1           Manipal Academy of Higher Education (MAHE), Manipal

1.1          Objectives and Methodology:

 

 

OBJECTIVE 1: Establishment of Chikungunya clinical cohorts and a CHIKV clinical sample biobank OBJECTIVE 2: Characterization of immune response during acute CHIKV infection

1.2          OBJECTIVE 1: Establishment of Chikungunya clinical cohorts and a CHIKV clinical sample biobank

Sample collection:

Samples will be collected from the following sites;

 

2.2.1.1        Acute febrile illness sites clinical sites

1.       Jayachamarajendra Hospital, Thirthahalli, Shimoga, Karnataka

2.       District Hospital, Mananthwady, Wayanad, Kerala,

3.       District Hospital, Sonapur, Kamrup Metropolitan, Assam

4.       Community Health Centre, Idar, Sabarkantha, Gujarat

5.       Government Hospital, Denkanikottai, Krishnagiri, Tamil Nadu

6.       Indira Gandhi Memorial Hospital (IGMH), Agartala, Tripura

7.       Kasturba Medical College Hospital, Manipal

 

2.2.1.2        Chikungunya outbreaks reported from anywhere in India during the study period

Chikungunya often appear in a geographical area as outbreak/ epidemic. Hence, if necessary, to achieve the required sample size MIV, MAHE will also make efforts to recruit cases from these outbreaks whenever it is feasible.

Case definition:

Suspected Chikungunya case: An acute febrile illness case with arthralgia and or arthritis within 14 days of onset of illness and belonging to 12 to 75 years of age.

Confirmed Chikungunya case: A case which is laboratory confirmed positive for Chikungunya Realtime PCR or Chikungunya IgM ELISA and belonging to 12 to 75 years of age.

Acute Chikungunya: A acute febrile case with arthralgia and or arthritis within 14 days of onset of illness which is laboratory confirmed positive for Chikungunya Realtime PCR or Chikungunya IgM ELISA and belonging to 12 to 75 years of age.

Recovered chikungunya case: A laboratory confirmed case of Chikungunya case without any residual clinical signs or symptoms as on 60th day of post onset of illness.

Chronic Chikungunya case: A case of laboratory confirmed Chikungunya with persistent arthralgia even after 2 months of the onset of illness.

Enrolment to the study: Retrospective:

Serum samples of laboratory confirmed Chikungunya detected and identified through acute febrile illness surveillance will be used in this study to isolate Chikungunya virus and create Chikungunya serum bank.

Prospective:

 

Once the suspected case is identified, the study is briefly explained to the patient and the bystander. All questions and doubts regarding the study from the patient should be clearly answered. Following this, if the patient is willing to participate, the informed consent is obtained.

Data collection (Demographic, Clinical and epidemiological)

Demographic, clinical and epidemiological data will be collected from all enrolled cases at the time of enrollment using a standard data collection tool. Further, clinical data is collected during convalescent / follow up period at each visit 1st, 3rd, 6th, 12th, 18th, 24th, 30th and 36th month post of illness from all laboratory confirmed CHIKV cases.

Clinical specimen collection:

Acute sample collection: 8 ml blood in plain vacutainer will be collected at the time of enrollment to the study.

Convalescent / follow-up sample collection: 5-8ml blood in plain vacutainer will be collected at 1st, 3rd, 6th, 12th, 18th, 24th, 30th and 36th month post of illness from all laboratory confirmed (PCR positive or anti-CHIKV IgM antibody positive) CHIKV cases. On every follow up visit for providing clinical data and sample Rs. 500/- will be paid to compensate the persons travel and time.

Sample transportation:

All the clinical samples collected will be transported in cold chain to MAHE on the same day using courier services.

Sample processing and testing at Clinical sites (PGIMER, Chandigarh, AIIMS, Bhubaneswar and TNMC, Mumbai):

Serum is separated and aliquoted under aseptic conditions. All samples will be tested for CHIKV - Realtime PCR and anti-CHIKV IgM antibodies in acute blood samples using a CHIKV TRC standard protocol. All positive and negative sera will be transported to MAHE, Manipal on a daily basis.

