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Serum-derived IgG from coxsackievirus A6-infected patients can enhance the infection of peripheral blood mononuclear cells with coxsackievirus A6
Microbial Pathogenesis 125 (2018) 7–11
Contents lists available at ScienceDirect
Microbial Pathogenesis journal homepage: www.elsevier.com/locate/micpath
Serum-derived IgG from coxsackievirus A6-infected patients can enhance the infection of peripheral blood mononuclear cells with coxsackievirus A6
T
Sushama Aswathyraja,b, Famara Sanea, Chandrasekhar Raghuc, Sasidharanpillai Sabeenab, Enagnon Kazali Alidjinoua, Govindakarnavar Arunkumarb,∗∗, Didier Hobera,∗ a
Univ Lille Faculté de Médecine, CHU Lille Laboratoire de Virologie EA3610, F-59037, Lille, France Manipal Center for Virus Research (MCVR), Manipal Academy of Higher Education, Manipal, Karnataka, India c Department of Pharmaceutical Biotechnology, Manipal College of Pharmaceutical Sciences, Manipal Academy of Higher Education, Manipal, Karnataka, India b
A R T I C LE I N FO
A B S T R A C T
Keywords: Enterovirus IFNα Viral RNA In vitro ELISA RT-qPCR
Coxsackievirus A6 (CV-A6) has recently emerged as an enterovirus causing Hand Foot and Mouth Disease with severe complications. The pathogenic mechanisms of CV-A6- associated Hand foot and Mouth disease are largely unknown. In this study, it was investigated whether serum and IgG from patients with CV-A6 infection can enhance the infection of PBMC with the virus. Serum samples were obtained from five children with CV-A6 infection confirmed by RT-PCR and seven controls. IgG was isolated from serum by using affinity chromatography columns. CV-A6 was incubated with serum or IgG from controls and patients then the mixtures were added to PBMC cultures. The levels of IFNα in supernatants were measured by ELISA, and the levels of intracellular viral RNA were measured by RT-qPCR. It has been observed that there is an anti-CV-A6 enhancing activity in serum and serum-derived immunoglobulin G of children with CV-A6 infection but not in those of uninfected controls. Whether this activity has implications in the pathogenesis of CV-A6 associated diseases should be investigated.
1. Introduction Coxsackievirus A6 (CV-A6), a single-stranded RNA virus, belongs to Picornaviridae family (Genus Enterovirus). This enterically transmitted virus has emerged as a pathogen responsible for Hand, foot and mouth disease (HFMD) [1]. HFMD is a common infectious disease in children characterized by fever, vesicular rashes on hand, feet, buttock and ulcers in the oral mucosa. Even though HFMD is a self-limiting disease, a small proportion of children may experience severe complications such as meningitis, encephalitis, acute flaccid paralysis and neuro-respiratory syndrome. Until 2008, most of the HFMD outbreaks were mainly due to two enterovirus serotypes namely enterovirus 71 (EV-71) and coxsackievirus A16 (CV-A16). However, since 2008 coxsackievirusA6 (CV-A6) has been found associated with epidemics and outbreaks of HFMD in Europe, in Asia-Pacific region and in India [2–5]. Outbreak data revealed that CV-A6 could affect broader demographic regions and different age groups (both children and adults), resulting in most severe forms of disease compared to classic HFMD.
Primary multiplication of enteroviruses takes place in the respiratory or gastrointestinal tract, followed by the appearance of the infectious virus in the blood, facilitating further spread to secondary target organs. Though CV-A6 causes different clinical manifestations varying from mild asymptomatic infection to severe encephalitis to date. There has been no study unravelling the pathogenic mechanisms of CV-A6-associated disease. Serum-dependent enhancement of virus infection was reported in infection by other members of the Picornaviridae family such as EV 71, poliovirus, foot-and-mouth disease virus [6]. Our team has identified that coxsackievirus B4 (Genus Enterovirus), can infect monocytes by the antibody-dependent mechanism through interactions between the virus, antiviral antibodies, and specific receptors that result in increased IFN-α production [7,8]. In this study, it was investigated whether serum from CV-A6-infected children can enhance the infection of PBMC with CV-A6.
∗
Corresponding author. Laboratoire de Virologie EA3610, Centre Paul Boulanger Hôpital A Calmette CHRU Lille, 1 Boulevard du Pr J Leclerc, 59037, Lille, France. Corresponding author. Manipal Centre for Virus Research, Manipal Academy of Higher Education (Deemed to be University), Manipal, Karnataka, 576104, India. E-mail addresses: [email protected] (G. Arunkumar), [email protected] (D. Hober). ∗∗
https://doi.org/10.1016/j.micpath.2018.08.064 Received 29 March 2018; Received in revised form 30 August 2018; Accepted 31 August 2018 Available online 04 September 2018 0882-4010/ © 2018 Elsevier Ltd. All rights reserved.
Microbial Pathogenesis 125 (2018) 7–11
S. Aswathyraj et al.
2. Materials and methods
to manufacturer's instructions. RNA Extraction: The extraction of total RNA was performed using the Tri-reagent method. Quantitative Real-Time Reverse transcriptase PCR (rRT-PCR): Affinityscript® QPCR cDNA Synthesis (Agilent Technologies, USA) kit was used for the synthesis of cDNA from RNA. The reaction mix, with a total volume of 50 μL, included master mix, water, forward primer at a concentration of 10 mM/L and RT stratastript enzyme mix (Stratagene, USA). The reaction was performed in the Perkin Elmer 2400 thermocycler with the following program: 25 °C for 5 min, 42 °C for 15 min, and 95 °C for 5 min. Pan enterovirus quantitative PCR was performed with the kit Brilliant® II QPCR (Agilent Technologies, USA) on the Mx3000p® (Stratagene, USA). The reaction mixture, with a total volume of 20 μL, contained 2× Brilliant Master mix, nuclease-free water, probe (2 mM/ L), forward primer, reverse primer (each primer concentration 10 mM/ L) and cDNA. The amplification program was set for 10 min at 95 °C and then 40 cycles of heating (30 s at 95 °C) and annealing (1 min at 60 °C). The primer sequences targeting the highly conserved 5′nontranslated region of enteroviruses were: forward primer CCC ATG TGA CGG CTA ATC ATT ACC ATA GTC AGC AGC, reverse primer ATT ACC ATA GTC AGC AGC, probe FAM-CGA AAC CTA CTT TCC TGG GTG GTG TTTROX. The standard for quantification of RNA was enterovirus 71 (VIRCELL, Granada, Spain) with 5-point range: 1.26 × 104; 1.26 × 103; 126; 12.6 and 1.26 copies/mL. Statistical analysis: performed with GraphPad Prism® V6.0 version. Mann–Whitney U Test was used when appropriate. The pvalue < 0.05 was considered statistically significant.
