Eur J Clin Microbiol Infect Dis (2015) 34:187–195 DOI 10.1007/s10096-014-2213-9
ARTICLE
Influence of HLA-DRB alleles on haemorrhagic fever with renal syndrome in a Chinese Han population in Hubei Province, China N. Zhu & F. Luo & Q. Chen & N. Li & H. Xiong & Y. Feng & Z. Yang & W. Hou
Received: 26 January 2014 / Accepted: 14 July 2014 / Published online: 30 August 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Specific human leucocyte antigen (HLA) alleles are considered a genetic risk factor for the progression of haemorrhagic fever with renal syndrome (HFRS) caused by hantaviruses. The aim of this study was to establish whether HLA-DRB alleles are associated with the severity of HFRS caused by different types of hantaviruses in a Chinese Han population from Hubei Province of central China. Twenty-two specific HLA-DRB alleles were analysed by sequencespecific primer–polymerase chain reaction (SSP-PCR) in 100 HFRS patients and 213 healthy volunteers. Associations of HLA-DRB alleles with the severity and clinical parameters of HFRS caused by Hantaan virus (HTNV) or Seoul virus (SEOV) infection were evaluated. Six alleles (HLADRB1*0401-0411, HLA-DRB1*1001, HLA-DRB1*11011105, HLA-DRB1*1201-1202, HLA-DRB1*1305 and DRB5*0101-0201) demonstrated strong associations with HFRS caused by HTNV and SEOV infections. Further comparison of these HLA-DRB1 allele frequencies between HFRS patients with differing severities and healthy controls
Ni Zhu and Fan Luo contributed equally to this work. Z. Yang (*) : W. Hou (*) State Key Laboratory of Virology, Institute of Medical Virology, School of Basic Medical Sciences, Wuhan University, 185 Donghu Road, Wuhan 430071, People’s Republic of China e-mail: [email protected] e-mail: [email protected] N. Zhu Department of Microbiology, School of Basic Medical Sciences, Hubei University of Science and Technology, Xianning 437100, China Present Address: N. Zhu : F. Luo : Q. Chen : N. Li : H. Xiong : Y. Feng State Key Laboratory of Virology, Institute of Medical Virology, School of Basic Medical Sciences, Wuhan University, 185 Donghu Road, Wuhan 430071, People’s Republic of China
demonstrated that the HLA-DRB1*0401-0411, HLADRB1*1001 and DRB1*1305 alleles were more frequent in the moderate course of HTNV-infected HFRS. Meanwhile, the DRB1*1101-1105 allele was more frequently observed in the severe course of HTNV-infected HFRS. We also found that the HLA-DRB1*1201-1202 allele frequency was higher in the moderate course of SEOV-infected HFRS, whereas the DRB5*0101-0201 allele may play a protective role in moderate HFRS caused by both HTNV and SEOV infections. These results provide evidence of the influence of HLA-DRB on the severity of HFRS and confirm the effect of HLA-DRB on HFRS during different types of hantavirus infection in a Chinese Han population in Hubei Province, China.