2.2.8    Sample processing and testing at clinical (surveillance) sites managed by MAHE:

Plain blood samples collected are transported daily under cold chain to MAHE, Manipal for further processing. No testing is done at these sites. All samples will be tested for CHIKV-real time PCR and anti-CHIKV IgM antibodies in acute blood samples using a CHIKV TRC standard protocol.

Sample processing and testing at MAHE, Manipal: Acute samples

All samples (both samples from MAHE surveillance sites and the clinical sites at PGIMER, Chandigarh, AIIMS, Bhubaneswar and TNMC, Mumbai will be tested tested for CHIKV-real time PCR and anti- CHIKV IgM antibodies in acute blood samples using a CHIKV TRC standard protocol.

Convalescent /Follow-up samples:

 

All the convalescent / follow-up samples will be tested for anti-CHIKV IgM ELISA.

 

Sample repository (Biobank):

MAHE will develop Chikungunya clinical sample bank / repository. Serum samples, plasma and PBMCs from laboratory confirmed cases of Chikungunya collected at acute and convalescent phases will be de- identified and will be banked at MIV as a national resource.

The biobank at MIV, MAHE aims to be a national facility. We propose to be a serum sample repository which would cater the academia and research industries.

Sample transportation to ICGEB, Delhi (For Virological characterization)

A portion of the acute, convalescent/follow up serum samples of laboratory confirmed CHIKV cases will be sent to the virology platform lead at ICGEB for virus isolation and further characterization and PRNT.

Revised status: We propose additional data and sample collection from laboratory confirmed CHIKV cases at regular intervals during the convalescent phase (1, 3, 6, 12, 18, 24, 30, 36 moths post onset of illness).

 

 

1.3          Objective 2: Characterization of adaptive human immune response during CHIKV infection and to identify biomarkers of CHIKV recovery / chronicity.

The current knowledge on the human adaptive immune responses to CHIKV and disease kinetics is very limited. Anti- CHIKV antibodies (both IgM and IgG type) are reported to persist for many weeks. Further it is reported that cytotoxic CD8+ T cells remains activated in the blood for several weeks post infection. However, mostly it is derived from limited sample size and is variable among studies.

Cytokines and chemokine are thought to play an important role in viral immunopathology and their response during Chikungunya infection and association with severity/ chronicity varies widely among studies. A study by Ng. et al., 2009 shows increase in IL-1β, IL-6 and a decrease in RANTES were associated with disease severity. A study by Wauquier et al. IL-6, IL-16, IL-17, IP-10, MCP-1, MIF, SDF-1a, IL-1ra, IL-2ra, G-CSF, GM-CSF, VEGF, IL-7, IL-12p40, and IFN-a2, ICAM1, VCAM1, and RANTES upregulated. And levels of IL-1a, MCP-3, HGF, M-CSF, b-NGF, SCF, CTACK, Eotaxin, IL-3, and TNF-b were significantly lower in patients than in controls. A study by the Reddy et al. IL-6, IL-8, IP-10, MCP1, RANTES and MIG were upregulated.

Little is known about the long-term sequelae or the pathogenesis of arthropathy, and the acquisition of protective immunity of CHIKV. Understanding the human immune responses against CHIKV and disease kinetics will give insight into determinants of disease.

Hence, we propose to characterise adaptive immune response during CHIKV infection by quantitating and immunophenotyping the lymphocytic response and exploring the possibility of identifying biomarkers of recovery / chronicity in CHIKV cases by analysing a battery of cytokines, chemokine and growth factors.

Activity 1: Characterization of adaptive immune response in acute and convalescent / follow up samples during CHIKV infection

2.3.1.1        Study subjects and recruitment:

We intend to recruit 50 Chikungunya real time PCR positive cases and 20 normal healthy individuals. For this activity we will be recruiting cases only from the clinical (surveillance site) directly managed by MAHE in the states of Tamil Nadu, Kerala and Karnataka for the logistic and case availability reasons.

