Journal of Medical Virology

Polymorphisms in Human Leukocyte Antigens, Human Platelet Antigens, and Cytokine Genes in Hantavirus Cardiopulmonary Syndrome Patients from Ribeira˜o Preto, Brazil Alessandra Abel Borges,1 Eduardo Antonio Donadi,2 Gelse Mazzoni Campos,1 Glauciane Garcia de Figueiredo,1 Fabiano Pinto Saggioro,3 Soraya Jabur Badra,1 Neifi Hassan Saloum Deghaide,2 and Luiz Tadeu Moraes Figueiredo1* 1

Virology Research Center, School of Medicine of Ribeira˜o Preto, University of Sa˜o Paulo, Ribeira˜o Preto, Brazil Molecular Immunology Laboratory, Hospital das Clı´nicas, School of Medicine of Ribeira˜o Preto, University of Sa˜o Paulo, Ribeira˜o Preto, Brazil 3 Death Verification Service (CEMEL) and Department of Pathology, School of Medicine of Ribeira˜o Preto, University of Sa˜o Paulo, Ribeira˜o Preto, Brazil 2

Hantavirus cardiopulmonary syndrome is a severe human disease associated with hantavirus infection. The clinical course of illness varies greatly among individuals, possibly due to viral and immunological elements and the influence of host genetic factors on clinical outcome. As the magnitude of immune activation has been associated with disease severity, polymorphisms in genes involved in the immune response that may affect the development of this syndrome were investigated. Polymorphisms in the TGF-b, IL-10, IL6, and IFN-g genes, human leukocyte antigens (HLA), and human platelet alloantigens (HPA) genes were investigated in 122 patients with Araraquara virus infection from Ribeira˜o Preto, Brazil. Patients were divided into two groups: hantavirus cardiopulmonary syndrome (HCPS group; n ¼ 26) and hantavirus seropositive only (n ¼ 96). The frequencies of HLA alleles, cytokines and platelet antigen genotypes were evaluated in both groups and compared to a control group. The data demonstrated no significant influence of the HLA alleles, HPA, IL-6, and IL-10 genotypes on susceptibility to hantavirus infection. However, the hantavirus seropositive group presented a significantly higher frequency of a polymorphism associated with a high IFN-g production than the HCPS group. In addition, a genotype associated with high TGF-b production was found more frequently in individuals infected with hantavirus than in the control group. This phenotype was associated with a less intense thrombocytopenia in the HCPS group and may be protective C 2013 WILEY PERIODICALS, INC. 

against the most severe form of hantavirus disease. More studies are required to quantify further the influence of the high TGF-b producer phenotype on the outcome of hantavirus infection. J. Med. Virol. # 2013 Wiley Periodicals, Inc.

KEY WORDS:

hantavirus; cardiopulmonary syndrome; genetic polymorphisms; TGF-b; HLA

INTRODUCTION The genus Hantavirus, family Bunyaviridae, includes rodent-borne viruses that can cause severe and lethal human diseases. In the Americas, human hantavirus infection frequently results in hantavirus cardiopulmonary syndrome (HCPS), a disease reported in many countries and with high mortality rates [Jonsson et al., 2010]. In Brazil, more than 1,600 cases of hantavirus cardiopulmonary syndrome

Grant sponsor: Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜ o Paulo (FAPESP) (Process Number 03/ 03682-3); Grant number: 02/14149-1.  Correspondence to: Luiz Tadeu Moraes Figueiredo, MD, Virology Research Center, School of Medicine of Ribeira˜o Preto, University of Sa˜o Paulo, Av. Bandeirantes 3900; Ribeira˜o Preto– SP, 14049-900, Brazil. E-mail: [email protected] Accepted 9 October 2013 DOI 10.1002/jmv.23836 Published online in Wiley Online Library (wileyonlinelibrary.com).

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have been recorded since 1993. In the Ribeira˜o Preto region, Sa˜o Paulo State, this syndrome is caused by the Araraquara virus (ARAV), where 65 cases of this disease were registered from 1998 to 2011, with a 44.6% fatality rate. To date, ARAV is considered by the International Committee on Taxonomy of Viruses (ICTV) as an Andes virus (ANDV) strain or as one of the ANDV-like viruses included in a monophyletic clade in the phylogenetic analysis of South American hantaviruses [Firth et al., 2012]. Hantavirus cardiopulmonary syndrome is associated with acute thrombocytopenia and changes in vascular permeability that can cause capillary leaking syndrome, leading to inflammatory pulmonary edema, myocarditis, and cardiogenic shock [Saggioro et al., 2007; Borges and Figueiredo, 2008]. Several studies have suggested that host genetic polymorphisms may influence the clinical outcome of hantavirus infection, resulting in milder or more severe illness [Ma¨kela¨ et al., 2002; Kilpatrick et al., 2004; Ferrer et al., 2007; Wang et al., 2009; Borges et al., 2010; Korva et al., 2011; Laine et al., 2012]. Furthermore, serological surveys have demonstrated anti-hantavirus antibodies in individuals without cardiopulmonary illness history [Campos et al., 2003; Pereira et al., 2012]. Some important immune system genes, such as those that encode human leukocyte antigens (HLA) and cytokines, may affect the outcome of hantavirus disease. Previous studies have shown that singlenucleotide polymorphisms (SNPs) in cytokine genes may affect the magnitude of cytokine production [Keen, 2002] and, therefore, could influence the severity of hantavirus disease [Maes et al., 2006; Borges et al., 2010]. Polymorphic genes that encode glycoproteins on endothelial cells and the platelet surface, such as the b3 chain of the aIIbb3 (CD41, CD61) and avb3 (CD51, CD61) integrins, characterize the human platelet alloantigens (HPA). These alloantigens are epitopes resulting from several biallelic systems, defined by distinct SNPs. Of these, those located at the CD61 gene could also contribute to the outcome of hantavirus infections, since b3 integrins mediate the cellular entry of pathogenic hantaviruses [Mackow and Gavrilovskaya, 2009]. The HPA-1, HPA-4, and HPA-6 epitopes are located on the N-terminal region of a 17 kDa polypeptide of the glycoprotein GPIIIa (chain b3, CD61) [Newman et al., 1985]. The HPA-1a, HPA4a, and HPA-6a antigens correspond to the same GPIIIa allele, presenting the phenotype Leu33Arg143Arg489. The variant forms of these alloepitopes are HPA-1b (phenotype Pro33), HPA-4b (phenotype Gln143) and HPA-6b (phenotype Gln489) [Newman, 1994]. In this study, human genetic polymorphisms in HLA, platelet alloantigens and cytokine genes were analyzed in patients with hantavirus infection from Ribeira˜o Preto, taking into consideration the subcliniJ. Med. Virol. DOI 10.1002/jmv

