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OPEN
received: 22 October 2015 accepted: 03 February 2016 Published: 26 February 2016
Serological detection and analysis of anti-VP1 responses against various enteroviruses (EV) (EV-A, EV-B and EV-C) in Chinese individuals Caixia Gao1,*, Yingying Ding1,*, Peng Zhou2, Jiaojiao Feng1, Baohua Qian3, Ziyu Lin2, Lili Wang1, Jinhong Wang1, Chunyan Zhao1, Xiangyu Li1, Mingmei Cao1, Heng Peng1, Bing Rui1 & Wei Pan1 The overall serological prevalence of EV infections based on ELISA remains unknown. In the present study, the antibody responses against VP1 of the EV-A species (enterovirus 71 (EV71), Coxsackievirus A16 (CA16), Coxsackievirus A5 (CA5) and Coxsackievirus A6 (CA6)), of the EV-B species (Coxsackievirus B3 (CB3)), and of the EV-C species (Poliovirus 1 (PV1)) were detected and analyzed by a NEIBM (novel evolved immunoglobulin-binding molecule)-based ELISA in Shanghai blood donors. The serological prevalence of anti-CB3 VP1 antibodies was demonstrated to show the highest level, with anti-PV1 VP1 antibodies at the second highest level, and anti-CA5, CA6, CA16 and EV71 VP1 antibodies at a comparatively low level. All reactions were significantly correlated at different levels, which were approximately proportional to their sequence similarities. Antibody responses against EV71 VP1 showed obvious differences with responses against other EV-A viruses. Obvious differences in antibody responses between August 2013 and May 2014 were revealed. These findings are the first to describe the detailed information of the serological prevalence of human antibody responses against the VP1 of EV-A, B and C viruses, and could be helpful for understanding of the ubiquity of EV infections and for identifying an effective approach for seroepidemiological surveillance based on ELISA. An increasing number of enteroviruses (EV), a genus of the Picornaviridae family, continue to be identified, and currently these viruses are phylogenetically divided into 12 species: enterovirus A, B, C, D, E, F, G, H and J (EV A–J), and rhinovirus A, B and C. Apart from the three rhinovirus species, four (EV-A, B, C and D) of the enterovirus species infect humans. EV-A consists of 25 types including CA, EV71, and simian (or baboon) enteroviruses. EV-B consists of 63 serotypes including CB, CA9, echoviruses (E), and simian enteroviruses. EV-C consists of 23 types including three PV, CA, and EV-C viruses. EV-D consists of five types including chimpanzee and gorilla viruses1,2 ([http://www.picornaviridae.com], September, 2015). The vast majority of enteroviruses (EV-A, B, C and D) infect the human gastrointestinal tract and can spread to other organs, such as the heart or the central nervous system. Most EV infections are asymptomatic, but over 20 clinically recognized syndromes, including poliomyelitis, encephalitis, meningitis, HFMD, myocarditis, respiratory illnesses, febrile illnesses, pleurodynia, herpangina, conjunctivitis, gastroenteritis, myopericarditis, hepatitis, and pancreatitis, have been frequently associated with many of the 103 EV types2. Among these diseases, HFMD has been the most common EV disease that usually affects children, particularly those less than 5 years old3–5. Several large outbreaks of HFMD have been reported in eastern and southeastern Asian countries and regions during the late 20th century6–9. Since 2008, a dramatic increase in HFMD prevalence has been reported in mainland China3,10–12. The HFMD etiological agents usually consist of viruses in EV-A and EV-B, and most of 1
Department of Medical Microbiology and Parasitology, School of Basic Medicine, Second Military Medical University. 2Department of Physiology, Anhui Medical University, Hefei, China. 3Department of Blood Transfusion, Changhai Hospital, Second Military Medical University, Shanghai, China. *These authors contributed equally to this work. Correspondence and requests for materials should be addressed to W.P. (email: [email protected]) Scientific Reports | 6:21979 | DOI: 10.1038/srep21979
1
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Figure 1. Expression and purification of the recombinant EV71, CA16, CA5, CA6, CB3, PV1 and HAV VP1 proteins. (a) Purified recombinant VP1 proteins were separated on 12% SDS-PAGE gel and stained with Coomassie blue. The expression of the recombinant EV71, CA16, CA5, CA6, CB3, PV1 and HAV VP1 proteins was induced with IPTG. The relative molecular weights (MW) of the expressed products were 50.37 kDa for EV71, 50.38 kDa for CA16, 50.20 kDa for CA5, 50.86 kDa for CA6, 50.45 kDa for CB3, 51.16 kDa for PV1 and 48.08 kDa for HAV. The expressed products were purified using Ni-NTA column affinity chromatography. (b) Western blot analysis of the expression of His-probe recombinant EV71, CA16, CA5, CA6, CB3, PV1 and HAV VP1 proteins.