2.3.1.2        Clinical specimen:

2ml EDTA blood samples will be obtained from all the participants on the day of recruitment and a convalescent /follow up sample will be collected at the end of the 4th week. Additionally, 2ml blood samples will be collected from chronic CHIKV cases at the end of 3, 6, 12, 18, 24, 30 and 36 months post onset of illness.

2.3.1.3        Detection and quantitation of CHIKV RNA

CHIKV RNA will be detected and quantitated in acute serum samples by a quantitative real time PCR using established primer/probe set that targets the CHIKV E1 gene.

2.3.1.4        Characterization of immune responses to acute chikungunya infection in human

A series of established flow cytometry panels (table 1 and table 2) will be run in order to characterize the population of B and T cell in human during infection, each panel is described in detail below. For all panels, whole blood (50-300μl) will be subjected against the indicated monoclonal antibody cocktail, followed by treatment with FACS Lyse and washed prior to acquiring the data on a BD Accuri™ C6 Cytometer. Data will be analysis by done using flowjo (Treestar, Ashland) software. A data summary will be recorded in Microsoft Excel and graphs will be made using Graphpad Prism. Approximately, ten healthy donor blood will be collected to derive a population based normal range1.

2.3.1.4.1                   Absolute quantification of lymphocytes

Peripheral blood absolute lymphocyte cell populations will be assessed by using a CD19/CD45/CD3/CD8 monoclonal antibody (mAb) cocktail in trucount tube (BD) following manufacturer’s instructions.

2.3.1.4.2                   Phenotyping of T and B cells

The panels of antibodies that will be used to define the naïve and memory populations of CD4 and CD8 T cells, as well as the activated populations of both B and T cells are given in Table 1. CD38 and HLADR are well established markers of T cells activation and are indicated of antigen stimulation. Frequencies of naïve and memory population are expected to change during acute illness, and this panel of selected antibodies will allow for quantification of these changes. Two distinct B cell populations, the plasmablasts, which are the antibody secreting cells, and the activated B cells, which are the cells destined to become long lived memory B cells, exist during acute B cell responses.

Table 1: Panel of antibodies targeting specific immune cell populations

 

Cell population	mAb cocktail	Fluorophore
Activated CD4 T cell	CD38	FITC
	HLADR	PE
	CD3	PE Cy7
	CD4	APC
Activated CD8 T cell	CD38	FITC
	HLADR	PE
	CD3	PE Cy7
	CD8	APC

Naïve vs Memory T cell	CCR7	FITC
	CD4	PE
	CD45RA	PE Cy7
	CD8	APC
Plasmablasts	CD19	FITC
	CD38	PE
	CD3 and CD20	PE Cy7
	CD27	FITC
Activated B	CD19	PE
	CD20	PE Cy7
	CD71	APC

 

2.3.1.4.3                   Characterizing effector CD8 T cells

CD8 T cells play a major role during any viral infection, so these cells will be examined in further detail. The three panels given in Table 2 are selected based on their specificity and application in defining the proliferating cells (by Ki-67 staining) and in evaluating certain important effector characteristics of these proliferating cells such as markers of cytotoxicity (Granzyme B and Perforin), inhibitory markers (PD-1 and CTLA-4), and memory markers (Bcl-2 and CD45RA).

Table 2: Panel of antibodies specific for CD8 cell population

 

Cell population	mAb cocktail	Fluorophore
Effector CD8 cells- I	Ki67	FITC
	Granzyme B	PE
	Perforin	PE Cy7
	CD8	APC
Effector CD8 cells –II	Ki 67	FITC
	PD-1	PE
	CD152 (CTLA-4)	PE Cy7
	CD8	APC
Effector CD8 cells- III	Ki-67	FITC
	Bcl-2	PE
	CD45RA	PE Cy7
	CD8	APC

2.3.1.4.4                   Humoral immune response to CHIKV

IgM and IgG levels against CHIKV infection will be measured in all acute and convalescent/follow up blood samples. Assay will be performed using Inbios - CHIKjj Detectâ„¢ IgM ELISA Kit and CHIKjj Detectâ„¢ IgG ELISA Kit and as per manufactures protocol.