Cells: Peripheral blood mononuclear cells (PBMC) were isolated from buffy coat cell pellet provided by The French Blood Establishment (EFS) Lille. Mononuclear cells have been separated on density gradients of Ficoll-Hypaque. Briefly the buffy coat was directly layered on top of Ficoll-Paque™ (GE Healthcare) (ratio 1:1), and centrifuged at 500 g for 30 min at 20 °C. PBMC layer was collected and washed twice with RPMI-1640 medium. The cells were then suspended in RPMI medium supplemented with 10% heat-inactivated fetal calf serum, 1% non-essential amino acid, 1% penicillin-streptomycin and 1% of L-glutamine. The cells were counted, and the cell number was adjusted to 5 × 106 cells/mL. Vero cells: (ATCC® CCL81™) were grown in Dulbecco's modified eagle's medium (DMEM; Gibco BRL, Invitrogen, Gaithersburg, MD, USA) supplemented with 10% fetal calf serum (FCS, Sigma St Louis, MO, USA), 2–6 mM L-Glutamine (Gibco BRL), 50 mg/ mL streptomycin, and 50 IU/mL penicillin (Bio Whittaker). Virus: CV-A6 isolated from a clinical sample was propagated in Vero cells (ATCC® CCL81™). The infectious titers of culture supernatants were assessed using the end-point dilution assay, and the ReedMuench statistical method was used to determine the tissue culture 50% infectious dose (TCID50). The virus isolate was aliquoted and stored at −80 °C. Serum samples: Obtained from children with CV-A6 infection (Hand Foot and mouth Disease/encephalitis/meningitis/myocarditis) confirmed by reverse transcriptase - polymerase chain reaction. Serum samples from age-matched control children with clinical symptoms of dengue-like illness or influenza-like illness received at Manipal Centre for Virus Research, Manipal, India were included. The consent of parents was obtained, and the study was approved by Institutional Ethical committee. Microneutralization Assay: Fifty microliters of sample dilutions (2 fold serial dilutions) and 50 μL of virus stock containing 200 TCID50 CVA6 were mixed and incubated in a microtiter plate with Vero cells at 37 °C for seven days. The cell control and virus control were run simultaneously. The neutralizing antibody titres were expressed as the reciprocal of highest dilution of serum that completely inhibited the cytopathic effect of the virus. Extraction of IgG from serum: Protein G HP Spintrap (GE healthcare UK limited) was used to isolate IgG from the serum samples. IgG-enriched fraction and IgG-depleted fractions were processed separately. Serum-dependent enhancement assay: Serum/eluted IgG was diluted with RPMI-1640 media in the ratio 1/10, 1/100 and 1/1000. Twenty-five microliters of medium or diluted serum/eluted IgG was incubated for 2 h at 37 °C with 25 μl of CV-A6 suspension (105 TCID50). Post-incubation, the mixtures were inoculated into PBMC cultures in microplates (5 × 105 cells/well). After 48 h of incubation, supernatants were collected for INFα detection by ELISA. After collection of the supernatant, the wells containing the cells were washed four times with cold 1× PBS (4 °C) by centrifuging at 1500 rpm for 10 min. The cells were removed easily by adding TRI Reagent® (Sigma- Aldrich), collected in a total volume of 900 μL tri-reagent (Sigma) in 1.5 ml tubes and subjected to RNA extraction. ELISA (sandwich ELISA): IFN-α was measured by using pan-specific INF-α ELISA kit (Mabtech® Human IFN-α ELISA PRO kit) according
3. Result 3.1. Children with CV-A6 infection Serum was collected from 5 children (less than ten year-old) with CV-A6 infection confirmed by detection of enteroviral RNA by reversetranscriptase real-time PCR (RT-PCR) in cerebrospinal fluid and/or throat swab and/or vesicle swab. Based on clinical symptoms associated with CV-A6 infection, these patients were further classified as acute encephalitis syndrome (n = 2), meningoencephalitis (n = 1), myocarditis (n = 1) and HFMD (n = 1) (Table 1). The titres of neutralizing anti-CV-A6 antibodies in serum were < 1:4 as displayed by micro neutralization assay. The serum samples were collected after the onset of the disease as soon as one day in the case of patients number 3, and 4–6 days in the case of other patients. Serum samples were obtained from age-matched controls without symptoms of infection (n = 1) or with symptoms of dengue-like illness (n = 3) or influenza-like illness (n = 3). The titres of neutralizing antiCVA6 antibodies in serum from 5 subjects were < 1:4 and from 2 others were 1:32 and 1:128 as displayed by micro neutralization assay. 3.2. Serum from children with CV-A6 infection can enhance the CVA6induced production of INFα by PBMC The levels of INF-α in supernatants of PBMC culture inoculated with CV-A6 mixed with medium was lower than 20 pg/ml. When CV-A6 was mixed with various dilutions of serum or serum-derived IgG from
Table 1 Demographic and clinical details of Coxsackie virus-A6 infected children (n = 5). No
Age(Y)
Sex
Clinical symptoms
Syndrome
Outcome
1 2 3 4 5
6.5 4 8 2 4
F M M M M
Fever, headache, vomiting, Seizure Fever,coryza,myalgia and altered sensorium Fever and Skin Lesions Hepatomegaly, Myocarditis, Vomiting Diarrhea, Mechanical Ventilation Fever,coryza,myalgia and altered sensorium
Acute Encephalitis Acute Encephalitis Hand Foot Mouth Disease Myocarditis Meningoencephalitis
Death Death Recovery Recovery Recovery
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Fig. 1. Serum and IgG from children with CV-A6 infection enhance the CV-A6 induced production of IFNα by PBMC. CV-A6 was incubated with serum or serum-derived IgG for 2 h then the mixtures were added to PBMC cultures. Two days later the supernatants were harvested to evaluate the level of IFNα by ELISA; the results were expressed as pg/ml. Individual representation of IFNα levels obtained when CVA6 was mixed with diluted serum samples (1/10; 1/100; 1/1000 dilution) from 7 controls (A) or IgG isolated from these samples (B). Individual representation of highest IFNα levels obtained when CV-A6 was mixed with serum samples from 5 children with CV-A6 infection or with serum-derived Ig G (IgG enriched serum) or IgG depleted serum (C). The values were obtained with dilution 1/1000 and 1/10 of serum in the case of patients 1 and 5 respectively; and dilution 1/100 in the case of patients 2-4.