Introduction Haemorrhagic fever with renal syndrome (HFRS) is an endemic zoonosis transmitted to humans from infected species of rodents, and is clinically characterised by fever, haemorrhage and acute renal dysfunction [1]. HFRS is caused by several serotypes of hantaviruses in the hantavirus genus of the family Bunyaviridae, primarily involving the Hantaan virus (HTNV) and Seoul virus (SEOV) in Asia and the Puumala virus (PUUV) and Dobrava virus (DOBV) in Europe [1]. Worldwide, HFRS affects thousands of people each year, with the majority occurring in China [2, 3]. Hubei Province is one of the most severe endemic areas of HFRS in central China, there have been more than 100,000 HFRS patients reported in this area since 1957 when the first HFRS patient was found [2]. Although integrated intervention measures, including rodent control, environmental management and vaccinations have been implemented, HFRS remains a serious public health problem in Hubei Province. In Hubei, HFRS is primarily caused by HTNVor SEOV [2], the clinical
188
severity of which varies greatly from the most commonly seen case where the patient is asymptomatic to a lethal outcome. However, the mechanisms underlying the varying severity of HFRS are not yet understood. It is generally known that human leucocyte antigen (HLA) molecules play an important role in influencing the progression of disease. HLA molecules including HLA class I molecules encoded by HLA-A, -B and -C genes and HLA class II molecules encoded by HLA-DR, -DQ and -DP genes can effectively present viral antigens to CD8+ T cells, CD4+ T cells and dendritic cells, leading to the stimulation and regulation of host innate and adaptive immune responses against viral pathogens which lead to viral clearance or persistence [4]. Many different HLA alleles that are associated with differing viral infectiousness, clinical courses and outcomes of viral disease have been identified in different populations of the world during the past several years. In hantavirus infections, the HLA-B8-DR3 extended haplotype and HLA-DRB1*0301 were observed to be associated with the severe course of nephropathia epidemica (NE, a milder form of HFRS) caused by PUUV infection [5, 6]. In contrast, the HLA-B27 molecule, a known marker for autoimmune diseases, was associated with the milder clinical course of NE with PUUV infection in Finland [7]. Additionally, there was a higher frequency of the HLA-B*35 allele in HFRS patients with DOBV infections compared with PUUV infections, and HLA-DRB1*13 was associated with the severe course of PUUV-caused HFRS, yet not with DOBV-caused HFRS in a Slovenian population [8]. In China, HLA-DRB1*09 was associated with HFRS in a Chinese Han population [9], whereas HLA-B*46, HLA-B*46-DRB1*09 and HLA-B*51DRB1*09 were known to associate with severe/critical HFRS patients caused by HTNV infection in a Chinese Han population in northwest China [10]. These studies demonstrate that several HLA alleles are risk factors for the clinical severity of HFRS. In the present study, we analysed 22 HLA-DRB allele frequencies in HFRS patients and in healthy controls, and assessed the clinical findings of HFRS caused by HTNV and SEOV infections in Hubei Province of central China to address the possibility of associations of HLA-DRB alleles with the clinical severity of HFRS disease. Despite intensive research that has focused on HLA alleles associated with HFRS or the disease severity of HTNV-caused HFRS in Chinese Han populations, the geographic distribution of Chinese Han populations, rodent reservoirs and differing climates, the association between HLADRB alleles and HFRS caused by SEOV infection has not been studied. In this study, several new HLA-DRB alleles were found to be susceptible or resistant to HFRS and correlated with disease severity and the clinical findings of HFRS infected with different serotypes of hantaviruses in a Chinese Han population.
Eur J Clin Microbiol Infect Dis (2015) 34:187–195
Materials and methods Subjects and clinical data collection A total of 100 patients hospitalised in Hubei Province with a clinical and serological diagnosis of HFRS were included in the study. The patients consisted of 76 males and 24 females, aged 33±13 years. Comprehensive clinical and laboratory data were retrospectively gathered from the patients’ records. The severity of HFRS disease was categorised into four different groups, including mild, moderate, severe and critical groups, which were based on clinical and laboratory parameters that were evaluated as described previously [9, 11]. The control group consisted of 213 randomly chosen healthy unrelated individuals. Of these, 138 subjects were male and 75 subjects were female (age 39±11 years). All subjects were recruited from 13 different cities’ hospitals in Hubei Province from 1986 to 2012. Peripheral blood samples (5 ml) were collected from all subjects and stored at −80 °C until use. Informed consent was obtained from each participant, and the research protocol was approved by the Medical Ethics Committee of Wuhan University, People’s Republic of China. Hantavirus genotyping and HLA typing Genomic RNA was isolated from the peripheral blood of patients by the one-step guanidinium isothiocyanate-phenolchloroform method [12]. Hantavirus genotypes were determined by reverse transcription (RT)-heminested polymerase chain reaction (PCR) with two pairs of HTNV/SEOV genotype-specific primers and one pair of hantavirus universal primers [13–15]. The epidemic viral strains of hantavirus, HTNV 76-118 and SEOV R22, were considered as the positive controls in this study. Genomic DNA was isolated from the peripheral blood of all subjects by the phenol-chloroform method [16]. Genotypes were determined by sequence-specific primer–polymerase chain reaction (SSP-PCR) with HLA-DRB1-specific primers as previously described [17]. The determination of HLA alleles was performed regardless of the severity of each HFRS patient. Statistical analysis The frequencies of HLA alleles were calculated by direct counting. The Chi-squared test or Fisher’s exact test, when an expected value was less than 5, was used for comparison of the frequencies of individual alleles between the controls and HFRS patients. The Kruskal–Wallis test, Mann–Whitney U-test, Chi-squared test or Student’s t-test were used to compare the clinical parameters among different groups. p-Values less than 0.05 were considered to be statistically significant. The odds ratio (OR), which reflects the likelihood
Eur J Clin Microbiol Infect Dis (2015) 34:187–195
of a subject carrying a specific allele, was calculated to identify the associated risk factors. All statistical analyses were performed using the Statistical Package for the Social Sciences (SPSS) version 17.0.