 

 

 

Activity 2: To Study the biomarkers associated with recovery / chronicity in CHIKV infection

2.3.2.1        Clinical specimens: Stored plasma samples from acute and convalescent phases obtained from the activity 1.

2.3.2.2        Classification of cases:

2.3.2.2.1                   Fully recovered chikungunya case: Person who was Chikungunya PCR positive on enrolment and has no residual chikungunya symptoms at 60 days from the onset of illness.

2.3.2.2.2                   Chronic chikungunya case: Person who was Chikungunya PCR positive on enrolment and has arthralgia even after 2-3 months from the onset of illness.

2.3.2.3        Cytokines, chemokines and growth factors analysis

On a chosen set of age matched selected 15 recovered cases and 15 chronic cases, plasma levels of a series of biomarkers in chikungunya infected individuals will be quantitated using bead-based immuno- assays following the manufacturers instruction. A multiplex panel is chosen based on the physiologic role of each biomarker in the human antiviral response and given in the table 6. The assay will be performed on a MAGPIX® system. Serum from ten healthy donors will be analyzed in each assay to derive a population based normal range2.

 

Cytokines

G-CSF, GM-CSF, IFN-∝, IFN-γ, IL-1β, IL-1RA, IL-2, IL-2R, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL- 12 (p40/p70), IL-13, IL-15, IL-17, TNF-∝, CCL2

Chemokines

Eotaxin, IP-10, MCP-1, MIG, MIP-1∝, MIP-1β, CXCL9, CXCL10, RANTES

Growth Factors

EGF, FGF-basic, HGF, VEGF

 

Table 6: Panel of cytokines, chemokines and growth factors

Revised status: In addition to acute and convalescent (4th week) samples, we propose to immunophenotype lymphocytic response during various time points in chronic CHIKV cases. Further, we propose to study the variations in cytokines, chemokines and growth factors during acute and chronic Chikungunya cases to identify possible biomarkers that is associated with recovery or chronicity in CHIKV cases.

2           AIIMS, Bhubaneswar

2.1          OBJECTIVE1: ENROLLING OF SUSPECTED CHIKUNGUNYA CASES, DATA COLLECTION AND COLLECTION AND TRANSPORT OF SAMPLES TO MAHE

2.2          Methodology:

Study site: All India Institute of Medical Sciences (AIIMS), Bhubaneswar Case definition:

Acute suspected Chikungunya: An acute febrile illness case with arthralgia and or arthritis within 14

days of onset of illness and belonging to 12 to 75 years of age.

 

Acute confirmed Chikungunya: A case which is laboratory confirmed positive for Chikungunya Realtime PCR or Chikungunya IgM ELISA.

Chronic Chikungunya: A case of laboratory confirmed Chikungunya with persistent arthralgia even after 3 months of the onset of illness.

Enrolment to the study

Once the suspected case is identified, the study is briefly explained to the patient and the bystander. All questions and doubts regarding the study from the patient should be clearly answered. Following this, if the patient is willing to participate, the informed consent is obtained.

Data collection

 

The questionnaire in the case report form will be administered. The demographical, clinical history and epidemiological details of the patient will be collected using a tablet PC.

Clinical sample collection

Acute sample collection: 8 ml of blood in plain vacutainer will be collected at the time of enrollment to the study.

Convalescent/ follow-up sample collection: 5-8ml blood in plain vacutainer will be collected at 1st, 3rd, 6th, 12th, 18th, 24th, 30th and 36th month post of illness from all laboratory confirmed (PCR positive or anti-CHIKV IgM antibody positive) CHIKV cases. On every follow up visit for providing clinical data and sample Rs. 500/- will be paid to compensate the persons travel and time.