these include CA2-8, 10, 12, 14, 16 and EV71, which are EV-A viruses13,14. CA16 and EV71 are major etiological agents for HFMD. EV virions are comprised of 60 copies of four capsid proteins (VP1, VP2, VP3 and VP4) that form a symmetrical icosahedral structure. The capsid proteins VP1, VP2 and VP3 are exposed on the virus surface, and the smallest protein, VP4, is arranged inside the icosahedral lattice15–18. The VP1 protein is highly exposed and has been suggested to play an important role in viral pathogenesis and virulence19–21. The viral structural proteins VP1, VP2, VP3 are highly structured, comprising a beta sheet, and are likely the principal targets for the host’s humoral immunity responses16,18,19,22–24. The neutralizing antibody assay has been the only effective serological assay to successfully assess human EV epidemics25–28. However, this technique has provided little cumulative evidence to evaluate the overall serological prevalence levels of various EV types and species. Our previous study, through the use of a NEIBM-based ELISA, demonstrated that the non-neutralizing antibody responses against EV71 were predominantly in response to VP129. In this present study, the VP1 proteins of the major etiological agents for HFMD in EV-A (EV71, CA16, CA5 and CA6) and in EV-B (CB3) and for poliomyelitis in EV-C (PV1) were prepared and used to detect and analyze the anti-VP1 antibody responses by a NEIBM-based ELISA and a competitive ELISA in Shanghai blood donors in August 2013 and May 2014. Different and related antibody responses against VP1 from EV-A, B and C were revealed, which demonstrated different levels of serological prevalence among the various EV types and species. These results could be helpful for understanding ubiquitous EV infections and the establishment of EV seroepidemiological surveillance based on a convenient ELISA.
Results
Production of various recombinant VP1 proteins. In our previous study, an anti-VP1 response was found to be the predominant antibody response against EV71 capsid proteins29. In this present study, the VP1 of various viruses from three species of the Enterovirus genus, EV71, CA16, CA5 and CA6 from EV-A, CB3 from EV-B, PV1 from EV-C, and HAV from a Hepatovirus genus virus (as a control) were expressed as fusion proteins in E. coli and verified by SDS-PAGE (Fig. 1a). The size of each recombinant protein was in agreement with the expected molecular weight. All of the fusion proteins were found in inclusion bodies. Although these proteins
Scientific Reports | 6:21979 | DOI: 10.1038/srep21979
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Figure 2. Comparison of the antibody reactivity against VP1 of EV71, CA16, CA5, CA6, CB3, PV1 and HAV in serum samples in August 2013 (a) and May 2014 (b). Each symbol represents an individual sample, and the lines indicate X25%, X50% and X75% value of the group from the bottom to the top, respectively. “NS” represents no significant difference between the two groups. The differences were significantly different between the other groups.
were insoluble, they could be easily solubilized in 8 M urea and conveniently purified under denaturing conditions. Western blot analysis confirmed the expression of the purified proteins (Fig. 1b).