Results Patient characteristics The study determined that 49 out of 100 HFRS patients were diagnosed with HTNV infection, 35 patients had SEOV infection and 16 patients had an unknown type of hantavirus infection. After classification according to their clinical and laboratory parameters during hospitalisation, there were 18 mild HFRS, 60 moderate HFRS, 19 severe HFRS and three critical HFRS patients. Due to the limited number of critical clinical courses of HFRS, critical and severe HFRS patients were combined and referred as the severe course group. In the 49 HFRS patients with HTNV infection, eight were clinically diagnosed as mild HFRS, 27 as moderate HFRS and 14 as severe and critical HFRS. Moreover, 35 SEOV-caused HFRS patients were categorised into three groups comprising seven mild HFRS patients, 23 moderate HFRS patients and five patients with severe and critical HFRS. In addition, the common clinical symptoms and several abnormal laboratory findings of HFRS patients were observed (Table 1). Frequencies of HLA-DRB alleles in healthy controls and HFRS patients with HTNV and SEOV infections Altogether, 19 different HLA-DRB1, HLA-DRB3, HLADRB4 and HLA-DRB5 alleles from 100 HFRS patients and 213 healthy controls in a Chinese Han population from Hubei Province of China were determined in this study, as shown in Table 2. It was found that there were significantly different (p
ARTICLE
Influence of HLA-DRB alleles on haemorrhagic fever with renal syndrome in a Chinese Han population in Hubei Province, China N. Zhu & F. Luo & Q. Chen & N. Li & H. Xiong & Y. Feng & Z. Yang & W. Hou
Received: 26 January 2014 / Accepted: 14 July 2014 / Published online: 30 August 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Specific human leucocyte antigen (HLA) alleles are considered a genetic risk factor for the progression of haemorrhagic fever with renal syndrome (HFRS) caused by hantaviruses. The aim of this study was to establish whether HLA-DRB alleles are associated with the severity of HFRS caused by different types of hantaviruses in a Chinese Han population from Hubei Province of central China. Twenty-two specific HLA-DRB alleles were analysed by sequencespecific primer–polymerase chain reaction (SSP-PCR) in 100 HFRS patients and 213 healthy volunteers. Associations of HLA-DRB alleles with the severity and clinical parameters of HFRS caused by Hantaan virus (HTNV) or Seoul virus (SEOV) infection were evaluated. Six alleles (HLADRB1*0401-0411, HLA-DRB1*1001, HLA-DRB1*11011105, HLA-DRB1*1201-1202, HLA-DRB1*1305 and DRB5*0101-0201) demonstrated strong associations with HFRS caused by HTNV and SEOV infections. Further comparison of these HLA-DRB1 allele frequencies between HFRS patients with differing severities and healthy controls
Ni Zhu and Fan Luo contributed equally to this work. Z. Yang (*) : W. Hou (*) State Key Laboratory of Virology, Institute of Medical Virology, School of Basic Medical Sciences, Wuhan University, 185 Donghu Road, Wuhan 430071, People’s Republic of China e-mail: [email protected] e-mail: [email protected] N. Zhu Department of Microbiology, School of Basic Medical Sciences, Hubei University of Science and Technology, Xianning 437100, China Present Address: N. Zhu : F. Luo : Q. Chen : N. Li : H. Xiong : Y. Feng State Key Laboratory of Virology, Institute of Medical Virology, School of Basic Medical Sciences, Wuhan University, 185 Donghu Road, Wuhan 430071, People’s Republic of China
demonstrated that the HLA-DRB1*0401-0411, HLADRB1*1001 and DRB1*1305 alleles were more frequent in the moderate course of HTNV-infected HFRS. Meanwhile, the DRB1*1101-1105 allele was more frequently observed in the severe course of HTNV-infected HFRS. We also found that the HLA-DRB1*1201-1202 allele frequency was higher in the moderate course of SEOV-infected HFRS, whereas the DRB5*0101-0201 allele may play a protective role in moderate HFRS caused by both HTNV and SEOV infections. These results provide evidence of the influence of HLA-DRB on the severity of HFRS and confirm the effect of HLA-DRB on HFRS during different types of hantavirus infection in a Chinese Han population in Hubei Province, China.