controls the levels of IFNα in supernatants of PBMC cultures were lower than 26 pg/ml (Fig. 1 A and B). In contrast when CV-A6 was mixed with various dilutions of serum (final dilution 1/10 to 1/1000) or serumderived IgG from patients, the levels of IFNα in supernatants of PBMC cultures were up to 65 pg/ml (Fig. 1C). The highest level of IFNα was obtained with CV-A6 mixed with serum (dilution 1/1000) from the child with acute encephalitis (patient 1 in Fig. 1C). The mean level of IFNα obtained with serum samples from children with CV-A6 infection was significantly higher than the one obtained with serum samples from controls (37.5 ± 16 vs 15.5 ± 2.6 pg/ml, p-value < 0.002). 3.3. Serum-derived IgG from children with CV-A6 infection can increase the level of intracellular CV-A6 RNA in PBMC CV-A6 was incubated for 2 h at 37 °C in presence of medium or various dilutions (1/10; 1/100 and 1/1000) of serum-derived IgG from children with CV-A6 infection and controls. Then the mixtures were added to PBMC cultures, and the plates were incubated. After 24 h, the cells were recovered to determine the level of intracellular viral RNA. There was no enteroviral RNA, or the level was under the limit of detection of the assay when CV-A6 was incubated in presence of medium. The enteroviral RNA level was 3 × 102 copy/ng total RNA when CV-A6 was incubated with serum-derived IgG from one control. The neutralizing activity of serum from this subject was < 1:4. The enteroviral RNA level was under the limit of detection of the assay with serumderived IgG from the six other controls. The neutralizing activity of serum from 4 out of these subject was < 1:4 and from 2 others was 1:32 and 1:128 respectively (data not shown). In contrast, when PBMC was inoculated with CV-A6 mixed with serum-derived IgG from children with CV-A6 infection, the levels of intracellular viral RNA were much higher (Fig. 2). The enteroviral RNA levels were at least around 4 × 104 copy/ng of total RNA when CVA6 was mixed with serum derived IgG from patient 1 and patient 4
Fig. 2. Individual representation of levels of enteroviral RNA in PBMC inoculated with CV-A6 mixed with serum-derived IgG of children with CVA6 infection. CV-A6 was incubated with serum-derived IgG (1/10, 1/100 and 1/1000 dilution) of each patient (patient 1 to 5), then the mixture was added to PBMC cultures as described in the legend of Fig. 1. After two days of incubation, the cultures were washed four times with PBS and extraction of total RNA was carried out using tri-reagent technique. The level of viral RNA was evaluated by real-time RT-qPCR and the results expressed as copy/ng of total RNA.
(dilution 1/100). Meanwhile, enteroviral RNA levels were up to 105, 6 × 104, 5 × 104 copy/ng of total RNA when CVA6 was mixed with serum-derived IgG from patient 5 (dilution 1/10), patient 2 (dilution 1/ 100) and patient 3 (dilution 1/1000) respectively. There was no relationship between the outcome of the infection and the serum/IgG –dependent enhancement of CV-A6 infection in our system.
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4. Discussion
individuals as a result of previous infections. An anti-CV-A6 enhancing activity was observed in patients with HFMD whom disease outcome was favourable (patient number 3) and in patients with the more severe disease. There was no clear relationship between the pattern of the CV-A6 infection or its outcome and the anti-CV-A6 enhancing activity of serum. Nevertheless, the role of the anti-CV-A6 enhancing activity in the course of the infection is an open question. Whether this activity of serum observed in vitro can reflect a favourable condition for deleterious outcome in some individuals remains to be determined. Our data suggest that non-neutralizing antibodies appear early during the infection. The enhancement is due to non-neutralizing antibodies or antibodies at subneutralizing levels produced during the infection. There was no correlation between the viral copy number and the dilution of IgG in our experiments. Furthermore it cannot be excluded that antibodies directed against more broadly expressed epitopes on other enteroviruses bound CV-A6 particles in our system. Further studies are needed to understand this phenomenon, which will improve the knowledge of CV-A6-associated disease. In conclusion, there is an anti-CV-A6 enhancing activity in serum and serum-derived IgG of children with CV-A6 infection. The anti-CVA6 enhancing activity of serum should be evaluated in larger cohorts. Further investigation are needed to determine whether this activity has implications in the pathogenesis of CV-A6 induced diseases and in the design of vaccines aimed to fight HFMD that is associated with a high morbidity rate and can lead to severe complications.