Borges et al.

cal profile of hantavirus infection observed commonly in this region [Campos et al., 2003]. MATERIALS AND METHODS Study Groups The participants of the study were divided into three groups. The main group (HCPS) comprised 26 patients with hantavirus cardiopulmonary syndrome— 19 males and seven females aged from 14 to 63 years old, admitted to hospitals in the city of Ribeira˜o Preto and neighboring towns. Six of these were fatal cases. Acute hantavirus infection diagnosis was performed by RT-PCR and confirmed by EIA [Figueiredo et al., 2009; Borges et al., 2010]. The second group of subjects (seropositive) comprised 96 individuals with IgG antibodies to the N protein of ARAV, but with no previous history of acute respiratory illness. Individuals in this group were selected from participants of a previous serological survey carried out in Jardinopolis County, in the Ribeira˜o Preto region, Brazil [Campos et al., 2003]. One hundred blood donors from the Hemocentro of Ribeira˜o Preto were included in a control group for cytokine genotyping analyses. Of these, 50 were selected at random for platelet alloantigen genotyping analyses. Bone-marrow donors (n ¼ 1,252) were included as a control group for HLA analyses. The study was performed in compliance with relevant laws and institutional guidelines and in accordance with the ethical standards of the Declaration of Helsinki. All patients gave written informed consent, as approved by the Ethics Committee in Research at the Hospital of the School of Medicine, University of Sa˜o Paulo in Ribeira˜o Preto (process number 2618/03) and by the National Commission of Research Ethics (CONEP) of the Ministry of Health, Brazil (register 7830, process number 25000.057239/ 2003-33). HLA, Cytokines, and Platelet Alloantigen Genotyping Blood samples were drawn into tubes containing EDTA; genomic DNA was isolated by the salting-out method. The HLA-A, -B, -Cw, and -DRB1 alleles and alleles of the TGF-b (codons 10, 25), IL-10 promoter (1082, 819, and 592), IL-6 (174), and IFN-g (874) cytokine genes were determined by SSP-PCR using commercially available kits (Micro SSP HLA DNA Typing and Cytokine Genotyping Tray; One Lambda, Canoga Park, Los Angeles, CA). SNPs in codons 10 (þ869T/C) and 25 (þ915G/C) of the TGFb1 gene were determined and the haplotypes were defined as the following TGF-b1 producer phenotypes: low (CC [codon 10]/CC [codon 25], CC/GC, TT/ CC, and TC/CC), intermediate (CC/GG, TC/GC, and TT/GC), or high (TC/GG and TT/GG) [Perrey et al., 1998]. The presence of a T or A nucleotide at position þ874 of the IFNg gene was analyzed for three possible genotypes: TT, TA, and AA. The AA

Human Polymorphisms in Hantavirus Disease

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group and the control group. The P values in HLA analysis were corrected by multiplying the number of alleles tested in each locus. In the HCPS group, the comparison of the TGF-b genotype (phenotype producer) between fatal cases and survivors was also analyzed by a Fisher’s exact test. Comparisons between the TGF-b high producer phenotype and the low and intermediate producer phenotype, in relation to clinical and laboratory parameters (mean arterial pressure, PO2, hematocrit, leukocyte and platelet counts, creatinine and C-reactive protein serum levels, and the number of days of hospitalization) in the HCPS group were made by a Mann–Whitney’s Utest. The comparison of the TGF-b producer phenotypes with the presence or the absence of clinical bleedings was analyzed by a Fisher’s exact test. The a significance level was set at 0.05 for all statistical tests. Statistical analyses were carried out using GraphPad InStat version 3.01 (GraphPad Software, Inc., La Jolla, San Diego, CA).

genotype is associated with low production, TA with intermediate production and TT with high production of cytokine [Pravica et al., 1999]. SNPs at position 174 in the IL-6 promoter were determined. Both the GG and GC genotypes are associated with increased levels of IL-6, while the CC genotype leads to decreased expression of cytokine [Fishman et al., 1998]. Three SNPs in the IL-10 promoter were investigated: 1082 A/G, 819 T/C, and 592 A/C. The different haplotypes that have been associated with production of IL-10 are: high IL-10 producer haplotype (GCC/GCC), intermediate producer haplotypes (GCC/ACC, GCC/ATA), and low IL-10 producer haplotypes (ATA/ATA, ACC/ATA, ACC/ACC) [Turner et al., 1997]. Human platelet alloantigen genotyping was performed with specific primers by PCR-SSP [Skogen et al., 1994] for three systems, including HPA-1a/ HPA-1b (serologic designation PlA1/PlA2), HPA-4a/ HPA-4b (Pena/Penb), and HPA-6a/HPA-6b (Ca/Tub/ Ca/Tua), which are located on b3 (CD61).