Introduction Haemorrhagic fever with renal syndrome (HFRS) is an endemic zoonosis transmitted to humans from infected species of rodents, and is clinically characterised by fever, haemorrhage and acute renal dysfunction [1]. HFRS is caused by several serotypes of hantaviruses in the hantavirus genus of the family Bunyaviridae, primarily involving the Hantaan virus (HTNV) and Seoul virus (SEOV) in Asia and the Puumala virus (PUUV) and Dobrava virus (DOBV) in Europe [1]. Worldwide, HFRS affects thousands of people each year, with the majority occurring in China [2, 3]. Hubei Province is one of the most severe endemic areas of HFRS in central China, there have been more than 100,000 HFRS patients reported in this area since 1957 when the first HFRS patient was found [2]. Although integrated intervention measures, including rodent control, environmental management and vaccinations have been implemented, HFRS remains a serious public health problem in Hubei Province. In Hubei, HFRS is primarily caused by HTNVor SEOV [2], the clinical
188
severity of which varies greatly from the most commonly seen case where the patient is asymptomatic to a lethal outcome. However, the mechanisms underlying the varying severity of HFRS are not yet understood. It is generally known that human leucocyte antigen (HLA) molecules play an important role in influencing the progression of disease. HLA molecules including HLA class I molecules encoded by HLA-A, -B and -C genes and HLA class II molecules encoded by HLA-DR, -DQ and -DP genes can effectively present viral antigens to CD8+ T cells, CD4+ T cells and dendritic cells, leading to the stimulation and regulation of host innate and adaptive immune responses against viral pathogens which lead to viral clearance or persistence [4]. Many different HLA alleles that are associated with differing viral infectiousness, clinical courses and outcomes of viral disease have been identified in different populations of the world during the past several years. In hantavirus infections, the HLA-B8-DR3 extended haplotype and HLA-DRB1*0301 were observed to be associated with the severe course of nephropathia epidemica (NE, a milder form of HFRS) caused by PUUV infection [5, 6]. In contrast, the HLA-B27 molecule, a known marker for autoimmune diseases, was associated with the milder clinical course of NE with PUUV infection in Finland [7]. Additionally, there was a higher frequency of the HLA-B*35 allele in HFRS patients with DOBV infections compared with PUUV infections, and HLA-DRB1*13 was associated with the severe course of PUUV-caused HFRS, yet not with DOBV-caused HFRS in a Slovenian population [8]. In China, HLA-DRB1*09 was associated with HFRS in a Chinese Han population [9], whereas HLA-B*46, HLA-B*46-DRB1*09 and HLA-B*51DRB1*09 were known to associate with severe/critical HFRS patients caused by HTNV infection in a Chinese Han population in northwest China [10]. These studies demonstrate that several HLA alleles are risk factors for the clinical severity of HFRS. In the present study, we analysed 22 HLA-DRB allele frequencies in HFRS patients and in healthy controls, and assessed the clinical findings of HFRS caused by HTNV and SEOV infections in Hubei Province of central China to address the possibility of associations of HLA-DRB alleles with the clinical severity of HFRS disease. Despite intensive research that has focused on HLA alleles associated with HFRS or the disease severity of HTNV-caused HFRS in Chinese Han populations, the geographic distribution of Chinese Han populations, rodent reservoirs and differing climates, the association between HLADRB alleles and HFRS caused by SEOV infection has not been studied. In this study, several new HLA-DRB alleles were found to be susceptible or resistant to HFRS and correlated with disease severity and the clinical findings of HFRS infected with different serotypes of hantaviruses in a Chinese Han population.
