Chikungunya fever outbreak in Guntur, Andhra Pradesh, India.

Sir,

Chikungunya virus (CHIKV) infection is an Aedes mosquito borne illness caused by an Alphavirus of the family Togaviridae1. CHIKV was first isolated from Tanzania, Africa, in 19532. Since then it has caused numerous outbreaks in continental Africa, the Indian Ocean region, and Southeast Asia. The first outbreak of CHIKV in India was reported in 1963 in Kolkata followed by epidemics in Chennai, Puducherry and Vellore in 1964; Vishakapatnam, Rajahmudry, Kakinada, Nagpur in 1965; and Barsi in 19733. Subsequently, in the absence of either active or passive surveillance it seemed that the virus had disappeared from the country until the end of 20054.

Several States in India experienced massive outbreaks of CHIK infection during 2005-2006. Andhra Pradesh, the most affected State was first to report the CHIKV epidemic in December 2005 in India5. Several districts of Karnataka State have also recorded large number of CHIKV related fever cases36. The outbreak continued with reports of a large number of cases from several other States (Rajasthan, Gujarat, Tamil Nadu, Orissa and Madhya Pradesh)6. A change in the CHIKV genotype, enhanced efficiency of mosquitoes to transmit the virus, an immunologically naive population, rapid means of trade and travel, global warming and lack of an efficient public health system are some of the important factors that influenced the explosive re-emergence of CHIKV7.

In Andhra Pradesh, though the outbreak was controlled by the end of 2006, sporadic cases continued to be reported from different districts in the following years8910. During September-October 2013, a massive outbreak (with >1000 cases) was reported from Guntur district, with CHIK like symptoms. The present study was conducted to confirm the CHIKV infection among suspected patients using serology and molecular techniques.

A total of 1905 people from a population of 37,439 were affected with fever and poly-arthralgia in Thurakaplem, Dasaripalem, Rayapudi, Chintamotu, Thurupupslem, Donepodi, Vellataru, Kollaplem, Bhattiprolu, Addepalli, Illavarem and Pesarlanka villages of Guntur district. To investigate the causative agent, 60 serum samples (2 ml each) were selected randomly to represent all the above villages, transported to National Institute of Virology (NIV), Pune, on dry ice and screened for CHIKV using IgM enzyme linked immunosorbent assay (ELISA) (NIV kit), nested RT-PCR (reverse transcriptase-polymerase chain reaction) and real time PCR. The age of the patients from whom the samples were collected ranged from 5-65 yr. CHIKV IgM test was carried out as per manufacturer's instructions. For nested RT-PCR and real time PCR, RNA was isolated using QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany) as per manufacturer's instructions. Superscript II (Invitrogen, USA) was used for reverse transcription (42°C for 1 h). Nested RT-PCRs targeting the E1 and NSP 3 genes were carried out as described earlier3. Nested RT-PCR targeting the E3 gene was also done by using in-house designed primers: (F1,5’- CAG ATA CCC GTG CAC ATG AAGT-3’ and R1, 5’- TGA GCT AAG TAT GGT CTT GT-3’) that produced a 534 base pairs (bp) fragment and (F 2, 5’-CAG ACC GAT CTT CGA CAA CA-3’ and R 2, 5’-TCA TGA CGT TGT CCT CAA GC-3’) that produced a 271-bp product. Cycling conditions were one cycle at 94°C for 5 min; 35 cycles each of 94°C (1 min), 47°C (1 min), and 72°C (1 min); followed by final extension for 7 min at 72°C. The products were visualized on 1.2 per cent agarose gel11. Standard CHIKV from NIV repository (African genotype, Strain No. 061573; Andhra Pradesh 2006; Accession Number EF027134) was used as positive control and phosphate buffer saline (PBS) as negative control to compare the results.

Viral load in the serum samples was determined by real time-PCR targeting the E3 structural protein region using the standard curve method111213. One step real time RT-PCR was performed in 25 μl reaction mixture containing 5μl RNA, 12.5μl TaqMan One-Step RT-PCR 2X Master Mix, 1 μl 40X (RT + RNAasin) (Applied Biosystems, USA) each 1 μl sense (μM), 1 μl anti-sense (μM) primer and 1μl TaqMan probe. Real-time one step RT-PCR was performed in a 96-well format using 7300 real time PCR system and SDS software V 1.0.2 (Applied Biosystems). The amplification programme included: reverse transcription at 48°C for 30 min, initial denaturation at 95°C for 10 min, and 50 cycles of denaturation (95°C for 15 sec) and annealing and extension (60°C for 1 min)111213.

Samples were also processed for virus isolation3 but virus could not be isolated. Nucleotide sequencing was performed for the partial E1 gene of four clinical samples. The PCR products were purified by using QIAquickPCR Purification Kit (Qiagen) and sequenced using Big Dye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, USA) in an automatic sequencer (ABI PRISM 3100 Genetic Analyzer, Applied Biosystems). Multiple sequence alignment of the nucleotide sequences of the E1 gene was performed using Clustal X, version 1.8314.