Sample processing and testing:

Serum is separated and aliquoted under aseptic conditions. All samples will be tested for CHIKV - Realtime PCR and anti-CHIKV IgM antibodies in acute blood samples using a CHIKV TRC standard protocol. All positive and negative sera will be transported to MAHE, Manipal on a daily basis.

Sample shipment:

All samples collected (acute sample from suspected CHIKV cases and, convalescent and follow up samples from laboratory confirmed CHIKV cases) will be transported to MAHE, Manipal on a daily basis.

3           PGIMER, Chandigarh

3.1          OBJECTIVE1: ENROLLING OF SUSPECTED CHIKUNGUNYA CASES, DATA COLLECTION AND COLLECTION AND TRANSPORT OF SAMPLES TO MAHE

3.2          Methodology:

Study site: Post Graduate Institute of Medical Education & Research, Chandigarh Case definition:

Acute suspected Chikungunya: An acute febrile illness case with arthralgia and or arthritis within 14

days of onset of illness and belonging to 12 to 75 years of age.

 

Acute confirmed Chikungunya: A case which is laboratory confirmed positive for Chikungunya Realtime PCR or Chikungunya IgM ELISA.

Chronic Chikungunya: A case of laboratory confirmed Chikungunya with persistent arthralgia even after 3 months of the onset of illness.

Enrolment to the study

Once the suspected case is identified, the study is briefly explained to the patient and the bystander. All questions and doubts regarding the study from the patient should be clearly answered. Following this, if the patient is willing to participate, the informed consent is obtained.

Data collection

 

The questionnaire in the case report form will be administered. The demographical, clinical history and epidemiological details of the patient will be collected using a tablet PC.

Clinical sample collection

 

Acute sample collection: 8 ml of blood in plain vacutainer will be collected at the time of enrollment to the study.

Convalescent/ follow-up sample collection: 5-8ml blood in plain vacutainer will be collected at 1st, 3rd, 6th, 12th, 18th, 24th, 30th and 36th month post of illness from all laboratory confirmed (PCR positive or anti-CHIKV IgM antibody positive) CHIKV cases. On every follow up visit for providing clinical data and sample Rs. 500/- will be paid to compensate the persons travel and time.

Sample processing and testing:

Serum is separated and aliquoted under aseptic conditions. All samples will be tested for CHIKV - Realtime PCR and anti-CHIKV IgM antibodies in acute blood samples using a CHIKV TRC standard protocol. All positive and negative sera will be transported to MAHE, Manipal on a daily basis.

Sample shipment:

All samples collected (acute sample from suspected CHIKV cases and, convalescent and follow up samples from laboratory confirmed CHIKV cases) will be transported to MAHE, Manipal on a daily basis.

4           TNMC, Mumbai

4.1          OBJECTIVE1: ENROLLING OF SUSPECTED CHIKUNGUNYA CASES, DATA COLLECTION AND COLLECTION AND TRANSPORT OF SAMPLES TO MAHE

4.2          Methodology:

Study site: T.N. Medical College & B.Y.L. Nair Ch. Hospital, Mumbai Case definition:

Acute suspected Chikungunya: An acute febrile illness case with arthralgia and or arthritis within 14

days of onset of illness and belonging to 12 to 75 years of age.

 

Acute confirmed Chikungunya: A case which is laboratory confirmed positive for Chikungunya Realtime PCR or Chikungunya IgM ELISA.

Chronic Chikungunya: A case of laboratory confirmed Chikungunya with persistent arthralgia even after 3 months of the onset of illness.

Enrolment to the study

Once the suspected case is identified, the study is briefly explained to the patient and the bystander. All questions and doubts regarding the study from the patient should be clearly answered. Following this, if the patient is willing to participate, the informed consent is obtained.

Data collection

 

The questionnaire in the case report form will be administered. The demographical, clinical history and epidemiological details of the patient will be collected using a tablet PC.

Clinical sample collection

 

Acute sample collection: 8 ml of blood in plain vacutainer will be collected at the time of enrollment to the study.