RESULTS Statistical Analyses

Alleles of HLA-A, -B, -Cw, and -DRB1 genes were determined only in 26 patients with hantavirus cardiopulmonary syndrome (Table I) and their frequencies were compared with those of the control group. Differences in allele frequencies did not retain statistically

Fisher’s exact test was used to define the significance of differences among gene frequencies of HLA alleles, platelet alloantigens and cytokine genotypes detected between the HCPS group, the seropositive

TABLE I. Characteristics of Patients With Hantavirus Cardiopulmonary Syndrome (HCPS) and Their HLA-A, -B, Cw, DRB1, and HPA-1 Alleles Alleles N 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

Gender M M M M M M M F M M M M F F F M M M F F M M F M M M

Age 38 35 16 27 17 42 37 43 49 40 25 31 21 30 24 39 37 63 34 15 30 14 55 42 31 54

HCPS outcome Cure Cure Cure Cure Cure Cure Cure Cure Cure Cure Cure Cure Cure Cure Cure Cure Cure Cure Cure Cure Death Death Death Death Death Death

HLA-A

HLA-B

HLA-Cw









A 02, A 24 A 29, A 31 A 23, A 24 A 36, A 24 A 02, A 66 A 11, A 24 A 02, A 24 A 02, A 03 A 01, A 02 A 02, A 68 A 01, A 02 A 01, A 80 A 02, A 29 A 03, A 24 A 11, A 26 A 01, A 26 A 02, A ya A 01, A 11 A 01, A 68 A 02, A 11 A 01, A 02 A 02, A 03 A 02, A 24 A 30, A 32 A 26, A 68 A 30, A 32



B 07, B 44 B 35, B 39 B 15, B 49 B 15, B 15 B 35, Bya B 51, Bya B 44, B 35 B 57, Bya B 14, B 57 B 51, B 58 B 58, B 35 B 44, B 08 B 44, Bya B 15, B 44 B 38, B 27 B 08, B 38 B 07, B 50 B 35, B 51 B 07, B 37 B 44, B 51 B 51, B 15 B 35, B 50 B 27, B 35 B 40, B 53 B 38, B 78 B 07, B 35



Cw 07, Cw 07 Cw 04, Cw 07 Cw 03, Cw 07 Cw 03, Cw 08 Cw 04, Cwya Cw 01, Cw 15 Cw 04, Cw 05 Cw 02, Cw 07 Cw 05, Cw 07 Cw 03, Cw 16 Cw 04, Cw 07 Cw 07, Cw 04 Cw 16, Cwya Cw 03, Cw 05 Cw 02, Cw 12 Cw 07, Cw 12 Cw 04, Cw 06 Cw 01, Cw 04 Cw 06, Cw 07 Cw 05, Cw 15 Cw 03, Cw 15 Cw 04, Cw 07 Cw 02, Cw 03 Cw 04, Cw 02 Cw 16, Cw 12 Cw 04, Cw 07

HLA-DR 

HPA-1 

DRB1 3, DRB1 16 DRB1 9, DRB1 16 DRB1 8, DRB1 11 DRB1 4, DRB1 15 DRB1 4, DRya DRB1 13, DRB1 14 DB1 16, DRB1 11 DRB1 03, DRB1 15 DRB1 01, DRB1 04 DRB1 13, DRB1 11 DRB1 13, DRB1 07 DRB1 03, DRB1 07 DRB1 07, DRAy a DRB1 01, DRB1 11 DRB1 11, DRy a DRB1 03, DRB1 08 DRB1 03, DRB1 07 DRB1 01, DRB1 11 DRB1 09, DRB1 10 DRB1 13, DRB1 14 DRB1 16, DRB1 04 DRB1 11, DRB1 07 DRB1 14, DRB1 13 DRB1 11, DRB1 13 DRB1 04, DRB1 13 DRB1 03, DRB1 04

1A, 1B 1A 1A, 1B 1A 1A 1A 1A 1A 1A 1A 1A 1A 1A, 1B 1A 1A 1A, 1B n.d. n.d. n.d. n.d. 1A 1A 1A, 1B 1A, 1B 1A n.d.

n.d., not determined; HPA-1, human platelet alloantigens-1. a y denotes that it was not possible to type the second allele for the indicated HLA or denotes the occurrence of the homozygosis for the first allele shown.