The current study is different in many respects from those of other investigators; it is reported for the first time that the serum from individuals with CV-A6 infection can enhance the infection of PBMC with CV-A6 in vitro. Several considerations are noteworthy. The issue of the anti-CV-A6 enhancing activity of serum has been addressed in vitro in a system based on human PBMC. In this system, the levels of intracellular enteroviral RNA were measured by quantitative real-time RT-PCR, which allowed comparisons between the levels of enteroviral RNA in PBMC inoculated with CV-A6 or with the virus mixed with serum from individuals. Moreover, the anti-CV-A6 enhancing activity of serum has been addressed through the levels of IFNα in supernatants of PBMCs measured by a sensitive ELISA. The same batch of PBCM was used to test the enhancing activity of all serum samples from patients and controls which allowed inter-individual comparisons. The production of IFNα by PBMC associated with the presence of enteroviral RNA in the cells when CV-A6 was mixed with serum from patient supported the idea that cells were infected with CV-A6 in these experiments. The enhanced CV-A6-induced release of IFNα by PBMC and the increased level of enteroviral RNA in these cells obtained with diluted serum from patients were due to IgG as shown by the activity of serumderived IgG from CV-A6 infected individuals. IgG was obtained from serum by affinity chromatography using Protein G, an enhancing effect was observed with the eluted fraction but not with the filtrate fraction. In contrast serum and IgG from individuals without CV-A6 infection did not enhance the CV-A6-induced release of IFNα by PBMC nor the infection of these cells. The level of intracellular enteroviral RNA was under the limit of detection of the assay when CV-A6 was incubated with medium or serum-derived IgG of controls. In contrast the level of intracellular enteroviral RNA was high when CV-A6 was incubated with serum-derived IgG of individuals with CV-A6 infection. At optimal dilution of serum the amount of viral RNA was at least 40000 copy/ng of total RNA, which is more consistent with the hypothesis of CV-A6 replication than just internalization. This is in agreement with previous studies demonstrating that viral replication was not detected in PBMC inoculated with enteroviruses (CV-B3, Echovirus 1, poliovirus 1) unless viral RNA was transfected [9]. In the same vein high levels of CVB4 RNA were detected in PBMC when the virus was previously incubated with IgG containing anti-CV-B4 antibodies. The enhancement is associated with intracellular viral replication, as previously shown by Alidjinou et al. [10]. The strong increase in viral load before and after incubation with enhancing serum is clearly compatible with intracellular viral replication. It was reported that the infection of PBMCderived monocytes with CV-B4 was enhanced by serum/IgG, which resulted in the release of infectious particles by these cells [7]. Whether the scenario is similar for CV-A6 remains to be determined. This is reminiscent of previous studies describing that human serum harboured an enhancing anti-CV-B4 activity, due to IgG, in human PBMC or cell line cultures, provided the serum was diluted [7]. It was shown that immune serum-mediated enhancement of infection with CV-B4 in vitro in human PBMCs was due to anti-CV-B4 antibodies [11,12]. We assume that anti-CV-A6 antibodies, contained in immune serum and IgG obtained from CV-A6 infected individuals, enhanced the infection of PBMC with CV-A6 in vitro in the present study. In serum from patients there was an anti-CV-A6 enhancing activity, due to serum-derived IgG as shown in this study, although the anti-CVA6 neutralizing activity was under the limit of detection of the assay (< 1/4). The anti-CV-A6 enhancing activity was observed as soon as day one after the onset of the disease in patients. Furthermore, there was no enhancing activity in serum from controls although a neutralizing activity was displayed (titre values up to 1/32 and 1/128). Longitudinal studies are needed to determine whether the enhancing activity appears early in the course of the infection before the neutralizing activity or whether this activity is preexisting in some
Conflicts of interest None. Acknowledgement This work has been supported by the Ministère de l’Education Nationale de la Recherche et de la Technologie, Université de Lille (EA 3610) and Centre Hospitalier Régional et Universitaire de Lille France. The authors thank referring clinicians and Indian Council of Medical Research Government of India, New Delhi for the funding of the project: “Distribution of non-polio enterovirus in children with HFMD in Kerala, Karnataka and Goa” file No.VIR/NP/66/2013-ECD-1. References [1] S. Aswathyraj, G. Arunkumar, E.K. Alidjinou, D. Hober, Hand, foot and mouth disease (HFMD): emerging epidemiology and the need for a vaccine strategy, Med. Microbiol. Immunol. 205 (2016) 397–407. [2] R. Osterback, T. Vuorinen, M. Linna, P. Susi, T. Hyypia, M. Waris, Coxsackievirus A6 and hand, foot, and mouth disease, Finland. Emerg Infect Dis. 15 (2009) 1485–1488. [3] W. Xing, Q. Liao, C. Viboud, J. Zhang, J. Sun, J.T. Wu, et al., Hand, foot, and mouth disease in China, 2008-12: an epidemiological study, Lancet Infect. Dis. 14 (2014) 308–318. [4] N. Sarma, A. Sarkar, A. Mukherjee, A. Ghosh, S. Dhar, R. Malakar, Epidemic of hand, foot and mouth disease in West Bengal, India in August, 2007: a multicentric study, Indian J. Dermatol. 54 (2009) 26. [5] V. Gopalkrishna, P.R. Patil, G.P. Patil, S.D. Chitambar, Circulation of multiple enterovirus serotypes causing hand, foot and mouth disease in India, J. Med. Microbiol. 61 (2012) 420–425. [6] J.-F. Han, R.-Y. Cao, Y.-Q. Deng, X. Tian, T. Jiang, E.-D. Qin, et al., Antibody dependent enhancement infection of Enterovirus 71 in vitro and in vivo, Virol. J. 8 (2011) 106. [7] D. Hober, W. Chehadeh, A. Bouzidi, P. Wattré, Antibody-dependent enhancement of coxsackievirus B4 infectivity of human peripheral blood mononuclear cells results in increased interferon-alpha synthesis, J. Infect. Dis. 184 (2001) 1098–1108. [8] P. Sauter, D. Hober, Mechanisms and results of the antibody-dependent enhancement of viral infections and role in the pathogenesis of coxsackievirus B-induced diseases, Microb. Infect. 11 (2009) 443–451. [9] T. Vuorinen, R. Vainionp, J. Heino, T. Hyypi, Enterovirus receptors and virus replication in human leukocytes, J. Gen. Virol. 80 (1999) 921–927. [10] E.K. Alidjinou, F. Sané, I. Engelmann, D. Hober, Serum-dependent enhancement of coxsackievirus B4-induced production of IFNα, IL-6 and TNFα by peripheral blood mononuclear cells, J. Mol. Biol. 425 (2013) 5020–5031.
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[11] W. Chehadeh, P.-E. Lobert, P. Sauter, A. Goffard, B. Lucas, J. Weill, et al., Viral protein VP4 is a target of human antibodies enhancing coxsackievirus B4-and B3induced synthesis of alpha interferon, J. Virol. 79 (2005) 13882–13891.
[12] P. Sauter, P.-E. Lobert, B. Lucas, R. Varela-Calvino, G. Alm, P. Wattre, et al., Role of the capsid protein VP4 in the plasma-dependent enhancement of the Coxsackievirus B4E2-infection of human peripheral blood cells, Virus Res. 125 (2007) 183–190.