Antibody responses against VP1 of EV-A, EV-B and EV-C demonstrated different prevalence levels and high correlations. To characterize the antibody reactions against VP1 of the various viruses,
a NEIBM-based ELISA was performed to determine the sample reactivities against VP1 of EV71, CA16, CA5 and CA6 from EV-A, CB3 from EV-B and PV1 from EV-C in August 2013 and May 2014. As shown in Fig. 2a, the anti-VP1 reactivities of the samples from August 2013 demonstrated four reactivity levels. The highest level was against CB3 VP1, which was significantly stronger than that against PV1 VP1, HAV VP1 and the VP1 of EV71, CA16, CA5 and CA6. The second highest level was against PV1 VP1, which was significantly stronger than against HAV VP1 and the VP1 of EV71, CA16 and CA6. A low level was observed against the VP1 of the EV-A members EV71, CA16, CA5 and CA6, which was only significantly stronger than that against HAV VP1, while the lowest level observed was against HAV VP1. Moreover, the anti-VP1 responses against EV-A members EV71, CA16, CA5 and CA6 also demonstrated different reactivity levels, with the anti-CA5 VP1 response significantly stronger than that of the anti-EV71 VP1 and the anti-CA6 VP1 responses. Similar to these results from August 2013, the anti-VP1 reactivities from the May 2014 samples demonstrated three response levels (Fig. 2b): the highest level was against CB3 VP1 and PV1 VP1, the second highest level was against EV-A VP1, and the lowest level was against HAV VP1. However, the anti-VP1 reactivities against the different EV-A viruses demonstrated the same levels, which was different from what was observed in the samples from August 2013. These results could suggest different levels of serological prevalence of various EV infections. In addition, the significantly higher sample reactivity against the VP1 of CB3 or CA5 than against the VP1 of PV1 or EV71, respectively, was found in the samples from August 2013 but not in the samples from May 2014 (Fig. 2). To further characterize the antibody reactions against these enteroviruses, a correlation analysis of the antibody reactions was performed. As shown in Fig. 3, the reactions against VP1 for all of the detected viruses showed significant correlations but at different correlation levels. The correlations between reactions against VP1 of the various EV-A viruses, except for EV71, were the highest, with correlation coefficients above 0.8 at both the August 2013 and May 2014 time points (Fig. 3a,b). The correlations between reactions against VP1 of EV71 and that of other EV-A viruses were the second highest, with correlation coefficients between 0.612 and 0.708. The correlations between reactions against the VP1 of viruses in various EV species (EV71, CA16, CA5 and CA6 in EV-A, CB3 in EV-B and PV1 in EV-C) were also generally the second highest, with correlation coefficients between 0.5 and 0.8 with two exceptions: the correlation between reactions against EV71 VP1 and CB3 VP1 in August 2013 had a correlation coefficient of 0.469, and the correlation between reactions against CA5 VP1 and CB3 VP1 in May 2014 had a correlation coefficient of 0.835. The correlations between reactions against the VP1 of Enterovirus genus viruses and the VP1 of HAV from the Hepatovirus genus were the lowest, with correlation coefficients of less than 0.5. Very interestingly, these results revealed that the correlation levels between the reactions against VP1 of the various viruses were roughly proportional to their sequence similarities, with the exception of EV71 (Fig. 3). These results suggest that the homologous amino acids sequences contribute to the highly significant correlations between the antibody responses against VP1 of the various enteroviruses.
