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Summer Research Fellowship Programme of India's Science Academies

The interleukin-6 expression in Japanese encephalitis virus-infected human microglial cells

Rinkal Sundriyal

BVSc. & A.H., G.B. Pant University of Agriculture and Technology, Pantnagar Uttarakhand 263145

Professor Sunit K. Singh

Professor of Molecular Immunology, Molecular Biology Unit, Institute of Medical Sciences, Banaras Hindu University, Varanasi 221005

Abstract

Japanese Encephalitis (JE) is caused by Japanese Encephalitis virus (JEV), which is transmitted through mosquitoes (Culex sp.). The cases of the JE have been frequently reported in Southeast Asian Countries. The JEV infection in brain cells leads to the release of several cytokines and chemokines; which activate the microglial cells. Microglial cells are the resident macrophages of CNS. The production of Interleukin-6 by microglial cells play an important role in the host defence mechanism as it can act as both pro-inflammatory and anti-inflammatory cytokine depending upon the conditions. IL-6 can also induce the acute phase response during chronic inflammation. The microglial cells were infected by JEV and confirmed by qPCR. The JEV infection to microglial cells suppresses the IL-6 expression, which was confirmed by qPCR. The suppression of IL-6 may be the strategy adopted by JEV to evade from host’s innate immune response.

Keywords: RNA isolation, real-time PCR, inflammation, cDNA synthesis, IL-6

Abbreviations

List of Abbrevations
JEVJapanese Encephalitis Virus
DENVDengue Virus 
ZIKVZika Virus
YFVYellow Fever Virus
WNVWest Nile Fever Virus
BBBBlood-Brain Barrier
IL-6 Interleukin-6  
NS3 geneNon-Structural 3 gene 
GAPDHGlyceraldehyde-3-Phosphate Dehydrogenase 
NIVNational Institute of Virology
SVZSub-Ventricular Zone
NPCsNeural Progenitor Cells
qPCRReal-time Polymerase Chain Reaction 
NVBDCPNational Vector Borne Disease Control Programme
WHOWorld Health Organization
CNSCentral Nervous System 

INTRODUCTION

Japanese Encephalitis (JE) is a mosquito-borne arboviral disease and its etiological agent is Japanese Encephalitis Virus (JEV) which belongs to Flaviviridae family. Other members of this family are Dengue virus (DENV), Zika Virus (ZIKV), Yellow Fever virus (YFV) and West Nile virus (WNV). The JEV circulates in enzootic cycle where the pigs are the reservoir for infection, water birds and mosquitoes are the vectors and humans are accidental dead-end hosts ​​​1​​ . JEV is endemic in Southeast Asia and around 68,000 cases are reported annually. The new-borns and the old-age people are at higher risk for JEV infection. The incubation period of JEV usually lasts for 4 -14 days with mild fevers and headache which progresses to vomiting and gastrointestinal pain. The severity of disease is characterized by the rapid onset of high-grade fever, headache, seizures, spastic paralysis and neck stiffness, which ultimately lead to death. The symptomatic treatment regime is followed during JE. Moreover, there is no therapeutics to prevent the encephalitis during JEV infection due to which, 30%-50% case fatality rate has been reported and the survivors suffer from permanent neurological and psychiatric sequelae ​2.

JEV is a positive ssRNA virus with the genome size of ~11kb. The positive-strand directly translates into a polyprotein, which encodes three structural (Capsid, Pre-Membrane and Envelope proteins) and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5) 3.

JEV primarily infects and kills neurons which release the pro-inflammatory cytokines and activate the microglial cells. The activated microglial cells further kill the neurons in bystander fashion 4. Microglial cells are resident macrophages of CNS, which play a very important role during infection. The JEV infection to microglial cells results in the production of pro-inflammatory cytokines (TNF-α, IL-1β and IL-6) 5. IL-6 is a soluble pleiotropic cytokine which is produced during the acute phase of infection, where it can act as an anti-inflammatory and pro-inflammatory cytokine 6. Therefore, in the present study, we have shown JEV infects microglial cells and suppressed the pro-inflammatory cytokine IL-6 during JEV infection.

LITERATURE REVIEW

Japanese Encephalitis

Japanese Encephalitis (JE) is caused by Japanese Encephalitis Virus (JEV), an arbovirus which belongs to the Flaviviridae family. It is a vector-borne disease spread by mosquitoes of Culex sp. (mostly by Culex tritaeniorhynchus). JEV was first reported in 1871 in Japan and today JE is frequently reported from Southeast Asian countries. Humans are considered as ‘dead-end host’ and thus disease does not transmit through physical contact. The JEV undergoes enzootic cycle among mosquitoes, pigs and/or water birds, where the pigs are the amplifying hosts, water birds and mosquitoes are the vectors and humans are accidental-dead end hosts 7. The JEV transmission enhances during the rainy season when mosquito’s population increases.