Convalescent/ follow-up sample collection: 5-8ml blood in plain vacutainer will be collected at 1st, 3rd, 6th, 12th, 18th, 24th, 30th and 36th month post of illness from all laboratory confirmed (PCR positive or anti-CHIKV IgM antibody positive) CHIKV cases. On every follow up visit for providing clinical data and sample Rs. 500/- will be paid to compensate the persons travel and time.

Sample processing and testing:

Serum is separated and aliquoted under aseptic conditions. All samples will be tested for CHIKV - Realtime PCR and anti-CHIKV IgM antibodies in acute blood samples using a CHIKV TRC standard protocol.

Sample shipment:

All samples collected (acute sample from suspected CHIKV cases and, convalescent and follow up samples from laboratory confirmed CHIKV cases) will be transported to MAHE, Manipal on a daily basis.

5           ICGEB, New Delhi

 

Objective 1-Characterization of CHIKV isolates from various clinical cohorts

 

Isolation of CHIKV virus from clinical samples provided by MAHE will be performed. The samples will be processed for target E1 gene amplification and the amplified products will be sequenced, and phylogenetic analysis will be performed on them. We propose to perform E1 gene sequencing from at least 100 isolates/samples received form MAHE. Further, we will identify unique strains based on the phylogeny and clinical spectrum and subject to next generation sequencing of approximately 20 isolates. All viruses will be purified and the strains will be used to generate a CHIKV repository on fee-for-service model and suitable provision for accessibility. The repository will have continued access on fee for service model even after the end of the project.

Status: No revision required

 

Objective 2 - Evaluation of sero-conversion and neutralization of clinical isolates.

 

 

 

Through this objective, we propose to establish NT/ PRNT assay with convalescent samples in a medium throughput manner. Paired serum samples acute and convalescent from laboratory confirmed CHIKV cases will be subjected to micro neutralization and neutralization assays do determine the sero conversion and antibody titres. The high titre sera thus identified will be used to study cross neutralization pattern of selected CHIKV isolates. We will also develop and standardize high throughput antibody detection assays.

Expected outcome: We anticipate that this study will help us understand the percentage of chikungunya infection in the Indian population, the nature of binding antibodies participating during chikungunya infection in the India population and might give directions for better disease management. We also want to correlate the neutralization status of the binding antibodies with the molecular signatures and their involvement in disease progression.

Revised status: We propose to study the seroconversion and neutralisation capacity of the clinical samples for the additional time-points that are being collected in the revised protocol. The status of the binding antibodies, IgM especially, will be evaluated for all the timepoints namely, 1, 2, 6, 12, 18, 24, 30, 36 months and tested for their neutralisation capacity. Further, PRNTs for all the animal samples will

be performed at ICGEB. Efforts will be taken to use tagged CHIKV for establishing Sero-conversion assays (micro NT/PRNT).

Justification for using tagged CHIKV for neutralisation assays

 

Currently, PRNTs are being standardised using reference viruses and is done in a 96-well format with the readout being counting the plaques which is time consuming and prone to individual-to-individual counting errors thereby affecting results. We propose to use tagged CHIKV for neutralising assays where the readouts can be automated using cell biology techniques. We propose that this method will enable us to speed up the PRNT time and also make the assays more robust. We also propose to make the assay medium to high throughput.

In this regard, Jing Jin and Graham Simmons from Vitalant Research Institute, USA has kindly agreed to share the m-Cherry-CHIKV (WA) clone with consortium. MTA has been done and submitted to RCGM for approval. Upon receiving the approval, the tagged CHIKV will be imported and the assays will be standardised.3

Reference: Jin J, et al. (2018) PLoS Negl Trop Dis 12(7): e0006693.