Obvious differences between the anti-VP1 reactions were revealed by inhibition analysis. To
further characterize the antibody reactions in detail, each of the 48 samples that were strongly reactive against VP1 of the various viruses (the serum samples were sorted by the OD value of the anti-VP1 reactivity, and the ones with highest reactivities were chosen) was analyzed in a competitive inhibition ELISA. The results are shown in Fig. 4. The anti-CB3 VP1 samples were completely inhibited by CB3 VP1, partially inhibited by VP1 of PV1 and the EV-A viruses, except for EV71, and poorly inhibited by VP1 of HAV and EV71 (Fig. 4e). The anti-PV1 VP1 samples were completely inhibited by the VP1 of both PV1 and CB3, strongly inhibited by the VP1 of CA5,
Scientific Reports | 6:21979 | DOI: 10.1038/srep21979
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Figure 3. Correlation coefficients of antibody reactions, percent similarity of amino acid sequences and phylogenetic dendrogram between seven VP1 proteins. **represents p
OPEN
received: 22 October 2015 accepted: 03 February 2016 Published: 26 February 2016
Serological detection and analysis of anti-VP1 responses against various enteroviruses (EV) (EV-A, EV-B and EV-C) in Chinese individuals Caixia Gao1,*, Yingying Ding1,*, Peng Zhou2, Jiaojiao Feng1, Baohua Qian3, Ziyu Lin2, Lili Wang1, Jinhong Wang1, Chunyan Zhao1, Xiangyu Li1, Mingmei Cao1, Heng Peng1, Bing Rui1 & Wei Pan1 The overall serological prevalence of EV infections based on ELISA remains unknown. In the present study, the antibody responses against VP1 of the EV-A species (enterovirus 71 (EV71), Coxsackievirus A16 (CA16), Coxsackievirus A5 (CA5) and Coxsackievirus A6 (CA6)), of the EV-B species (Coxsackievirus B3 (CB3)), and of the EV-C species (Poliovirus 1 (PV1)) were detected and analyzed by a NEIBM (novel evolved immunoglobulin-binding molecule)-based ELISA in Shanghai blood donors. The serological prevalence of anti-CB3 VP1 antibodies was demonstrated to show the highest level, with anti-PV1 VP1 antibodies at the second highest level, and anti-CA5, CA6, CA16 and EV71 VP1 antibodies at a comparatively low level. All reactions were significantly correlated at different levels, which were approximately proportional to their sequence similarities. Antibody responses against EV71 VP1 showed obvious differences with responses against other EV-A viruses. Obvious differences in antibody responses between August 2013 and May 2014 were revealed. These findings are the first to describe the detailed information of the serological prevalence of human antibody responses against the VP1 of EV-A, B and C viruses, and could be helpful for understanding of the ubiquity of EV infections and for identifying an effective approach for seroepidemiological surveillance based on ELISA. An increasing number of enteroviruses (EV), a genus of the Picornaviridae family, continue to be identified, and currently these viruses are phylogenetically divided into 12 species: enterovirus A, B, C, D, E, F, G, H and J (EV A–J), and rhinovirus A, B and C. Apart from the three rhinovirus species, four (EV-A, B, C and D) of the enterovirus species infect humans. EV-A consists of 25 types including CA, EV71, and simian (or baboon) enteroviruses. EV-B consists of 63 serotypes including CB, CA9, echoviruses (E), and simian enteroviruses. EV-C consists of 23 types including three PV, CA, and EV-C viruses. EV-D consists of five types including chimpanzee and gorilla viruses1,2 ([http://www.picornaviridae.com], September, 2015). The vast majority of enteroviruses (EV-A, B, C and D) infect the human gastrointestinal tract and can spread to other organs, such as the heart or the central nervous system. Most EV infections are asymptomatic, but over 20 clinically recognized syndromes, including poliomyelitis, encephalitis, meningitis, HFMD, myocarditis, respiratory illnesses, febrile illnesses, pleurodynia, herpangina, conjunctivitis, gastroenteritis, myopericarditis, hepatitis, and pancreatitis, have been frequently associated with many of the 103 EV types2. Among these diseases, HFMD has been the most common EV disease that usually affects children, particularly those less than 5 years old3–5. Several large outbreaks of HFMD have been reported in eastern and southeastern Asian countries and regions during the late 20th century6–9. Since 2008, a dramatic increase in HFMD prevalence has been reported in mainland China3,10–12. The HFMD etiological agents usually consist of viruses in EV-A and EV-B, and most of 1
Department of Medical Microbiology and Parasitology, School of Basic Medicine, Second Military Medical University. 2Department of Physiology, Anhui Medical University, Hefei, China. 3Department of Blood Transfusion, Changhai Hospital, Second Military Medical University, Shanghai, China. *These authors contributed equally to this work. Correspondence and requests for materials should be addressed to W.P. (email: [email protected]) Scientific Reports | 6:21979 | DOI: 10.1038/srep21979
1
www.nature.com/scientificreports/
Figure 1. Expression and purification of the recombinant EV71, CA16, CA5, CA6, CB3, PV1 and HAV VP1 proteins. (a) Purified recombinant VP1 proteins were separated on 12% SDS-PAGE gel and stained with Coomassie blue. The expression of the recombinant EV71, CA16, CA5, CA6, CB3, PV1 and HAV VP1 proteins was induced with IPTG. The relative molecular weights (MW) of the expressed products were 50.37 kDa for EV71, 50.38 kDa for CA16, 50.20 kDa for CA5, 50.86 kDa for CA6, 50.45 kDa for CB3, 51.16 kDa for PV1 and 48.08 kDa for HAV. The expressed products were purified using Ni-NTA column affinity chromatography. (b) Western blot analysis of the expression of His-probe recombinant EV71, CA16, CA5, CA6, CB3, PV1 and HAV VP1 proteins.