JE involves neurological signs and symptoms which primarily affects children between the age of 0-15 years and old-age people 8. The symptoms include headache, fever, convulsions along with gastrointestinal pain and vomiting. There are no prescribed anti-viral drugs against JE and vaccination is the only option. As a result, about 30%-50% case fatality rate has been reported in JE patients and the survivors suffer from permanent nervous defects and psychological problems.

Japanese Encephalitis in India

In India, JE was first reported in 1955 from Southern states of Madras, now Tamil Nadu. At present, it is endemic in 171 districts of 19 states mainly in Uttar Pradesh. The vectors for JEV transmission in India are Culex tritaeniorhynchus, C. vishnui and C. pseudovishnui. These mosquitoes breed in the water with luxuriant vegetation mainly in paddy fields. According to the census of NVBDCP (National Vector Borne Disease Control Programme), 1678 JE cases with 182 deaths were recorded in 2018, while 321 cases and 30 deaths have been reported till June 2019. The Symptomatic treatment regime is followed for JE patients, along with supportive clinical management. The fluid and electrolytes are administered to manage the initial acute phase of infection. Steps taken by the government include the establishment of physical, medical and rehabilitation (PMR) department and a Vector-Borne Disease Surveillance Unit (VBDSU) at BRD Medical College, UP; supplying free of cost diagnostic kits from NIV, Pune, etc 9.

Live attenuated vaccine of strain SA 14-14-2 was developed by China against JEV which was approved by WHO in 2013 10. In India, JENVAC, prepared by Bharat Biotech (Indian strain Kolar-821564XY), is an inactivated JEV vaccine propagated in Vero cells 11. In addition, inactivated mouse brain-derived JE vaccine is used which was developed by using Nakayama strain of JEV in Central Research Institute Kasauli, Himanchal Pradesh 9.

Role of Human Microglial Cells in JEV infection

Microglial cells are the resident macrophages of the nervous system, involved in immune surveillance and phagocytosis. Microglial cells play an important role in both innate and adaptive immune response in CNS, by migrating to the site of infection and injury 5. The sub-ventricular zone (SVZ) from where neural progenitors cells (NPCs) originate are infected by JEV. The production of pro-inflammatory cytokine (IL-6) activates the microglial cells and promotes neuronal killing in bystander fashion. Further, it was reported microglial cells act as a reservoir for JEV infection 12.

Role of IL-6 in Inflammation

The expression of pro-inflammatory cytokines (IL-1β, IL-6, TNF-α) increases during inflammation. Interleukin-6 (IL-6) is a glycosylated soluble pleiotropic cytokine (molecular weight- 26KDa) involved in immune response, inflammation, acute phase response and haematopoiesis 13. It is also involved in B and T cell differentiation 14. At the initial phase, IL-6 along with chemokines help in chemotaxis 15. IL-6 increases lymphocytic response during viral infection. It can act as both pro-inflammatory and anti-inflammatory cytokine. The IL-6 level helps in maintain the immunological haemostasis through classical-signalling pathway whereas the excessive production triggers a series of inflammatory response through trans-signalling pathway 6. Therefore, IL-6 activates the immune response against the etiological agent.

METHODOLOGY

RNA Isolation using TRIzol Reagent

The human microglial cells (CHME3) infected with JEV for 12hrs, 24hrs and 48hrs were used. The total RNA was extracted from JEV mock-infected and infected human microglial cells by using manual method. TRIzol reagent (a mixture of phenol and guanidinium iso-thiocyanate) has been used to homogenize the cells. The chloroform was added and centrifuged at 14,000 x g for 15 min at 4°C. The three phases were separated, the upper phase contains RNA, the intermediate phase contains DNA and the lower phase contains organic solvents. The upper aqueous phases were collected in a fresh tube and an equal volume of isopropanol were added and kept at 4°C for 20 min. The precipitated RNA was centrifuged at 14,000 x g for 15 min at 4°C. The pellets were washed twice with 75% ethanol and centrifuged at 7500 x g for 5 min at 4°C. The pellets were air-dried and the RNA was resuspended in 30µl nuclease-free water. RNA quantification was done by using Nanodrop Spectrophotometer.

cDNA Synthesis

The total RNA (500ng) for each sample was converted into cDNA by using SuperScript First-strand synthesis kit (Invitrogen).

First step was the addition of following reagents (Table-1):

Table-1: Components for the cDNA synthesis
Sample Template RNA + Nuclease-free water (µl) dNTPs(µl) Random Hexamers (µl) Total volume (µl)
12C 8 1 1 10
12I 8 1 1 10
24I 8 1 1 10
48I 8 1 1 10

Tubes were short spinned and incubated at 65°C for 5 min in the thermocycler.

The master mix was prepared by adding following reagents (Table-2):

Table-2: Master mix for cDNA synthesis
Reagents Volume (µl/tube)
10X RT Buffer2
MgCl24
Dithioreitol (DTT)2
RNase out1
Total volume9

The 9µl master mix (10X RT Buffer, MgCl2, DTT, RNase out) and 1µl Reverse Transcriptase enzyme were added to each tube and were given a quick spin.

The tubes were kept in the thermocycler for cDNA synthesis with the following thermal profile (Table-3).