11
Contents lists available at ScienceDirect
Microbial Pathogenesis journal homepage: www.elsevier.com/locate/micpath
Serum-derived IgG from coxsackievirus A6-infected patients can enhance the infection of peripheral blood mononuclear cells with coxsackievirus A6
T
Sushama Aswathyraja,b, Famara Sanea, Chandrasekhar Raghuc, Sasidharanpillai Sabeenab, Enagnon Kazali Alidjinoua, Govindakarnavar Arunkumarb,∗∗, Didier Hobera,∗ a
Univ Lille Faculté de Médecine, CHU Lille Laboratoire de Virologie EA3610, F-59037, Lille, France Manipal Center for Virus Research (MCVR), Manipal Academy of Higher Education, Manipal, Karnataka, India c Department of Pharmaceutical Biotechnology, Manipal College of Pharmaceutical Sciences, Manipal Academy of Higher Education, Manipal, Karnataka, India b
A R T I C LE I N FO
A B S T R A C T
Keywords: Enterovirus IFNα Viral RNA In vitro ELISA RT-qPCR
Coxsackievirus A6 (CV-A6) has recently emerged as an enterovirus causing Hand Foot and Mouth Disease with severe complications. The pathogenic mechanisms of CV-A6- associated Hand foot and Mouth disease are largely unknown. In this study, it was investigated whether serum and IgG from patients with CV-A6 infection can enhance the infection of PBMC with the virus. Serum samples were obtained from five children with CV-A6 infection confirmed by RT-PCR and seven controls. IgG was isolated from serum by using affinity chromatography columns. CV-A6 was incubated with serum or IgG from controls and patients then the mixtures were added to PBMC cultures. The levels of IFNα in supernatants were measured by ELISA, and the levels of intracellular viral RNA were measured by RT-qPCR. It has been observed that there is an anti-CV-A6 enhancing activity in serum and serum-derived immunoglobulin G of children with CV-A6 infection but not in those of uninfected controls. Whether this activity has implications in the pathogenesis of CV-A6 associated diseases should be investigated.
1. Introduction Coxsackievirus A6 (CV-A6), a single-stranded RNA virus, belongs to Picornaviridae family (Genus Enterovirus). This enterically transmitted virus has emerged as a pathogen responsible for Hand, foot and mouth disease (HFMD) [1]. HFMD is a common infectious disease in children characterized by fever, vesicular rashes on hand, feet, buttock and ulcers in the oral mucosa. Even though HFMD is a self-limiting disease, a small proportion of children may experience severe complications such as meningitis, encephalitis, acute flaccid paralysis and neuro-respiratory syndrome. Until 2008, most of the HFMD outbreaks were mainly due to two enterovirus serotypes namely enterovirus 71 (EV-71) and coxsackievirus A16 (CV-A16). However, since 2008 coxsackievirusA6 (CV-A6) has been found associated with epidemics and outbreaks of HFMD in Europe, in Asia-Pacific region and in India [2–5]. Outbreak data revealed that CV-A6 could affect broader demographic regions and different age groups (both children and adults), resulting in most severe forms of disease compared to classic HFMD.
Primary multiplication of enteroviruses takes place in the respiratory or gastrointestinal tract, followed by the appearance of the infectious virus in the blood, facilitating further spread to secondary target organs. Though CV-A6 causes different clinical manifestations varying from mild asymptomatic infection to severe encephalitis to date. There has been no study unravelling the pathogenic mechanisms of CV-A6-associated disease. Serum-dependent enhancement of virus infection was reported in infection by other members of the Picornaviridae family such as EV 71, poliovirus, foot-and-mouth disease virus [6]. Our team has identified that coxsackievirus B4 (Genus Enterovirus), can infect monocytes by the antibody-dependent mechanism through interactions between the virus, antiviral antibodies, and specific receptors that result in increased IFN-α production [7,8]. In this study, it was investigated whether serum from CV-A6-infected children can enhance the infection of PBMC with CV-A6.
∗
Corresponding author. Laboratoire de Virologie EA3610, Centre Paul Boulanger Hôpital A Calmette CHRU Lille, 1 Boulevard du Pr J Leclerc, 59037, Lille, France. Corresponding author. Manipal Centre for Virus Research, Manipal Academy of Higher Education (Deemed to be University), Manipal, Karnataka, 576104, India. E-mail addresses: [email protected] (G. Arunkumar), [email protected] (D. Hober). ∗∗
https://doi.org/10.1016/j.micpath.2018.08.064 Received 29 March 2018; Received in revised form 30 August 2018; Accepted 31 August 2018 Available online 04 September 2018 0882-4010/ © 2018 Elsevier Ltd. All rights reserved.
Microbial Pathogenesis 125 (2018) 7–11
S. Aswathyraj et al.
2. Materials and methods
to manufacturer's instructions. RNA Extraction: The extraction of total RNA was performed using the Tri-reagent method. Quantitative Real-Time Reverse transcriptase PCR (rRT-PCR): Affinityscript® QPCR cDNA Synthesis (Agilent Technologies, USA) kit was used for the synthesis of cDNA from RNA. The reaction mix, with a total volume of 50 μL, included master mix, water, forward primer at a concentration of 10 mM/L and RT stratastript enzyme mix (Stratagene, USA). The reaction was performed in the Perkin Elmer 2400 thermocycler with the following program: 25 °C for 5 min, 42 °C for 15 min, and 95 °C for 5 min. Pan enterovirus quantitative PCR was performed with the kit Brilliant® II QPCR (Agilent Technologies, USA) on the Mx3000p® (Stratagene, USA). The reaction mixture, with a total volume of 20 μL, contained 2× Brilliant Master mix, nuclease-free water, probe (2 mM/ L), forward primer, reverse primer (each primer concentration 10 mM/ L) and cDNA. The amplification program was set for 10 min at 95 °C and then 40 cycles of heating (30 s at 95 °C) and annealing (1 min at 60 °C). The primer sequences targeting the highly conserved 5′nontranslated region of enteroviruses were: forward primer CCC ATG TGA CGG CTA ATC ATT ACC ATA GTC AGC AGC, reverse primer ATT ACC ATA GTC AGC AGC, probe FAM-CGA AAC CTA CTT TCC TGG GTG GTG TTTROX. The standard for quantification of RNA was enterovirus 71 (VIRCELL, Granada, Spain) with 5-point range: 1.26 × 104; 1.26 × 103; 126; 12.6 and 1.26 copies/mL. Statistical analysis: performed with GraphPad Prism® V6.0 version. Mann–Whitney U Test was used when appropriate. The pvalue < 0.05 was considered statistically significant.