J. Med. Virol. DOI 10.1002/jmv

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significance after correction (data not shown). The HLA-B 35 allele was present in 30.7% (8/26) of subjects with cardiopulmonary syndrome (Table I), and in 22.8% (286/1,252) of subjects of the control group, and this difference was not statistically significant (P ¼ 0.3496; data not shown). Of note, three of the HLA-B 35-carriers were fatal cases (Table I). Among the five surviving patients carrying HLA-B 35, two experienced mild disease (patients ID: 5 and 11), while three had severe illness (patients ID: 2, 7, and 18). One of these patients presented with pulmonary hemorrhage (patient ID: 2), and another (patient ID: 7) displayed dyspnea until Day 25 after illness onset; the RT-PCR was positive for the viral S and M segments in peripheral blood at that time. In summary, 75% (6/8) of individuals carrying HLA-B 35 included in this study had severe illness. Polymorphisms in the genes encoding the IL-6, IFN-g, IL-10, and TGF-b cytokines were analyzed, and their frequencies compared for the HCPS group, the seropositive group and the control group (Table II). No statistical differences were found for the IL-6 and IL-10 genotypes between the HCPS and seropositive groups, or between the HCPS and control groups (Table II). Just one patient (3.8%) with the cardiopulmonary syndrome presented the high IFN-g producer phenotype, compared to 23 (23.9%) seropositive individuals (P ¼ 0.0246; Table II). The high TGF-b producer phenotype (genotypes T/ T-G/G; T/C-G/C) was significantly more frequent in the HCPS and seropositive groups, compared to the other TGF-b phenotypes (intermediate or low). The former was present in 76.9% of patients with cardiopulmonary syndrome and in 83.3% of seropositive individuals, whereas only 48.5% of the control group presented this phenotype (P ¼ 0.0336 and P ¼ 0.0002, respectively; Table II). Corroborating these results, one patient (3.8%) with the cardiopulmonary syndrome was genotyped as a low TGF-b producer, while nine (27.3%) subjects in the control group presented this phenotype (Table II). The effect of TGF-b genotype upon outcome of illness in the HCPS group was also investigated. However, no significant differences were found between survivors (15 were high producers; 5 were low or intermediate producers) and fatal cases (five were high producers; one was an intermediate producer) in relation to TGF-b genotypes (P ¼ 0.9999; data not shown). In order to analyze whether TGF-b genotype could affect the severity of cardiopulmonary syndrome, clinical and laboratory data from 24 patients was used (Table III) to compare the high TGF-b producer group and the low þ intermediate TGF-b producer group. There were no significant differences between these groups with respect to leukocyte count, hematocrit, mean arterial pressure, oxygen pressure, creatinine, and C-reactive protein levels, or days of hospitalization (data not shown). However, the thrombocytopenia was significantly less intense in patients with the high TGF-b producer phenotype J. Med. Virol. DOI 10.1002/jmv

Borges et al.

(mean 87,400 platelets/mm3; P ¼ 0.0356) than in patients with the low or intermediate TGF-b producer phenotype (mean 66,833 platelets/mm3; Fig. 1A). Of the six patients with the low or intermediate TGF-b producer phenotype, four (66.7%) had some bleeding during acute ARAV infection, such as: pulmonary hemorrhage (patient ID: 2); epistaxis (patient ID: 9); placenta previa and abortion (patient ID: 13); and the presence of a red-colored orotracheal secretion (patient ID: 25) (Table III). On the other hand, of the 18 patients with the high TGF-bproducer phenotype, and with the data available, only three patients presented with some bleeding during hantavirus disease (Table III); this difference was significant (P ¼ 0.0381), according to a Fisher’s exact test (Fig. 1B). The polymorphisms of three platelet alloantigens systems were determined in patients with hantavirus cardiopulmonary syndrome, in the seropositive group, and in the control group. However, there were no significant differences in the genotype frequencies of HPA-1, HPA-4, and HPA-6 (Table II). The genotypes HPA 1a/1a and HPA 4a/4a were the most frequent in all groups. In addition, HPA-6a homozygosis was observed in all individuals. DISCUSSION This study evaluated the frequencies of polymorphisms in relevant genes involved in the immune response and their associations with the development of the hantavirus cardiopulmonary syndrome. Firstly, the frequencies of the HLA-A, -B, -Cw, and -DRB1 alleles, carried by Brazilian patients presenting acute illness for hantavirus, were compared with those of the control group. No significant differences were found in the frequencies of these genes between groups, indicating there is no relevant effect of HLA alleles on the development of cardiopulmonary syndrome in individuals infected with ARAV. In addition, these results illustrate the heterogeneous composition of the Brazilian population (including Caucasian immigrants from many regions of Europe, African descendant populations predominantly from the Niger-Congo of Equatorial Africa, and native Amerindian populations [Louzada-Junior et al., 2001]) and raises questions about the relevance of genetic polymorphism studies in populations with high levels of racial admixture. By contrast, HLA studies in more homogeneous populations have shown interesting results. For example, Finnish patients presenting with nephropathia epidemica have shown genetic susceptibility to this disease linked to the HLA-B8-DR3 haplotype [Mustonen et al., 1996; Ma¨kela¨ et al., 2002]. A genetic susceptibility to hemorrhagic fever with renal syndrome (HFRS) of Chinese individuals carrying HLA-B 46 -DRB1 09 has also been demonstrated [Wang et al., 2009]. Data from North American patients indicate that HLA-B 3501 could be associated with severe

1a/1a 1a/1b 1b/1b 4a/4a 4a/4b 4b/4b 6a/6a 6a/6b 6b/6b

15 (71.4%) 5 (23.8%) 1 (4.8%) 21 (100%) 0 0 21 (100%) 0 0

HCPS, n ¼ 21 62 (64.6%) 32 (33.3%) 2 (2.1%) 96 (100%) 0 0 96 (100%) 0 0

Seropositive, n ¼ 96

13 (13.6%) 3 (3.1%)

5 (19.2%) 1 (3.8%)

37 (74%) 13 (26%) 0 49 (98%) 1 (2%) 0 50 (100%) 0 0

9 (27.3%)

8 (24.2%)

16 (48.5%)

6 (6.0%)

94 (94.0%)

35 (35.0%)

47 (47.0%)

18 (18.0%)

47 (47.0%)

44 (44.0%)

9 (9.0%)

Controls, n ¼ 100

0.6198 0.6033 0.4507 — — — — — —

P (HCPS vs. seropositive)

80 (83.3%)

7 (7.3%)

1 (3.8%) 20 (76.9%)

89 (92.7%)

30 (31.3%)

11 (42.3%) 24 (92.3%)

43 (44.8%)

14 (53.8%)

44 (45.8%)

7 (26.9%) 23 (23.9%)

46 (47.9%)

17 (65.4%)

1 (3.8%)

6 (6.3%)

Seropositive, n ¼ 96

2 (7.7%)

HCPS, n ¼ 26

Controls, n ¼ 50

High (GCC/GCC) Intermediate (GCC/ACC; GCC/ATT) Low (ACC/ACC; ACC/ATA; ATA/ATA) High (T/T) Intermediate (T/A) Low (A/A) High (G/G; G/C) Low (C/C) High (T/T-G/G; T/C-G/C) Intermediate (T/C-G/C; C/C-G/G; T/T-G/C) Low (C/C-G/C; C/C-C/C; T/T-C/C; T/C-C/C)

Phenotype producer (genotype)

SNP, single-nucleotide polymorphism; HCPS, hantavirus cardiopulmonary syndrome. a In HCPS group, 25 individuals were genotyping for IL-6. b In control group, 33 individuals were genotyping for TGF-b.

