Chikungunya

Background & objectives: Chikungunya (CHIK) virus has caused numerous large outbreaks in India. No active or passive surveillance has been carried out since the last epidemic which occurred in 1971. For active surveillance, it is necessary to have a test, which can detect the virus from a large number of field-collected mosquitoes.

Methods: The present study describes the standardization of monoclonal antibody (MAb) based antigen capture ELISA to detect chikungunya virus antigen from the mosquitoes. CHIK virus antigen from suspension of experimentally infected mosquitoes and their progeny was captured on mouse polyclonal antibody, while biotinylated CHIK Mab was used as a probing antibody. CHIK virus antigen in the head squashes of virus inoculated mosquitoes was detected using indirect immunofluorescence antibody (IFA) test for confirmation of ELISA results.

Results: The ELISA test was sensitive enough to detect antigen even if a small fraction of a single infected mosquito homogenate was incorporated in the test. The IFA test failed to detect CHIK antigen in 10 and 25 (mu)l of suspension whereas with ELISA it was detected in all the samples. Progeny of Aedes aegypti and Ae. albopictus mosquitoes infected with chikungunya virus did not show the possibility of existence of transovarial transmission.

Interpretation & conclusion: This test is rapid and simple since it can be completed in two days as compared to the conventional mosquito inoculation and IFA techniques, which require at least 10 days. There is an additional advantage with this test that a large number of samples can be processed, and the remaining homogenate of the mosquitoes can be used for screening other viruses. Experimental data raised using this test showed that transovarial transmission of this virus does not occur in these vector species.

Key words Antigen capture ELISA - chikungunya - monoclonal antibody - transovarial transmission

Chikungunya (CHIK) virus belongs to genus Alphavirus in the family Alphaviridae. It is single stranded positive sense RNA virus and Aedes aegypti has been incriminated as the principal vector in India . The virus is of considerable public health importance since it is known to have caused a large number of outbreaks and epidemics.

Epidemiology of this virus shows that it causes large-scale epidemics and disappears abruptly from the affected area. Although there are no recent reports of outbreaks in India, the re-emergence of CHIK viral infection in the form of an outbreak in Thailand2 is alarming. There are no reports of transovarial transmission (TOT) of this virus in mosquitoes in nature as well there is no experimental evidence available3,4. It is suggested that during the inter-epidemic period it might be maintained in nature in an occult cycle3. However, it has also been suggested that CHIK virus might be maintained in nature at a low level and cases are probably misdiagnosed as dengue, due to similarity in the clinical symptoms5.

India has experienced CHIK epidemics in the past6-9 from Kolkata, Vellore, Barsi and Nagpur. However, after the Barsi epidemic serological surveillance has not been done and tests for CHIK antigen were not included in routine tests. Recently, in October 2001 there has been isolation of this virus from a town in Maharashtra state, India, from Ae. aegypti mosquitoes (NIV, unpublished data). With a view to detect CHIK virus activity in the vector mosquitoes, we have developed an monoclonal antibody based antigen detection ELISA. This method was employed to rule out the possibility of occurrence of TOT in vector mosquitoes. The present communication reports these findings.

Material & Methods

Mosquitoes: The Ae. aegypti and Ae. albopictus mosquitoes used in the present study were taken from the laboratory colony maintained at the National Institute of Virology (NIV), Pune. Mosquitoes were maintained in the insectary maintained at 28 +/- 1 deg C temperature and 85 +/- 5 per cent relative humidity (RH).

Chikungunya virus: (Kolkata strain, 634029) isolated from a patient during an epidemic which occurred at Kolkata, India in 1963, was used at 7th mouse passage level.

Infection of mosquitoes by intrathoracic inoculation (ITI): Four to five day old female mosquitoes of both the species were inoculated intrathoracically with approximately 0.2 (mu)I of virus suspension (2.1 log/0.2 (mu)l MID^sub 50^), following the method described by Rosen and Gubler10.

Standardisations of CHIK antigen capture ELISA: (i) Polyclonal ascitic fluid was developed as per the method described by Tung11.

(ii) CHIK monoclonal antibody (MAb) was generated against the El glycoprotein of CHIK virus African strain (8915223) using standard protocol*2. After repeated cloning, El - II antibody secreting clone was found to be stable and showed specificity to CHIK virus. MAb ascitic fluid was obtained from Department of Virology, NIV, Pune.

(iii) CHIK polyclonal as well as monoclonal ascitic fluids were used as starting material for IgG purification using Protein-G matrix (Amersham Company, USA) following the instruction manual supplied with the product.

(iv) Biotinylation of CHIK MAb IgG was carried out as described by Nerurkar el al13.

The antigen capture ELISA was essentially adapted from the method described by Joshi et al14. CHIK polyclonal IgG was used as a coating/ capturing antibody while biotinylated CHIK MAb IgG was used as a probing antibody. At every step chequer-board titration was carried out to arrive at the optimum concentration of each reagent. Bodies of the mosquitoes, which were found to be positive for CHIK virus in the head squashes were used as positive controls while clean mosquitoes from the colony were used as negative controls in each test. The samples having the optical density (OD) value double or more than that of the negative control, positive/negative (P/N) ratio > 2 were considered as positive.

Detection of virus in the mosquitoes by immunofluorescence: CHIK viral antigen in the head squashes of the virus-inoculated mosquitoes was detected using indirect immunofluorescence antibody (IFA) technique for confirmation of the ELISA results10. Affinity purified anti-rabbit (FITC) conjugate used in the study was procured from Sigma, USA. Infected mosquitoes used in the test were taken after day 10 post infection (PI).