Cells: Peripheral blood mononuclear cells (PBMC) were isolated from buffy coat cell pellet provided by The French Blood Establishment (EFS) Lille. Mononuclear cells have been separated on density gradients of Ficoll-Hypaque. Briefly the buffy coat was directly layered on top of Ficoll-Paque™ (GE Healthcare) (ratio 1:1), and centrifuged at 500 g for 30 min at 20 °C. PBMC layer was collected and washed twice with RPMI-1640 medium. The cells were then suspended in RPMI medium supplemented with 10% heat-inactivated fetal calf serum, 1% non-essential amino acid, 1% penicillin-streptomycin and 1% of L-glutamine. The cells were counted, and the cell number was adjusted to 5 × 106 cells/mL. Vero cells: (ATCC® CCL81™) were grown in Dulbecco's modified eagle's medium (DMEM; Gibco BRL, Invitrogen, Gaithersburg, MD, USA) supplemented with 10% fetal calf serum (FCS, Sigma St Louis, MO, USA), 2–6 mM L-Glutamine (Gibco BRL), 50 mg/ mL streptomycin, and 50 IU/mL penicillin (Bio Whittaker). Virus: CV-A6 isolated from a clinical sample was propagated in Vero cells (ATCC® CCL81™). The infectious titers of culture supernatants were assessed using the end-point dilution assay, and the ReedMuench statistical method was used to determine the tissue culture 50% infectious dose (TCID50). The virus isolate was aliquoted and stored at −80 °C. Serum samples: Obtained from children with CV-A6 infection (Hand Foot and mouth Disease/encephalitis/meningitis/myocarditis) confirmed by reverse transcriptase - polymerase chain reaction. Serum samples from age-matched control children with clinical symptoms of dengue-like illness or influenza-like illness received at Manipal Centre for Virus Research, Manipal, India were included. The consent of parents was obtained, and the study was approved by Institutional Ethical committee. Microneutralization Assay: Fifty microliters of sample dilutions (2 fold serial dilutions) and 50 μL of virus stock containing 200 TCID50 CVA6 were mixed and incubated in a microtiter plate with Vero cells at 37 °C for seven days. The cell control and virus control were run simultaneously. The neutralizing antibody titres were expressed as the reciprocal of highest dilution of serum that completely inhibited the cytopathic effect of the virus. Extraction of IgG from serum: Protein G HP Spintrap (GE healthcare UK limited) was used to isolate IgG from the serum samples. IgG-enriched fraction and IgG-depleted fractions were processed separately. Serum-dependent enhancement assay: Serum/eluted IgG was diluted with RPMI-1640 media in the ratio 1/10, 1/100 and 1/1000. Twenty-five microliters of medium or diluted serum/eluted IgG was incubated for 2 h at 37 °C with 25 μl of CV-A6 suspension (105 TCID50). Post-incubation, the mixtures were inoculated into PBMC cultures in microplates (5 × 105 cells/well). After 48 h of incubation, supernatants were collected for INFα detection by ELISA. After collection of the supernatant, the wells containing the cells were washed four times with cold 1× PBS (4 °C) by centrifuging at 1500 rpm for 10 min. The cells were removed easily by adding TRI Reagent® (Sigma- Aldrich), collected in a total volume of 900 μL tri-reagent (Sigma) in 1.5 ml tubes and subjected to RNA extraction. ELISA (sandwich ELISA): IFN-α was measured by using pan-specific INF-α ELISA kit (Mabtech® Human IFN-α ELISA PRO kit) according
3. Result 3.1. Children with CV-A6 infection Serum was collected from 5 children (less than ten year-old) with CV-A6 infection confirmed by detection of enteroviral RNA by reversetranscriptase real-time PCR (RT-PCR) in cerebrospinal fluid and/or throat swab and/or vesicle swab. Based on clinical symptoms associated with CV-A6 infection, these patients were further classified as acute encephalitis syndrome (n = 2), meningoencephalitis (n = 1), myocarditis (n = 1) and HFMD (n = 1) (Table 1). The titres of neutralizing anti-CV-A6 antibodies in serum were < 1:4 as displayed by micro neutralization assay. The serum samples were collected after the onset of the disease as soon as one day in the case of patients number 3, and 4–6 days in the case of other patients. Serum samples were obtained from age-matched controls without symptoms of infection (n = 1) or with symptoms of dengue-like illness (n = 3) or influenza-like illness (n = 3). The titres of neutralizing antiCVA6 antibodies in serum from 5 subjects were < 1:4 and from 2 others were 1:32 and 1:128 as displayed by micro neutralization assay. 3.2. Serum from children with CV-A6 infection can enhance the CVA6induced production of INFα by PBMC The levels of INF-α in supernatants of PBMC culture inoculated with CV-A6 mixed with medium was lower than 20 pg/ml. When CV-A6 was mixed with various dilutions of serum or serum-derived IgG from
Table 1 Demographic and clinical details of Coxsackie virus-A6 infected children (n = 5). No
Age(Y)
Sex
Clinical symptoms
Syndrome
Outcome
1 2 3 4 5
6.5 4 8 2 4
F M M M M
Fever, headache, vomiting, Seizure Fever,coryza,myalgia and altered sensorium Fever and Skin Lesions Hepatomegaly, Myocarditis, Vomiting Diarrhea, Mechanical Ventilation Fever,coryza,myalgia and altered sensorium
Acute Encephalitis Acute Encephalitis Hand Foot Mouth Disease Myocarditis Meningoencephalitis
Death Death Recovery Recovery Recovery
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Fig. 1. Serum and IgG from children with CV-A6 infection enhance the CV-A6 induced production of IFNα by PBMC. CV-A6 was incubated with serum or serum-derived IgG for 2 h then the mixtures were added to PBMC cultures. Two days later the supernatants were harvested to evaluate the level of IFNα by ELISA; the results were expressed as pg/ml. Individual representation of IFNα levels obtained when CVA6 was mixed with diluted serum samples (1/10; 1/100; 1/1000 dilution) from 7 controls (A) or IgG isolated from these samples (B). Individual representation of highest IFNα levels obtained when CV-A6 was mixed with serum samples from 5 children with CV-A6 infection or with serum-derived Ig G (IgG enriched serum) or IgG depleted serum (C). The values were obtained with dilution 1/1000 and 1/10 of serum in the case of patients 1 and 5 respectively; and dilution 1/100 in the case of patients 2-4.