6           ILS, Bhubaneshwar

6.1          OBJECTIVE 1: Development and characterization of animal model for Acute CHIKV infection

6.2          Methodology/Experimental Design:

Briefly, around 10-14 days old C57BL/6 mice / 4-6 weeks old AG129 mice will be divided into groups and injected either subcutaneously at the flank region with different doses of reference CHIKV strains / unique identified CHIKV isolates suspended in PBS/DMEM solution. Control mice will be injected with DMEM alone. Thereafter mice will be observed and scored at 24 hrs interval for identifying any phenotypic or behavioral changes lethargic, ruffled fur, hindered movement etc. in infected mice with respect to control. After identification of behavioral changes, both mock and CHIKV infected mice will be sacrificed through anesthesia followed by cardiac puncture and the blood as well as tissue samples will be collected and carried forward for further downstream processing in estimating CHIKV specific RNA or protein in infected mice.

Status: No change proposed

 

 

 

6.3          OBJECTIVE 2: Development and characterisation of animal model (acute and chronic) for CHIKV infection

Activity 1: Establishment of acute CHIKV infection in adult mice using reference as well as consortium generated CHIKV strains.

7.3.1.1        Timeline: 13- 24 months

To establish and assess acute infection of Chikungunya (CHIKV), age matched adult C57BL/6J mice, 4- 6 weeks old, will be divided into different groups and inoculated subcutaneously on the left ankle near the footpad into the footpadwith high titre viz. (106_108) PFU CHIKV virus [S27/DRDE-06] suspended in DMEM solution with final volume of 50µl-100µl. Control group mice will be injected with DMEM alone. Thereafter, establishment of acute infection of CHIKV in mice model will be assessed by the following parameters.

7.3.1.1.1                   Morbidity assessment:

For assessing morbidity, the CHIKV infected mice will be observed and scored at 1, 3, 5, 7 dpi for identifying any physiological or behavioural changes (footpad swelling and oedema, movement disability, inflammatory changes in musculoskeletal tissue near the site of injection and/or systemic, lethargy, feeding patterns, isolation etc.) with respect to the control group.

7.3.1.1.2                   Viral load detection

From tissue samples - At the times indicated above, mice will be sacrificed and tissues (brain, muscle, spleen, kidney, liver and lymph nodes) will be dissected, weighed, and homogenized. The total RNA will be extracted using trizol reagent. Quantification of viral RNA will be done through qRT-PCR using CHIKV E1 gene specific primers to estimate the viral load at different sites over specific time period.

From serum samples - The viral RNA will be extracted from serum samples using QIAamp Viral RNA Mini Kit and qRT-PCR will be performed using CHIKV E1 gene specific primers for the purpose mentioned above.

7.3.1.1.3                   Inflammatory Marker assessment

The expression of pro-inflammatory mediators like TNF-�’, MCP-1 (CCL2), IFN-γ, interleukin-6 (IL-6), and IFN-α/β, will be analysed along with a global mouse cytokine/chemokine panel of from the serum of infected and mock mice harvested at different dpi through Bioplex assays to correlate with pathogenicity and disease progression.

7.3.1.1.4                   Histology and Immunohistochemistry using different organs

The harvested tissues (Muscle, brain, spleen, liver & kidney) from both the mock and CHIKV infected mice will be fixed and stained with hematoxylin-eosin to determine the presence of viral protein and to estimate the extent of inflammation and tissue damage. Neurological complications in CHIKV infection have been reported earlier. Therefore immunohistochemistry stainingwill be performed using brain tissues to check for the viral load in the brain and assess the extent of tissue damage.

Once the acute model is established with reference strains, selected consortium strains (as per availability) will be taken for testing in the acute model.

Activity 2: Establishment of chronic CHIKV infection in adult mice using reference as well as consortium generated CHIKV strains

7.3.2.1        Timeline: 25-36 months

To establish and assess chronic infection of Chikungunya (CHIKV), age matched adult C57BL/6J mice, (6-8 weeks old), will be divided into different groups and inoculated subcutaneously on the left ankle near the footpad/ into the footpad with high titre viz. (102-104PFU) CHIKV virus using the above- mentioned reference strains suspended in DMEM solution with final volume of 50µl. Control group mice will be injected with DMEM alone. Thereafter, CHIKV infected mice will be observed and sacrificed at 1, 3, 7, 10, 14, 21 and 28 dpi and blood as well as organs will be processed as mentioned below.