Detection of virus in the mosquitoes by ELISA: Ten mosquitoes from the IFA confirmed CHIK positive batch were triturated in 1.0 ml PBS containing 0.1 per cent 3-[(3-cholamidopropyl) dimethylammonio] -2- hydroxy -I- propane sulphonate (CHAPSO) (Sigma, USA). These suspensions were centrifuged at 10,000g for 30 min. The supernatants (50(mu)l/well) were tested for CHIK virus antigen by capture ELISA. Inactivated mouse-brain antigen15 was used as positive control in the test. To determine the sensitivity of the test 10, 20, 30, 40 and 50(mu)l of the supernatants of infected mosquito suspensions were tested.

Detection of vertical transmission of virus in the mosquitoes: Mosquitoes of both the species were infected with virus by inoculation as mentioned above. The inoculated mosquitoes were first allowed to have a blood meal on clean uninfected chickens on day 3 PI and allowed to lay eggs. Similarly, mosquitoes were given 2nd, 3rd and 4th bloodmeal and eggs of G2, G3 and G4 cycles were obtained and conditioned for 3 to 4 days. In some of the experiments eggs obtained from the infected females were conditioned for 30 days prior to hatching. These were then allowed to hatch by immersing in water. The pools of III to IV instar larvae and emerging adults were triturated in PBS containing 0.1 per cent CHAPSO (Sigma Chemical Company, USA). These suspensions were centrifuged at 10,000 g for 30 min. The supernatants were tested for CHIK virus antigen by capture ELISA as described above.

Results & Discussion

The virus infected individual mosquitoes when homogenised without detergent CHAPSO, did not give satisfactory results. These homogenates of virus infected mosquitoes showed P/N ratio less than two when tested by antigen capture ELISA. However, the incorporation of detergent CHAPSO (0.1%) in the homogenizing buffer increased the efficacy of the test and P/N ratio of two or more was obtained. It appears that the incorporation of non-ionic detergents in the homogenizing solution might have cleared the associated cellular membrane proteins from the virus particles, which perhaps resulted in better binding of the antigen-antibody complex. To determine the sensitivity of this test, ten CHIK infected mosquitoes were triturated in 500 (mu)I of buffer and after centrifugation 10, 25, 50 and 100 (mu)l were used for the test in individual wells. The results showed that up to 10 (mu)I of homogenate the OD values were high enough to discriminate from the negative control (Fig.). IFA test failed to detect the virus with 10 and 25 (mu)l of homogenate. The ELISA test could pick up the infected mosquito even on day 2 PI when 50 (mu)l homogenates of individual mosquitoes (which were infected by intrathoracic inoculation) were tested from days I to 10 PI.

Earlier studies by Mourya4, showed that TOT of CHIK virus does not occur in Ae. aegypti and Ae. albopictus mosquitoes. Since, this test is found to be very sensitive to detect Chikungunya antigen in the mosquitoes further experiments were carried out to reconfirm these observations. Antigen capture ELISA and IFA tests carried out on the progeny obtained from intrathoracically infected mosquitoes showed negative results, which reconfirmed the absence of TOT in these two species of mosquitoes. Recently, it has been shown that when batches of eggs obtained from dengue infected Ae. aegypti mosquitoes are allowed to hatch after two months, the rate of vertical transmission increases and it is suggested that at room temperature the virus get an opportunity to multiply and increase its copy number in the quiescent embryos16. With this view, in further experiments the eggs obtained from the Chikungunya virus infected mosquitoes were allowed to hatch after one month of conditioning and antigen detection in the progeny was carried out using this test. The results indicated that phenomenon of TOT of CHIK virus does not occur in this, species of mosquitoes (Table).

This is the first report that CHIK MAb against El glycoprotein is employed for antigen detection in ELISA test. Results of the IFA technique corroborate those with this test, however as compared to IFA technique, this test seems to be sensitive and simple. Although there was no direct comparison between the IFA and ELISA results, the ELISA test was able to detect virus even from 10 VLI of homogenate when individual mosquito was triturated in 100 pd. The present findings also show the rapidity of the test since in a short time of two days, the test can be completed as compared to mosquito Inoculation and IFA techniques, which requires at least 10 days. There is an additional advantage with this test that a large number of samples can be processed and the remaining homogenate of the mosquitoes can be used for screening other viruses.

References

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13. Nerurkar LS, Namba N, Brashears G, Jacob AJ, Lee YJ, Sever JL. Rapid detection of herpes simplex virus in clinical specimens by use of a capture biotin-streptavidin enzyme-linked immunosorbent assay. J Clin Microbiol 1984; 20 : 109-14.

14. Joshi AM, Walimbe AM, Banerjee K. Potency testing of inactivated tissue culture vaccine against Japanese encephalitis using antigen capture ELISA. Acta Virol 1997; 41 : 157-9.

15. Clarke DH, Casals J. Techniques for haemagglutination and haemagglutination-inhibition with arthropod borne viruses. Am J Trop Med Hyg 1958; 7 : 561-73.

16. Mourya DT, Gokhale MD, Basu A, Barde PV, Sapkal GN, Padbidri VS, et al. Horizontal and vertical transmission of dengue virus type-2 in highly and lowly susceptible strains of Aedes aegypti mosquitoes. Acta Virol 2001; 45: 67-71.

S.L. Hundekar, J.P. Thakare, M.D. Gokhale, P.V. Barde, S.V. Argade & D.T. Mourya

National Institute of Virology (ICMR), Pune, India

Received September 20, 2001

Reprint requests:

Dr D.T. Mourya, Deputy Director, Microbial Containment Complex, Indian Council of Medical Research Sus Road, Pashan, Pune 411021, India

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