these include CA2-8, 10, 12, 14, 16 and EV71, which are EV-A viruses13,14. CA16 and EV71 are major etiological agents for HFMD. EV virions are comprised of 60 copies of four capsid proteins (VP1, VP2, VP3 and VP4) that form a symmetrical icosahedral structure. The capsid proteins VP1, VP2 and VP3 are exposed on the virus surface, and the smallest protein, VP4, is arranged inside the icosahedral lattice15–18. The VP1 protein is highly exposed and has been suggested to play an important role in viral pathogenesis and virulence19–21. The viral structural proteins VP1, VP2, VP3 are highly structured, comprising a beta sheet, and are likely the principal targets for the host’s humoral immunity responses16,18,19,22–24. The neutralizing antibody assay has been the only effective serological assay to successfully assess human EV epidemics25–28. However, this technique has provided little cumulative evidence to evaluate the overall serological prevalence levels of various EV types and species. Our previous study, through the use of a NEIBM-based ELISA, demonstrated that the non-neutralizing antibody responses against EV71 were predominantly in response to VP129. In this present study, the VP1 proteins of the major etiological agents for HFMD in EV-A (EV71, CA16, CA5 and CA6) and in EV-B (CB3) and for poliomyelitis in EV-C (PV1) were prepared and used to detect and analyze the anti-VP1 antibody responses by a NEIBM-based ELISA and a competitive ELISA in Shanghai blood donors in August 2013 and May 2014. Different and related antibody responses against VP1 from EV-A, B and C were revealed, which demonstrated different levels of serological prevalence among the various EV types and species. These results could be helpful for understanding ubiquitous EV infections and the establishment of EV seroepidemiological surveillance based on a convenient ELISA.
Results
Production of various recombinant VP1 proteins. In our previous study, an anti-VP1 response was found to be the predominant antibody response against EV71 capsid proteins29. In this present study, the VP1 of various viruses from three species of the Enterovirus genus, EV71, CA16, CA5 and CA6 from EV-A, CB3 from EV-B, PV1 from EV-C, and HAV from a Hepatovirus genus virus (as a control) were expressed as fusion proteins in E. coli and verified by SDS-PAGE (Fig. 1a). The size of each recombinant protein was in agreement with the expected molecular weight. All of the fusion proteins were found in inclusion bodies. Although these proteins
Scientific Reports | 6:21979 | DOI: 10.1038/srep21979
2
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Figure 2. Comparison of the antibody reactivity against VP1 of EV71, CA16, CA5, CA6, CB3, PV1 and HAV in serum samples in August 2013 (a) and May 2014 (b). Each symbol represents an individual sample, and the lines indicate X25%, X50% and X75% value of the group from the bottom to the top, respectively. “NS” represents no significant difference between the two groups. The differences were significantly different between the other groups.
were insoluble, they could be easily solubilized in 8 M urea and conveniently purified under denaturing conditions. Western blot analysis confirmed the expression of the purified proteins (Fig. 1b).