HPA HPA HPA HPA HPA HPA HPA HPA HPA

Genotype HPA

TGF-b (codons 10 and 25)

b

IL-6 (174)

a

IFN-g (intron 1 þ874)

IL-10 (1,082, 819, 592)

Cytokine gene (SNP position)

0.0323

0.7570

0.0336

1.0000

1.0000

0.5012

0.8233

0.1200

0.0775

0.0770

1.0000

P (HCPS vs control)

1.0000 1.0000 0.2958 1.0000 1.0000 — — — —

P (HCPS vs. control)

1.000

0.5340

0.5650

1.0000

1.0000

0.3506

0.5075

0.0246

0.1163

0.1272

0.6782

P (HCPS vs. seropositive)

0.0002

0.1749

0.0002

1.0000

0.7797

0.6495

0.7760

0.3801

0.8870

0.6673

0.5937

P (seropositive vs control)

0.2689 0.4508 0.5465 0.3425 0.3425 — — — —

P (seropositive vs. control)

TABLE II. Comparison of Cytokines and HPA Genotype Frequencies Among HCPS, Seropositive, and Control Groups

Human Polymorphisms in Hantavirus Disease 5

J. Med. Virol. DOI 10.1002/jmv

J. Med. Virol. DOI 10.1002/jmv

High Intermediate

High

24 25

26a

—

51 76

61

—

10,100 28,000

16,700

— 19,200

4,700 6,400 25,900 11,800 15,800 15,800 5,900

18,600 24,000 13,100 6,000 9,400 8,600 6,600 4,100 7,000 — 31,900

8,900 —

—

— 63,000

82,000

— 204,000

83,000 — — 71,000 43,000 34,000 93,000

111,000 72,000 66,000 100,000 84,000 75,000 83,000 70,000 66,000 87,000 59,000

102,000 64,000

—

— 88  44 —

76.6 39.0

49.4

70  50

99.0 87.0 81.0 75.0 88.0 84.0 86.0

107  72 60  40 — 130  80 120  80 80  50 80  40 — 78

48.6 82.0 — 95 128.0 73.4 56.7 — — 79.0 —

80  50 120  80 110  60 113  68 110  80 90  60 110  70 90  60 100  80 140  90 110  60

— 80  30

92 —

120  80 —

—, no data available; Ht, hematocrit; WBC, white blood cell; PO2, oxygen pressure. a Patients HLA-B 35-positive. b This patient presented sudden death.

High

— 62

Fatal cases 21b High High 22a

23a

44 44 58 48 60 53 50

High High High High High High High

14 15 16 17 18a 19 20

55 44 55 48 44 50 42 43 48 37.8 33.2

High High Intermediate High High High Intermediate High Low High Intermediate

38 50

1.7 1.4

—

— —

1.2 1.1 — 1.7 1.7 2.7 0.6

0.8 1.1 1.0 1.1 1.1 1.1 1.2 — — 1.4 —

1.3 —

—

0.56 8.66

3.2

— —

— — — — — — —

5.69 8.12 1.24 6.00 — 7.57 6.09 — 0.30 3.14 1.07

1.24 —

—

No No

No

— —

No Yes No Yes Yes Yes Yes

No No No No No No No — — No Yes

Yes —

Arterial WBC count Platelet count pressure Creatinine C-reactive Corticosteroid 3 3 Ht (%) (cells/mm ) (cells/mm ) (mmHg) PO2 (mmHg) (mg/dl) protein (mg/dl) therapy

3 4 5a 6 7a 8 9 10 11a 12 13

Survivors 1 High Intermediate 2a

Patient

TGF-beta phenotype producer

— Pulmonary and cerebral congestion Hematemesis and melena No Red—colored orotracheal secretion —

No Pulmonary hemorrhage No No No No No Hematuria Epistaxis No No No Placenta previa and abortion No No No No No No No

Clinical bleeding

—

1 1

1

— 1

5 7 7 6 12 9 15

9 10 8 9 10 5 9 7 7 8 8

4 17

Days of hospitalization

TABLE III. Clinical and Laboratory Features on Admission of Patients With Hantavirus Cardiopulmonary Syndrome and Their TGF-Beta Phenotypes

6 Borges et al.

Human Polymorphisms in Hantavirus Disease

7

Fig. 1. Mean platelet count in patients with hantavirus cardiopulmonary syndrome (HCPS), according to TGF-b phenotype (A). Patients with acute infection by Araraquara virus, divided according to TGF-b phenotype and the presence or absence of clinical bleedings during hantavirus cardiopulmonary syndrome (B).