Out the 60 samples (35 female and 25 males) processed, 42 (70%, 25 females and 17 males) were found to be positive for IgM antibodies. Age-wise distribution of CHIK fever cases did not vary significantly among the age groups starting from 5 to 65 in the affected areas (Fig. 1). All IgM positive samples were from 3-30 days post onset day (POD). Twelve samples were positive for nested RT-PCR (50% were IgM positive for CHIKV antibodies, POD between 2-9 days) targeting the NSP3 gene and six were positive for E3 gene region (all were negative for CHIKV antibodies, POD between 2-4 days) (Table, Fig. 2A, B). Only three samples (<3POD) which were IgM negative and nested RT-PCR positive were found to be positive by real time PCR. RNA load for positive samples was in the range of 104-105 copies/ml. A total of 12 samples were found to be negative for IgM antibodies, nested RT-PCR and real time PCR.

Fig. 1

Age-wise distribution of samples from cases and IgM positive status.

Table

Details of positive samples confirmed by nested RT- PCR and real time PCR

Fig. 2

(A). Nested RT-PCR of samples targeting NSP 3 gene region; Lane M: Molecular weight marker, Lanes 1, 3, 5-8: Negative control, Lanes 2, 4: Chikungunya positive samples, Lane 9: Positive control. (B). Nested RT-PCR of samples targeting E-3 gene region; Lane M: Molecular weight marker, Lanes 1, 3, 5: Negative control, Lane 2: Chikungunya positive samples, Lane 4: Positive control.

CHIKV outbreaks of the Asian genotype have been reported from Vijaywada, Rajahmundry, Visakhapatanam, etc. in Andhra Pradesh during 1964-1965 and re-emerged as the East Central South African (ECSA) genotype in 2005-2006 in an explosive form affecting 23 districts with approximately 77,533 cases1516. Since then, sporadic cases have been reported from different places of Andhra Pradesh358916171819202122. The present investigation showed 42 IgM positive cases out of the 60 samples (70% positivity) tested though all the patients had acute CHIK like symptoms such as high fever and polyarthralgia resulting in restricted movements. The IgM positivity in the serum samples demonstrated recent CHIKV infection in the area. However, using molecular methods, only a few samples were found positive as nested RT-PCR and real time PCR tests yielded 12 and 3 positives, respectively. This may be attributed to the high PODs of the samples as rate of detection decreases with increase in PODs. This result confirms the role of CHIKV in an outbreak form during the post monsoon season in Guntur district. Post monsoon CHIKV outbreak have also been reported from other parts of the country232425.

It is suggested that IgM antibodies develop after two days of the infection with CHIKV and in chronic cases virus persists upto three months26. Our results also support this as 42 samples were found to be positive for IgM antibodies. In the present study, positivity by both nested RT-PCR and real time PCR was found to be low in comparison with IgM antibody detection. Based on our earlier report11, the lower sensitivity that we observed in case of real time PCR in this study in comparison to nested RT-PCR was not expected. There might have been chances of RNA degradation as here the real time PCR was performed for those cases which were positive by nested RT- PCR.

Sequence analysis of the partial E1 gene of four samples identified the CHIKV strain as belonging to the currently circulating ECSA genotype and having an alanine residue at position 226 of the E1 protein (E1:226A). Notably none of the CHIKV isolates from Andhra Pradesh during 2005-2007 had A226V mutation that has been reported to provide a fitness advantage to Ae. albopictus mosquitoes627. Earlier studies based on the structural polyprotein gene have shown that the CHIKV isolates obtained from 2009-2010 outbreaks in Tamil Nadu and Andhra Pradesh formed a separate clade within the ECSA lineage with a few specific mutations including a novel E1-K211E mutation22. Several mutations in the CHIKV genome have been reported since 2005 that enable the virus to adapt to new vectors or help enhance the transmission of the virus28. The changes (M269V, D284E, and V322A) as noted in other CHIKV strains62930 were also observed in our CHIKV sequences. However, no unique mutation was observed in the partial E1 gene sequenced in this study. There may be a need for full E1 gene sequencing rather than partial gene sequencing along with sequencing of the structural genes. One of the other limitations of our study was the inability to isolate the CHIKV from serum samples. Improper maintenance of cold chain during collection and transportation of the samples could be the most probable reason.

Overall, the present study provided evidence for a CHIKV outbreak in Guntur district of Andhra Pradesh. This outbreak of CHIKV in Guntur district along with the prior ones since 2005-2006 demonstrates the endemicity of the State to the CHIKV infection. Surveillance of vectors and their breeding habitats and effective control measures are needed to contain spread of the virus. Comprehensive efforts from the health authorities along with community participation in source reduction, anti-larval and mosquito management as well as diagnostic facilities for early detection are warranted to control the disease.