Table-3: Thermal profile for cDNA synthesis
Time (minutes)Temperature (°C)
1025
5042
1570
4

At last, 1µl of RNase H were added to each tube and kept for 20 min at 37°C in thermocycler. After completion, the cDNA were stored at -20°C.

Real-time PCR

Real-time PCR was performed to check the infection of JEV in microglial cells at different time points (12hrs, 24hrs and 48hrs). The specific primers against JEV-NS3 (Non-structural 3) gene were used and the expression was normalized by using specific primers against Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene. The expression of IL-6 gene at 12hrs, 24hrs and 48hrs were checked by using IL-6 specific primers and SYBR green master mix.

The reverse and forward primers were diluted and a reaction mixture was prepared by adding the following reagents (Table-4).

Table-4: Reaction mixture for qPCR
ReagentsJEV NS3 gene (µl/tube)GAPDH gene (µl/tube)IL-6 gene (µl/tube)
2X Master mix101010
Forward primer332
Reverse primer332
Nuclease-free water779
Total Volume232323

The micro-titer plate was labelled and 23µl of the reaction mixture (prepared already) was added to well. The 2µl of cDNA was added to the well and gently mixed. The plate was sealed and wrapped with aluminium foil and centrifuged at 2500 x g for 5 min at 4°C. The plate set-up was done in the Agilent software and the thermocycles were set up as follow: hot start (3 min at 95°C), 3-step-Amplification (30 sec at 95°C, 1 min at 55°C, 40 sec at 72°C) and Melt curve (1 min at 95°C, 30 sec at 55°C, 30 sec at 95°C). The data were analysed by using Livak method.

RESULT AND DISCUSSION

RNA Isolation from JEV Infected Microglial Cells

The RNA were isolated from the mock-infected (control) sample and JEV infected samples at different time points (12hrs, 24hrs and 48hrs) and quantified by using Nanodrop Spectrophotometer (Table-5).

Table-5: RNA Concentration
SampleTotal RNA Concentration (ng/µl)Quality (A260/280)Purity (A260/230)
Control525.652.011.86
12I709.552.051.97
24I917.652.072.01
48I729.652.062.03

The total RNA (500ng) from all the samples was converted into total cDNA by using random hexamers and SuperScript first strand synthesis kit. The cDNA was used to perform qPCR and the expression of JEV NS3 and IL-6 gene were quantified in JEV infected microglial cells at different time points.

Real-Time PCR for JEV NS3 and IL-6 Expression during JEV Infection

The qPCR of control and JEV infected samples were performed and the expression of JEV NS3 gene has been observed maximum at 24hrs (Figure-1). The expression of IL-6 gene was found to decrease during JEV infection in microglial cells (Figure-2). Although JEV infection activates the immune response, in the present study the JEV infection suppressed the release of pro-inflammatory cytokine, IL-6. This may be the strategy adopted by JEV to actively replicate inside the host and evade from host immune response. The suppression of pro-inflammatory response has been reported to facilitate virus persistence in cells 16 .

Figure1.jpg
    Figure 2.jpg

      CONCLUSION

      The microglial cells were infected by JEV and the maximum replication has been observed at 24hrs post-infection. The suppression of pro-inflammatory response by JEV during infection may be the strategy adopted by JEV to evade the innate immune response. The evasion of host innate response and productive replication inside the host further prove the notion that microglial cells may be used as a reservoir for JEV infection. The molecular mechanism underlying the phenomenon of immune evasion by JEV has to be explored in order to develop drugs and therapeutics against JEV.

      ACKNOWLEDGEMENT

      First and foremost, I heartily thank Indian Academy of Sciences (IASc-INSA), Bangalore, for providing me a golden opportunity to carry out this project.

      It is my radiant sentiment to place on record my best regards, deepest sense of gratitude to Prof. Sunit K. Singh, Head-Molecular Biology Unit (MBU), Institute of Medical Sciences (IMS), Banaras Hindu University (BHU), for giving me constant support, advise and precious guidance which made me complete the project duly.

      I sincerely record my thanks to Ms. Meghana Rastogi for her kind advice that helped me to execute my work along with her expert assistance.

      I am equally thankful to Ms. Astha Shukla, Ms. Neha Pandey and Mr. Utkarsh Bhardwaj for their encouragement and moreover for their timely support and guidance until the completion of my project work.

      I owe my special thanks to the technical staff of Molecular Biology Unit, Institute of Medical Sciences (IMS), Banaras Hindu University (BHU) for their corporation and help.

      It is my privilege to express profound regards and a deep sense of gratitude to my teacher Dr. Anuj Tewari, Assistant Professor, Department of Veterinary Microbiology, College of Veterinary and Animal Sciences, Govind Ballabh Pant University of Agriculture and Technology, Pantnagar, for his guidance, valuable suggestions and encouragement. I am extremely thankful to him for forwarding my recommendation letter to IASc, Bangalore.

      Last but not least, I am so grateful to my beloved parents, colleagues and friends who have supported me with their valuable suggestions and guidance.

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