controls the levels of IFNα in supernatants of PBMC cultures were lower than 26 pg/ml (Fig. 1 A and B). In contrast when CV-A6 was mixed with various dilutions of serum (final dilution 1/10 to 1/1000) or serumderived IgG from patients, the levels of IFNα in supernatants of PBMC cultures were up to 65 pg/ml (Fig. 1C). The highest level of IFNα was obtained with CV-A6 mixed with serum (dilution 1/1000) from the child with acute encephalitis (patient 1 in Fig. 1C). The mean level of IFNα obtained with serum samples from children with CV-A6 infection was significantly higher than the one obtained with serum samples from controls (37.5 ± 16 vs 15.5 ± 2.6 pg/ml, p-value < 0.002). 3.3. Serum-derived IgG from children with CV-A6 infection can increase the level of intracellular CV-A6 RNA in PBMC CV-A6 was incubated for 2 h at 37 °C in presence of medium or various dilutions (1/10; 1/100 and 1/1000) of serum-derived IgG from children with CV-A6 infection and controls. Then the mixtures were added to PBMC cultures, and the plates were incubated. After 24 h, the cells were recovered to determine the level of intracellular viral RNA. There was no enteroviral RNA, or the level was under the limit of detection of the assay when CV-A6 was incubated in presence of medium. The enteroviral RNA level was 3 × 102 copy/ng total RNA when CV-A6 was incubated with serum-derived IgG from one control. The neutralizing activity of serum from this subject was < 1:4. The enteroviral RNA level was under the limit of detection of the assay with serumderived IgG from the six other controls. The neutralizing activity of serum from 4 out of these subject was < 1:4 and from 2 others was 1:32 and 1:128 respectively (data not shown). In contrast, when PBMC was inoculated with CV-A6 mixed with serum-derived IgG from children with CV-A6 infection, the levels of intracellular viral RNA were much higher (Fig. 2). The enteroviral RNA levels were at least around 4 × 104 copy/ng of total RNA when CVA6 was mixed with serum derived IgG from patient 1 and patient 4
Fig. 2. Individual representation of levels of enteroviral RNA in PBMC inoculated with CV-A6 mixed with serum-derived IgG of children with CVA6 infection. CV-A6 was incubated with serum-derived IgG (1/10, 1/100 and 1/1000 dilution) of each patient (patient 1 to 5), then the mixture was added to PBMC cultures as described in the legend of Fig. 1. After two days of incubation, the cultures were washed four times with PBS and extraction of total RNA was carried out using tri-reagent technique. The level of viral RNA was evaluated by real-time RT-qPCR and the results expressed as copy/ng of total RNA.
(dilution 1/100). Meanwhile, enteroviral RNA levels were up to 105, 6 × 104, 5 × 104 copy/ng of total RNA when CVA6 was mixed with serum-derived IgG from patient 5 (dilution 1/10), patient 2 (dilution 1/ 100) and patient 3 (dilution 1/1000) respectively. There was no relationship between the outcome of the infection and the serum/IgG –dependent enhancement of CV-A6 infection in our system.
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4. Discussion
individuals as a result of previous infections. An anti-CV-A6 enhancing activity was observed in patients with HFMD whom disease outcome was favourable (patient number 3) and in patients with the more severe disease. There was no clear relationship between the pattern of the CV-A6 infection or its outcome and the anti-CV-A6 enhancing activity of serum. Nevertheless, the role of the anti-CV-A6 enhancing activity in the course of the infection is an open question. Whether this activity of serum observed in vitro can reflect a favourable condition for deleterious outcome in some individuals remains to be determined. Our data suggest that non-neutralizing antibodies appear early during the infection. The enhancement is due to non-neutralizing antibodies or antibodies at subneutralizing levels produced during the infection. There was no correlation between the viral copy number and the dilution of IgG in our experiments. Furthermore it cannot be excluded that antibodies directed against more broadly expressed epitopes on other enteroviruses bound CV-A6 particles in our system. Further studies are needed to understand this phenomenon, which will improve the knowledge of CV-A6-associated disease. In conclusion, there is an anti-CV-A6 enhancing activity in serum and serum-derived IgG of children with CV-A6 infection. The anti-CVA6 enhancing activity of serum should be evaluated in larger cohorts. Further investigation are needed to determine whether this activity has implications in the pathogenesis of CV-A6 induced diseases and in the design of vaccines aimed to fight HFMD that is associated with a high morbidity rate and can lead to severe complications.