Antibody responses against VP1 of EV-A, EV-B and EV-C demonstrated different prevalence levels and high correlations. To characterize the antibody reactions against VP1 of the various viruses,
a NEIBM-based ELISA was performed to determine the sample reactivities against VP1 of EV71, CA16, CA5 and CA6 from EV-A, CB3 from EV-B and PV1 from EV-C in August 2013 and May 2014. As shown in Fig. 2a, the anti-VP1 reactivities of the samples from August 2013 demonstrated four reactivity levels. The highest level was against CB3 VP1, which was significantly stronger than that against PV1 VP1, HAV VP1 and the VP1 of EV71, CA16, CA5 and CA6. The second highest level was against PV1 VP1, which was significantly stronger than against HAV VP1 and the VP1 of EV71, CA16 and CA6. A low level was observed against the VP1 of the EV-A members EV71, CA16, CA5 and CA6, which was only significantly stronger than that against HAV VP1, while the lowest level observed was against HAV VP1. Moreover, the anti-VP1 responses against EV-A members EV71, CA16, CA5 and CA6 also demonstrated different reactivity levels, with the anti-CA5 VP1 response significantly stronger than that of the anti-EV71 VP1 and the anti-CA6 VP1 responses. Similar to these results from August 2013, the anti-VP1 reactivities from the May 2014 samples demonstrated three response levels (Fig. 2b): the highest level was against CB3 VP1 and PV1 VP1, the second highest level was against EV-A VP1, and the lowest level was against HAV VP1. However, the anti-VP1 reactivities against the different EV-A viruses demonstrated the same levels, which was different from what was observed in the samples from August 2013. These results could suggest different levels of serological prevalence of various EV infections. In addition, the significantly higher sample reactivity against the VP1 of CB3 or CA5 than against the VP1 of PV1 or EV71, respectively, was found in the samples from August 2013 but not in the samples from May 2014 (Fig. 2). To further characterize the antibody reactions against these enteroviruses, a correlation analysis of the antibody reactions was performed. As shown in Fig. 3, the reactions against VP1 for all of the detected viruses showed significant correlations but at different correlation levels. The correlations between reactions against VP1 of the various EV-A viruses, except for EV71, were the highest, with correlation coefficients above 0.8 at both the August 2013 and May 2014 time points (Fig. 3a,b). The correlations between reactions against VP1 of EV71 and that of other EV-A viruses were the second highest, with correlation coefficients between 0.612 and 0.708. The correlations between reactions against the VP1 of viruses in various EV species (EV71, CA16, CA5 and CA6 in EV-A, CB3 in EV-B and PV1 in EV-C) were also generally the second highest, with correlation coefficients between 0.5 and 0.8 with two exceptions: the correlation between reactions against EV71 VP1 and CB3 VP1 in August 2013 had a correlation coefficient of 0.469, and the correlation between reactions against CA5 VP1 and CB3 VP1 in May 2014 had a correlation coefficient of 0.835. The correlations between reactions against the VP1 of Enterovirus genus viruses and the VP1 of HAV from the Hepatovirus genus were the lowest, with correlation coefficients of less than 0.5. Very interestingly, these results revealed that the correlation levels between the reactions against VP1 of the various viruses were roughly proportional to their sequence similarities, with the exception of EV71 (Fig. 3). These results suggest that the homologous amino acids sequences contribute to the highly significant correlations between the antibody responses against VP1 of the various enteroviruses.
Obvious differences between the anti-VP1 reactions were revealed by inhibition analysis. To
further characterize the antibody reactions in detail, each of the 48 samples that were strongly reactive against VP1 of the various viruses (the serum samples were sorted by the OD value of the anti-VP1 reactivity, and the ones with highest reactivities were chosen) was analyzed in a competitive inhibition ELISA. The results are shown in Fig. 4. The anti-CB3 VP1 samples were completely inhibited by CB3 VP1, partially inhibited by VP1 of PV1 and the EV-A viruses, except for EV71, and poorly inhibited by VP1 of HAV and EV71 (Fig. 4e). The anti-PV1 VP1 samples were completely inhibited by the VP1 of both PV1 and CB3, strongly inhibited by the VP1 of CA5,
Scientific Reports | 6:21979 | DOI: 10.1038/srep21979
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Figure 3. Correlation coefficients of antibody reactions, percent similarity of amino acid sequences and phylogenetic dendrogram between seven VP1 proteins. **represents p