hantavirus cardiopulmonary syndrome and that HLA-B 35 and HLA-Cw 04 may be associated with a highly efficient presentation of Sin Nombre virus (SNV) antigen [Kilpatrick et al., 2004]. In this study, the HLA-B 35 allele was present in one third of patients with cardiopulmonary syndrome (Table I), most of these having severe illness, including three fatal cases. Thus, it is possible that, in individuals infected with ARAV, the HLA-B 35 allele may be associated with severe disease progression, such as previously reported for patients infected with SNV [Kilpatrick et al., 2004]. In accordance with this supposition, in another study carried out in Slovenia, higher frequencies of the HLA-B 35 allele were found among HFRS patients infected with Dobrava virus (DOBV) with a fatal outcome [Korva et al., 2011]. However, a previous study carried out with Chilean individuals infected with ANDV showed that the HLA-B 35 allele was more frequent among patients with mild disease (without respiratory failure and shock) than those with severe disease, but without statistical significance [Ferrer et al., 2007]. Furthermore, another study demonstrated that strong HLAB 35-restricted memory T cell responses to ANDV were associated with mild, rather than severe disease, outcome [Manigold et al., 2010]. Thus, reports on HLA polymorphisms and their associations with the severity of diseases caused by hantaviruses have shown discordant results, even involving the same species of hantavirus and its variants. The effect of HLA-B 35 on the severity of the hantavirus illness caused by the ANDV and the ANDV-like viruses

(including ARAV) remains unclear and further studies are necessary to resolve this question. A previous study showed that the polymorphic TNF2 allele (TNF-308G/A) was found more frequently in hantavirus cardiopulmonary syndrome patients from Ribeira˜o Preto, in comparison with hantavirus seropositive individuals without a history of acute respiratory illness, suggesting that the TNF2 allele could represent a risk factor for developing hantavirus cardiopulmonary syndrome [Borges et al., 2010]. However, other cytokine genes (besides the TNF-a gene) are polymorphic at specific sites and certain mutations located within coding/regulatory regions have been shown to affect the overall expression and secretion of cytokines, increasing the severity of various inflammatory and infectious diseases. For this reason, polymorphisms in the genes encoding the IL-6 and INF-g (pro-inflammatory) and IL-10 and TGF-b (anti-inflammatory) cytokines were analyzed in the participants of this study (Table II). No differences in the genotypes of IL-6 and IL-10 were observed among the groups studied (HCPS, seropositive, and control), suggesting no influence of the polymorphisms in the genes of these cytokines on hantavirus cardiopulmonary syndrome development. On the other hand, the high IFN-g producer phenotype was significantly less frequent in the HCPS group than in the seropositive group. IFN-g is a key cytokine in the development of Th1 responses, and it plays an essential role in antiviral immunity. The CD8þ T cells are a relevant source of IFN-g. A previous study demonstrated that the appearance of J. Med. Virol. DOI 10.1002/jmv

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virus-specific CD8þ T cells with the ability to produce IFN-g was associated with clearance of Hantaan virus (HTNV) in newborn mice [Araki et al., 2003]. Another study demonstrated that the elimination of ANDV from peripheral blood was associated with the appearance of CD8þ T cells, but not with the appearance of neutralizing antibodies [Manigold et al., 2008]. Likewise, although a previous report has shown significant correlations between other Th1 cytokines and clinical parameters (such as hypoxia, hypotension and hemoconcentration) in patients infected with ARAV presenting acute cardiopulmonary syndrome, the serum levels of IFN-g, specifically, were not associated with severity of disease [Borges et al., 2008]. In agreement with all these previous reports, in this study, the high IFN-g producer phenotype had a significantly higher frequency in the seropositive group (without any history of respiratory distress) than in the HCPS group. In human infections with ARAV and with other variants of ANDV, it is possible that the high IFN-g producer phenotype could represent a protective phenotype against cardiopulmonary syndrome development. However, the influence of IFN-g genotypes on hantavirus infections remains uncertain and merits further studies that include larger number of participants, to ascertain whether high IFN-g producer individuals could be indeed more protected against severe hantavirus diseases. In this study, a contingent tendency towards a high production of an essential suppressor cytokine, TGF-b, was demonstrated in both groups of individuals infected with hantavirus, where the high-producer phenotype was significantly more frequent in the HCPS and seropositive groups than in the control group. In addition, in patients presenting cardiopulmonary syndrome, the TGF-b genotype seems to affect the severity of illness, at least in relation to the risk of bleeding. The high TGF-b producer phenotype was associated with less intense thrombocytopenia in the HCPS group (Fig. 1A). In fact, bleedings in these individuals were significantly more frequent in patients with the low or intermediate TGF-b producer phenotype (66.7% of the group). Serum levels of TGFb have been reported to be significantly decreased in patients infected with ARAV presenting cardiopulmonary syndrome, within the first week of illness, possibly because immune regulatory activity is impaired [Borges et al., 2008]. Thus, the less intense thrombocytopenia observed, herein, in patients with the high TGF-b producer phenotype (who had a lower proportion of clinical bleedings), suggests that the high TGF-b producer phenotype may confer some protection against the most severe form of hantavirus disease. This phenotype may be of benefit to the patients, as a genetic tendency to produce higher amounts of TGF-b during hantavirus cardiopulmonary syndrome would reduce the exacerbated activation of the immune response. However, unexpectedly, the TGF-b genotype was not found to affect the J. Med. Virol. DOI 10.1002/jmv

Borges et al.

