigen (FITC,es demonstrates a majority of cells with co-localization of bothells of HT (yellow, original magnification � 600).

Table 2 Immunohistochemical expression of NF-kB and interleukin-6 and co-localization of B19 VP1/VP2-antigen and NF-kB.

Diagnosis No. of Patients(Positive cases/Total cases)

VP1/VP2-antigen NF-kB VP1/VP2-antigen/NF-kB Interleukin-6

Hashimoto’s thyroiditis 18/32 21/32 16/32 20/31Normal thyroid tissue 1/16a 2/14a 1/14 1/5Non-toxic multinodular goiter 2/19a ND ND NDFollicular thyroid carcinoma 0/10a 1/10a 0/10 1/7a

Thyroid medullary carcinoma 0/9a 2/9a 0/9 0/9a

a p < 0.05, p value was calculated by 2-tailed Fisher exact test comparing each group individually to the group of Hashimoto’sthyroiditis.

368 J. Wang et al.

in the thyroid follicular epithelial cells of HT. In general,the detection of a viral genome in human tissues only indi-cates previous infection of the virus and the viral proteinexpression is correlated with the effective replication ofthe virus. In this study, the detection of the persistence

Figure 5 Laser-capture microdissection and nested PCR amplifi(A1eA2) HT samples were immunolabeled for VP1/VP2-antigen aand microdissected (IHC, original magnification � 200). (A1) Tissu(B1eB2) Glands from normal thyroid were microdissected (IHC, orig(B2) Tissues after microdissection. (C) Products of nested PCR fromSpecimens from normal thyroid; Lane 1e3: Specimens from HT; Lanwere approximately 173 bp in size.

of B19 viral genome with expressed viral proteins in the thy-roid follicular epithelial cells indicated B19 replication andprotein synthesis,25 which means a productive infection ofthe virus that may lead to the cellular alterations in the dis-eased thyroid epithelium and stimulated autoimmune

cation for parvovirus B19 DNA in Hashimoto’s thyroiditis (HT).nd the glands that expressed VP1/VP2-antigen were selectedes before microdissection. (A2) Tissues after microdissection.inal magnification � 200). (B1) Tissues before microdissection.microdissected tissues. Lane M: 100 bp DNA Ladder. Lane 10e30:e N: Negative control; Lane P: Positive control. The amplicons

B19 infection and HT in adults 369

reaction of HT in some pathways. On the contrary, withoutthe expression of virus protein, the persistence of B19 ge-nome mainly denotes latent infection. In addition, the ex-pression of NF-kB and IL-6 responsible for inflammatoryand autoimmune disorders greatly increased in HT whencompared with control groups (Table 2) and the increasedexpression of NF-kB was significantly correlated with theexpression of B19 VP1/VP2-antigen (p < 0.001). In short,our study suggested that B19 infection may be linked toadult HT pathogenesis.

Most people are exposed to B19, as there is the presenceof antibodies specific for the virus in almost 60% of adults.6

In our present study, the positive rate of B19 DNA detectedby nPCR in NTT and TMC was similar to that in general pop-ulation (43.8% and 55.6%, respectively) while the positiverate in other diseased thyroid tissues was significantlyhigher than that in the general population (89.5% in NTMGand 100% in FTC, respectively). Generally, the virus B19 ina normal person has a relatively simple pathogenesis, andafter the acute phase, the virus can be eradicated fromthe blood by a specific humoral immune response.26 How-ever, the viral genomes can deposit and persist in humantissues in a latent state for lifelong after primary infec-tion,23 which explains why many normal and diseased thy-roid tissues although containing parvovirus DNA, do notproduce proteins (VP1/VP2-antigen negative). The differ-ent positive rates of B19 DNA in different thyroid diseasescould be attributed to the preference for the rapidly divid-ing cells in S phase for virus replication.27 It is reported thatthe proliferative activity of HT, NTMG and FTC is higherthan that in NTT.28 Therefore, it is comprehensible thatB19 infection rates in these groups are higher than that inNTT in this study. However, as mentioned above, only B19productive infection in adult HT is significant.

It is well known that parvovirus B19 can only infect thecells that have the proper receptors which the virus canattach to. Globoside, also known as the blood group Pantigen, has been confirmed to be the indispensablecellular receptor for B19 infection.29 According to someprevious studies, P antigen is present on erythroid progen-itors and a variety of other cells.30 Human thyroid follicularepithelial cells and lymphocytes have also been proved tohave globoside (blood group P antigen).31,32 Therefore,the expression of the specific cellular receptor of B19 inthese cells might account for their susceptibility to B19 in-fection. In our present study, by IHC-ISH double staining as-say, both thyroid epithelia and lymphocytes were found tobe target cells in HT for B19 infection. However, by IHC wefound the virus protein expressed mainly in thyroid epithe-lial cells (Fig. 2F). So the different distribution of specificreceptor for B19 infection could explain why the positiverate of detected B19 DNA in thyroid disorders originatingfrom thyroid follicle epithelial cells was significantly higherthan that in TMC originating from C-cells (p Z 0.006).

The autoimmune destruction of the HT-afflicted thyroid,which involves both cellular and humoral immunity,2 is socomplex, that it remains to determine the etiology andpathogenesis of HT. During the process of the initiation ofHT, active T helper cells are central to the generation ofan immune response to foreign and self-antigens.2 Interest-ingly, B19 can activate helper T cells through its VP1 andVP2 antigen stimulation.12,13 A phospholipase A2 activity

is located in the VP1u region of the B19 VP1 protein33 andthe proteins with VP1u may participate in the inflammatoryresponse linked to B19 infection.34 In our study, the expres-sion of VP1/VP2-antigen was extremely higher in HT sam-ples, which indicated the potential role of viral protein inthe pathogenesis of HT. In addition, previous studies indi-cated that the infiltrating mononuclear cells of HT inducean enhanced expression of NF-kB and IL-6,35 which playan important role in the inflammatory response and tissueinjury in HT. Our study showed that B19 can also activateNF-kB and IL-6 expression and NF-kB was well co-localizedwith B19 protein in thyroid epithelial cells (Fig. 4). There-fore, their increased expression may lead to the inflamma-tory and autoimmune disorder.16 Furthermore, it isreported that anti-parvovirus antibodies such as anti-VP1IgG can specifically recognize and react with human cyto-keratin,36,37 which is a protein widely distributed in epithe-lial cells. The expression of cytokeratin 8 in the thyroidfollicular epithelial cells was identified by the immunohis-tochemical analysis in our study. So the antibodies againstparvovirus B19 viral capsid antigen can react with the cyto-keratin in thyroid follicular epithelial cells and then it maycause thyroid cell damage and autoimmune disorder. Com-bination of different factors is responsible for the develop-ment of HT and B19 infection may play a role in thepathogenesis of adult HT.

In summary, we found for the first time that both B19genome and viral capsid proteins exist in a high proportionof thyroid tissues from adult HT patients. Our studyprovides meaningful evidence for B19 infection in adultHT tissues and suggests a potential role of B19 in thedevelopment of adult HT. However, further work is requiredto investigate whether and how human parvovirus B19participates in the pathogenesis of HT.

Conflict of interest

The authors state that there is no conflict of interest.

Acknowledgments

This work was supported by the National Natural ScienceFoundation of China (grants 30870947 and 30470825).

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