Eur J Clin Microbiol Infect Dis (2015) 34:187–195
Materials and methods Subjects and clinical data collection A total of 100 patients hospitalised in Hubei Province with a clinical and serological diagnosis of HFRS were included in the study. The patients consisted of 76 males and 24 females, aged 33±13 years. Comprehensive clinical and laboratory data were retrospectively gathered from the patients’ records. The severity of HFRS disease was categorised into four different groups, including mild, moderate, severe and critical groups, which were based on clinical and laboratory parameters that were evaluated as described previously [9, 11]. The control group consisted of 213 randomly chosen healthy unrelated individuals. Of these, 138 subjects were male and 75 subjects were female (age 39±11 years). All subjects were recruited from 13 different cities’ hospitals in Hubei Province from 1986 to 2012. Peripheral blood samples (5 ml) were collected from all subjects and stored at −80 °C until use. Informed consent was obtained from each participant, and the research protocol was approved by the Medical Ethics Committee of Wuhan University, People’s Republic of China. Hantavirus genotyping and HLA typing Genomic RNA was isolated from the peripheral blood of patients by the one-step guanidinium isothiocyanate-phenolchloroform method [12]. Hantavirus genotypes were determined by reverse transcription (RT)-heminested polymerase chain reaction (PCR) with two pairs of HTNV/SEOV genotype-specific primers and one pair of hantavirus universal primers [13–15]. The epidemic viral strains of hantavirus, HTNV 76-118 and SEOV R22, were considered as the positive controls in this study. Genomic DNA was isolated from the peripheral blood of all subjects by the phenol-chloroform method [16]. Genotypes were determined by sequence-specific primer–polymerase chain reaction (SSP-PCR) with HLA-DRB1-specific primers as previously described [17]. The determination of HLA alleles was performed regardless of the severity of each HFRS patient. Statistical analysis The frequencies of HLA alleles were calculated by direct counting. The Chi-squared test or Fisher’s exact test, when an expected value was less than 5, was used for comparison of the frequencies of individual alleles between the controls and HFRS patients. The Kruskal–Wallis test, Mann–Whitney U-test, Chi-squared test or Student’s t-test were used to compare the clinical parameters among different groups. p-Values less than 0.05 were considered to be statistically significant. The odds ratio (OR), which reflects the likelihood
Eur J Clin Microbiol Infect Dis (2015) 34:187–195
of a subject carrying a specific allele, was calculated to identify the associated risk factors. All statistical analyses were performed using the Statistical Package for the Social Sciences (SPSS) version 17.0.
Results Patient characteristics The study determined that 49 out of 100 HFRS patients were diagnosed with HTNV infection, 35 patients had SEOV infection and 16 patients had an unknown type of hantavirus infection. After classification according to their clinical and laboratory parameters during hospitalisation, there were 18 mild HFRS, 60 moderate HFRS, 19 severe HFRS and three critical HFRS patients. Due to the limited number of critical clinical courses of HFRS, critical and severe HFRS patients were combined and referred as the severe course group. In the 49 HFRS patients with HTNV infection, eight were clinically diagnosed as mild HFRS, 27 as moderate HFRS and 14 as severe and critical HFRS. Moreover, 35 SEOV-caused HFRS patients were categorised into three groups comprising seven mild HFRS patients, 23 moderate HFRS patients and five patients with severe and critical HFRS. In addition, the common clinical symptoms and several abnormal laboratory findings of HFRS patients were observed (Table 1). Frequencies of HLA-DRB alleles in healthy controls and HFRS patients with HTNV and SEOV infections Altogether, 19 different HLA-DRB1, HLA-DRB3, HLADRB4 and HLA-DRB5 alleles from 100 HFRS patients and 213 healthy controls in a Chinese Han population from Hubei Province of China were determined in this study, as shown in Table 2. It was found that there were significantly different (p