outcome of illness in the HCPS group, since no significant differences were found between surviving and fatal cases. Thus, more studies are clearly required in order to definitively identify the role of the high TGF-b producer phenotype in the outcome of hantavirus infections. Finally, polymorphisms in the human platelet alloantigens were examined in this study. However, no significant differences were found in the genotypes of HPA-1, HPA-4, and HPA-6 among the three groups. These findings suggest that the HPA-1, -4, and -6 alleles are not risk factors for hantavirus cardiopulmonary syndrome development. A recent study with Finnish patients presenting Puumala virus (PUUV) infection also showed that HPA-1 polymorphism was not associated with the severity of nephropathia epidemica [Laine et al., 2012]. Similar results were reported in a study of HFRS patients, where no significant differences in HPA-1 genotype distributions were observed between patients and controls [Liu et al., 2009]. However, in this same study, a significant difference was observed in the distributions of genotype and allele frequencies of HPA-3 (located on glycoprotein aIIb; CD41) between HFRS patients and controls, suggesting that the HPA-3 polymorphism may be one of the inherited risk factors associated with susceptibility to hantavirus infection and disease severity of HFRS [Liu et al., 2009]. This is a surprising finding, especially as numerous reports have demonstrated that it is the b3 chain (CD61) that interacts with hantaviruses [Mackow and Gavrilovskaya, 2009]. Nevertheless, it seems that (beyond CD61 polymorphism) the levels of this receptor on the platelet surface could be associated with hantavirus disease severity, at least for HFRS [Liu et al., 2008]. The influence of the CD61 levels on endothelial cells and platelets in hantavirus cardiopulmonary syndrome remains to be investigated. In conclusion, results did not show a significant influence of the polymorphic sites of HLA, IL-6, IL10, and platelet alloantigens genes on hantavirus cardiopulmonary syndrome development. Nevertheless, individuals with IgG antibodies to ARAV and without a history of cardiopulmonary illness had a higher frequency of the high IFN-g-producer phenotype than patients with hantavirus disease. In line with this result, a potential protective role of this phenotype against cardiopulmonary syndrome development should be investigated further. Finally, most individuals that had suffered hantavirus infection, with or without cardiopulmonary syndrome development, were carriers of genotypes associated with a high production of TGF-b. This phenotype may be protective against the most severe form of cardiopulmonary syndrome, because it was associated with a less intense thrombocytopenia and minor proportion of bleedings than other TGF-b phenotypes. Further studies that include a higher sample number, distinct cases of severity and cardiopulmonary syndrome caused by different hantaviruses are still necessary

Human Polymorphisms in Hantavirus Disease

to definitively ascertain the influence of polymorphic sites involved in immune responses on hantavirus infection and outcomes. ACKNOWLEDGMENTS We are very grateful to Dr. Renato Cassiano Silveira for the enrollment of patients from the Santa Casa de Misericordia de Passos Hospital, and to Dr. Vagner de Castro (Hemocentro/UNICAMP) for kindly supplying the positive controls for HPA analyses. We are very grateful to Dra. Juliana Helena Cha´vez (UFU), Dra. Ana Cla´udia Mendes Malhado (UFAL), Dr. Richard James Ladle (UFAL), and Dra. Nicola Conran (Hemocentro/UNICAMP) for revising this manuscript. REFERENCES Araki K, Yoshimatsu K, Lee BH, Kariwa H, Takashima I, Arikawa J. 2003. Hantavirus-specific CD8(þ)-T-cell responses in newborn mice persistently infected with Hantaan virus. J Virol 77:8408–8417. Borges AA, Figueiredo LTM. 2008. Mechanisms of shock in hantavirus pulmonary syndrome. Curr Opin Infect Dis 21:293–297. Borges AA, Campos GM, Moreli ML, Souza RLM, Saggioro FP, Figueiredo GG, Livonesi MC, Figueiredo LTM. 2008. Role of mixed Th1 and Th2 serum cytokines on pathogenesis and prognosis of Hantavirus Pulmonary Syndrome. Microbes Infect 10:1150–1157. Borges AA, Donadi EA, Campos GM, Moreli ML, Sousa RLM, Saggioro FP, Figueiredo GG, Badra SJ, Deghaine NHS, Figueiredo LTM. 2010. Association of -308G/A polymorphism of tumor necrosis factor-a gene promoter with the susceptibility to development of hantavirus cardiopulmonary syndrome in Ribeira˜o Preto region, Brazil. Arch Virol 155:971–975. Campos GM, Sousa RLM, Badra SJ, Pane C, Gomes UA, Figueiredo LTM. 2003. Serological survey of hantavirus in Jardino polis county, Brazil. J Med Virol 71:417–422. Ferrer P, Vial PA, Ferre´s M, Godoy P, Cuiza A, Marco C, Castilho C, Umana˜ ME, Rothhammer F, Llop E. 2007. Susceptibilidad gene´tica a hantavirus Andes: Asociacio n entre la expresio n clı´nica de la infeccio n y alelos del sistema HLA en pacientes chilenos. Rev Chil Infect 24:351–359. Figueiredo LTM, Moreli ML, Sousa RLM, Borges AA, Figueiredo GG, Machado AM, Bisordi I, Nagasse-Sugahara TK, Suzuki A, Pereira LE, Souza RP, de Souza LTM, Braconi CT, Harsi CM, Zanotto PMA, VGDN Consortium. 2009. Hantavirus pulmonary syndrome, Central Plateau, Southeastern, and Southern Brazil. Emerg Infect Dis 15:561–567. Firth C, Tokarz R, Simith DB, Nunes MRT, Bhat M, Rosa EST, Medeiros DBA, Palacios G, Vasconcelos PFC, Lipkin WI. 2012. Diversity and distribution of hantaviruses in South America. J Virol 86:13756–13766. Fishman D, Faulds G, Jeffery R, Mohamed-Ali V, Yudkin JS, Humphries S, Woo P. 1998. The effect of novel polymorphism in the interleukin-6 (IL-6) gene on IL-6 transcription and plasma IL-6 levels, and an association with systemic onset juvenile chronic. J Clin Invest 102:1369–1376. Jonsson CB, Figueiredo LTM, Vapalahti O. 2010. A global perspective on hantavirus ecology, epidemiology and disease. Microbiol Rev 23:412–441. Keen LJ. 2002. The extent and analysis of cytokine and cytokine receptor gene polymorphism. Transpl Immunol 10:143–146. Kilpatrick ED, Terajima M, Koster FT, Catalina MD, Cruz J, Ennis FA. 2004. Role of specific CD8þ T cells in the severity of a fulminant zoonotic viral hemorrhagic fever, Hantavirus Pulmonary Syndrome. J Immunol 172:3297–3304. Korva M, Saksida A, Kunilo S, Jeras BV, Avsic-Zupanc T. 2011. HLA-associated hemorrhagic fever with renal syndrome disease progression in Slovenian patients. Clin Vaccine Immunol 18: 1435–1440.