The current study is different in many respects from those of other investigators; it is reported for the first time that the serum from individuals with CV-A6 infection can enhance the infection of PBMC with CV-A6 in vitro. Several considerations are noteworthy. The issue of the anti-CV-A6 enhancing activity of serum has been addressed in vitro in a system based on human PBMC. In this system, the levels of intracellular enteroviral RNA were measured by quantitative real-time RT-PCR, which allowed comparisons between the levels of enteroviral RNA in PBMC inoculated with CV-A6 or with the virus mixed with serum from individuals. Moreover, the anti-CV-A6 enhancing activity of serum has been addressed through the levels of IFNα in supernatants of PBMCs measured by a sensitive ELISA. The same batch of PBCM was used to test the enhancing activity of all serum samples from patients and controls which allowed inter-individual comparisons. The production of IFNα by PBMC associated with the presence of enteroviral RNA in the cells when CV-A6 was mixed with serum from patient supported the idea that cells were infected with CV-A6 in these experiments. The enhanced CV-A6-induced release of IFNα by PBMC and the increased level of enteroviral RNA in these cells obtained with diluted serum from patients were due to IgG as shown by the activity of serumderived IgG from CV-A6 infected individuals. IgG was obtained from serum by affinity chromatography using Protein G, an enhancing effect was observed with the eluted fraction but not with the filtrate fraction. In contrast serum and IgG from individuals without CV-A6 infection did not enhance the CV-A6-induced release of IFNα by PBMC nor the infection of these cells. The level of intracellular enteroviral RNA was under the limit of detection of the assay when CV-A6 was incubated with medium or serum-derived IgG of controls. In contrast the level of intracellular enteroviral RNA was high when CV-A6 was incubated with serum-derived IgG of individuals with CV-A6 infection. At optimal dilution of serum the amount of viral RNA was at least 40000 copy/ng of total RNA, which is more consistent with the hypothesis of CV-A6 replication than just internalization. This is in agreement with previous studies demonstrating that viral replication was not detected in PBMC inoculated with enteroviruses (CV-B3, Echovirus 1, poliovirus 1) unless viral RNA was transfected [9]. In the same vein high levels of CVB4 RNA were detected in PBMC when the virus was previously incubated with IgG containing anti-CV-B4 antibodies. The enhancement is associated with intracellular viral replication, as previously shown by Alidjinou et al. [10]. The strong increase in viral load before and after incubation with enhancing serum is clearly compatible with intracellular viral replication. It was reported that the infection of PBMCderived monocytes with CV-B4 was enhanced by serum/IgG, which resulted in the release of infectious particles by these cells [7]. Whether the scenario is similar for CV-A6 remains to be determined. This is reminiscent of previous studies describing that human serum harboured an enhancing anti-CV-B4 activity, due to IgG, in human PBMC or cell line cultures, provided the serum was diluted [7]. It was shown that immune serum-mediated enhancement of infection with CV-B4 in vitro in human PBMCs was due to anti-CV-B4 antibodies [11,12]. We assume that anti-CV-A6 antibodies, contained in immune serum and IgG obtained from CV-A6 infected individuals, enhanced the infection of PBMC with CV-A6 in vitro in the present study. In serum from patients there was an anti-CV-A6 enhancing activity, due to serum-derived IgG as shown in this study, although the anti-CVA6 neutralizing activity was under the limit of detection of the assay (< 1/4). The anti-CV-A6 enhancing activity was observed as soon as day one after the onset of the disease in patients. Furthermore, there was no enhancing activity in serum from controls although a neutralizing activity was displayed (titre values up to 1/32 and 1/128). Longitudinal studies are needed to determine whether the enhancing activity appears early in the course of the infection before the neutralizing activity or whether this activity is preexisting in some
Conflicts of interest None. Acknowledgement This work has been supported by the Ministère de l’Education Nationale de la Recherche et de la Technologie, Université de Lille (EA 3610) and Centre Hospitalier Régional et Universitaire de Lille France. The authors thank referring clinicians and Indian Council of Medical Research Government of India, New Delhi for the funding of the project: “Distribution of non-polio enterovirus in children with HFMD in Kerala, Karnataka and Goa” file No.VIR/NP/66/2013-ECD-1. References [1] S. Aswathyraj, G. Arunkumar, E.K. Alidjinou, D. Hober, Hand, foot and mouth disease (HFMD): emerging epidemiology and the need for a vaccine strategy, Med. Microbiol. Immunol. 205 (2016) 397–407. [2] R. Osterback, T. Vuorinen, M. Linna, P. Susi, T. Hyypia, M. Waris, Coxsackievirus A6 and hand, foot, and mouth disease, Finland. Emerg Infect Dis. 15 (2009) 1485–1488. [3] W. Xing, Q. Liao, C. Viboud, J. Zhang, J. Sun, J.T. Wu, et al., Hand, foot, and mouth disease in China, 2008-12: an epidemiological study, Lancet Infect. Dis. 14 (2014) 308–318. [4] N. Sarma, A. Sarkar, A. Mukherjee, A. Ghosh, S. Dhar, R. Malakar, Epidemic of hand, foot and mouth disease in West Bengal, India in August, 2007: a multicentric study, Indian J. Dermatol. 54 (2009) 26. [5] V. Gopalkrishna, P.R. Patil, G.P. Patil, S.D. Chitambar, Circulation of multiple enterovirus serotypes causing hand, foot and mouth disease in India, J. Med. Microbiol. 61 (2012) 420–425. [6] J.-F. Han, R.-Y. Cao, Y.-Q. Deng, X. Tian, T. Jiang, E.-D. Qin, et al., Antibody dependent enhancement infection of Enterovirus 71 in vitro and in vivo, Virol. J. 8 (2011) 106. [7] D. Hober, W. Chehadeh, A. Bouzidi, P. Wattré, Antibody-dependent enhancement of coxsackievirus B4 infectivity of human peripheral blood mononuclear cells results in increased interferon-alpha synthesis, J. Infect. Dis. 184 (2001) 1098–1108. [8] P. Sauter, D. Hober, Mechanisms and results of the antibody-dependent enhancement of viral infections and role in the pathogenesis of coxsackievirus B-induced diseases, Microb. Infect. 11 (2009) 443–451. [9] T. Vuorinen, R. Vainionp, J. Heino, T. Hyypi, Enterovirus receptors and virus replication in human leukocytes, J. Gen. Virol. 80 (1999) 921–927. [10] E.K. Alidjinou, F. Sané, I. Engelmann, D. Hober, Serum-dependent enhancement of coxsackievirus B4-induced production of IFNα, IL-6 and TNFα by peripheral blood mononuclear cells, J. Mol. Biol. 425 (2013) 5020–5031.
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[11] W. Chehadeh, P.-E. Lobert, P. Sauter, A. Goffard, B. Lucas, J. Weill, et al., Viral protein VP4 is a target of human antibodies enhancing coxsackievirus B4-and B3induced synthesis of alpha interferon, J. Virol. 79 (2005) 13882–13891.
[12] P. Sauter, P.-E. Lobert, B. Lucas, R. Varela-Calvino, G. Alm, P. Wattre, et al., Role of the capsid protein VP4 in the plasma-dependent enhancement of the Coxsackievirus B4E2-infection of human peripheral blood cells, Virus Res. 125 (2007) 183–190.
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