9 Laine O, Joutsi-Korhonen L, Ma¨kela¨ S, Mikkelsson J, Pessi T, Tuomisto S, Huhtala H, Libraty D, Vaheri A, Karhunen P, Mustonen J. 2012. Polymorphisms of PAI-1 and platelet GP Ia may associate with impairment of renal function and thrombocytopenia in Puumala hantavirus infection. Thromb Res 129: 611–615. Liu Z, Gao M, Han Q, Fang J, Zhao Q, Zhang N. 2008. Intensity of platelet b3 integrin in patients with hemorrhagic fever with renal syndrome and its correlation with disease severity. Viral Immunol 21:255–261. Liu Z, Gao M, Han Q, Lou S, Fang J. 2009. Platelet glycoprotein IIb/IIIa (HPA-1 and HPA-3) polymorphisms in patients with hemorrhagic fever with renal syndrome. Hum Immunol 70: 452–456. Louzada-Junior P, Smith AG, Hansen JA, Donadi EA. HLA-DRB1 and -DQB1 alleles in the Brazilian population of the northeastern region of the state of Sa˜o Paulo. Tissue Antigens 2001. 57: 158–162. Mackow ER, Gavrilovskaya IN. 2009. Hantavirus regulation of endothelial cell functions. Thromb Haemost 102:1030–1041. Maes P, Clement J, Groeneveld PHP, Colson P, Huizinga TWJ, Van Ranst M. 2006. Tumor necrosis factor-a genetic predisposing factors can influence clinical severity in nephropathia epidemica. Viral Immunol 19:558–564. Ma¨kela¨ S, Mustonen J, Ala-Hoihala I, Hurne M, Partanen J, Vapalahti O, Vaheri A, Pasternack A. 2002. Human leukocyte antigen-B8-DR3 is a more important risk factor for severe Puumala Hantavirus infection than the tumor necrosis factor-a(308) G/A polymorphism. J Infect Dis 186:843–846. Manigold T, Martinez J, Lazcano X, Ye C, Schwartz S, Cuiza A, Valdivieso F, Hjelle B, Vial P. 2008. Case report: T-cell responses during clearance of Andes virus from blood cells 2 months after severe hantavirus cardiopulmonary syndrome. J Med Virol 80:1947–1951. Manigold T, Mori A, Graumann R, Llop E, Simon V, Ferre´s M, Valdivieso F, Castillo C, Hjelle B, Vial P. 2010. Highly differentiated, resting Gn-specific memory CD8þ T cells persist years after infection by Andes hantavirus. PlOS Pathog 6:e-1000779. Mustonen J, Partanent J, Kanerva M, Pietila K, Valpalahti O, Pasternack A, Vaheri A. 1996. Genetic susceptibility to severe course of nephropathia epidemica caused by Puumala hantavirus. Kidney Int 49:217–221. Newman PJ. 1994. Nomenclature of human platelet alloantigens: A problem with the HPA system? Blood 83:1447–1451. Newman PJ, Martin LS, Knipp MA, Kahn RA. 1985. Studies on the nature of the human platelet alloantigen PL A1: Localization to a 17,000-dalton polypeptide. Mol Immunol 22:719–729. Pereira GW, Teixeira AM, Souza MS, Braga AD, Sabino-Santos G, Figueiredo GG, Figueiredo LTM, Borges AA. 2012. Prevalence of serum antibodies to hantavirus in a rural population from south of Santa Catarina State, Brazil. Rev Soc Bras Med Trop 45:117– 119. Perrey C, Pravica V, Sinnott PJ, Hutchinson IV. 1998. Genotyping for polymorphisms in interferongamma, interleukin-10, transforming growth factor-b1 and tumour necrosis factor-alpha genes: A technical report. Transpl Immunol 6:193–197. Pravica V, Asderakis A, Perry C, Hajeer A, Sinnott PJ, Hutchinson IV. 1999. In-vitro production of IFN-g correlates with CA repeat polymorphisms in the human IFN-g gene. Eur J Immunogenet 26:1–3. Saggioro FP, Rossi AM, Duarte MIS, Martin CCS, Alves VAF, Moreli ML, Figueiredo LTM, Moreira JE, Borges AA, Neder L. 2007. Hantavirus infection induces a typical myocarditis that may be responsible for myocardial depression and shock in Hantavirus Pulmonary Syndrome. J Infect Dis 195:1541–1549. Skogen B, Belissimo DB, Hessner MJ, Santoso S, Aster RH, Newman PJ, Mcfarland JG. 1994. Rapid determination of platelet alloantigen genotypes by polymerase chain reaction using allele-specific primers. Transfusion 34:955–960. Turner DM, Williams DM, Sankaran D, Lazarus M, Sinnott PJ, Hutchinson IV. 1997. An investigation of polymorphism in the interleukin-10 gene promoter. Eur J Immunogenet 24:1–8. Wang ML, Lai JH, Zhu Y, Zhang HB, Li C, Wang JP, Li YM, Yang AG, Jin BQ. 2009. Genetic susceptibility to haemorrhagic fever with renal syndrome caused by Hantaan virus in Chinese Han population. Int J Immunogenet 36:227–229.

J. Med. Virol. DOI 10.1002/jmv