NIV, Pune is responsible for investigation of viral disease outbreaks of human including Zoonosis in India. As per the mandate of our institute, collection of the clinical samples from different species of animals for viral isolation and detection is required. This institute has policy to take approval on the projects which involves animals from the national committee called “Committee for the Purpose of Control & Supervision of Experiments on Animals (CPCSEA) under the Ministry of Environment and Forests, Government of India. Our study project No. HCL01/NIV15/2010 is approved by the Institutional Animal Ethical Committee (IAEC) permitting the use of infant and adult mice as laboratory animals for isolation of virus and development of antibodies respectively.

The present nosocomial CCHF outbreak was informed to Institutional Human Ethical Committee, NIV Pune. As per policy of institute the work conducted during epidemic situations is exempt from prior approval from the IEC. Due to this policy, this study was not pre-approved, but the Committee was notified after the outbreak. All study participants provided informed consent. Informed consent was in written format, both in English and local language (Guajarati). All the record analyzed was anonymized. Every sample was registered in the central registry of the institute and allotted a NIV number, which was used throughout the study.

After the request from the Gujarat Government Public Health authorities, a team from the NIV, Pune visited the private tertiary care hospital in Ahmadabad, along with local public health authorities. Medical records of hospitalised patients presented with HF manifestations were examined, and their family members or caretakers were interviewed. Blood and urine samples were collected from cases with HF manifestations and their contacts. All clinical samples were transported in cold chain to NIV, Pune. To ascertain whether additional CCHFV infections had occurred in contacts (hospital staff or family members) lacking CCHF infection symptoms, an additional 86 blood samples including CCHF suspected cases were tested by Vector-Best and indigenously developed ELISA for detection of IgM antibodies against CCHFV.

Earlier, there were two serum samples referred to NIV, Pune from Rajkot, Gujarat, from patients suspected for Hantaan and Nipah virus in the month of February 2010. These samples were from the patient and consulting physician. Both had succumbed to the unknown infection. These samples were also included in the present study.

Blood samples and ticks were collected from the buffalo, cattle, sheep and goat from the vicinity of the index case (case A), as well as from Kolat and surrounding villages, Jivanpura, Navapura and Changodar. A total of 138 ticks were collected from domestic animals from Kolat, were classified and pooled as per species. Eight tick pools were made and homogenized in MEM media. Tick homogenates were suspended in lysis buffer for viral RNA extraction. These were tested for CCHFV by qRT-PCR and nested RT-PCR. Hyalomma tick homogenates were also used for virus isolation in Vero E-6 cells and Swiss albino mice.

Rodents (n = 90) were also trapped from Kolat villege, morphologically identified and only blood samples from these animals were collected and transported to NIV, Pune.

RNA was extracted from human (serum and urine), and animal serum samples using Qiagen (Valencia, CA, USA) RNA extraction kit. Tick pools were homogenized in Minutesimum Essential Medium (MEM). This homogenate was used for RNA extraction and for virus isolation. In the initial screening CCHFV-specific TaqMan based qRT-PCR was carried out on the RNA as previously described [14]. RT-PCR was performed with the SuperScript One-Step RT-PCR kit with Platinum Taq (Invitrogen Corp., Carlsbad, CA, USA). Two sets of primers were used for initial RT-PCR. The primer set CCHF-F2 (TGG ACA CCT TCA CAA ACT C) and CCHF-R3 (GAC AAA TTC CCT GCA CCA) amplified a 530 nt region of the nucleocapsid (N) gene of CCHFV, while nested PCR using the primers CCHF-F3 (GAA TGT GCA TGG GTT AGC TC) and CCHF-R2 (GAC ATC ACA ATT TCA CCA GG) amplified a 226 nt region [15]. PCR products were analyzed on 2% agarose gel electrophoresis and Ethidium bromide straining. Cyclic sequencing was carried out at PCR condition 96°C - 1 minute, 96°C - 10 sec, 45°C - 5 sec and 60°C - 4 minutes for 25 cycles using ABI Big-Dye 3.1 dye chemistry (Applied Biosystems, Foster City, CA). These products were purified using Dyex 2.0 kit (Qiagen) according manufacturer's instructions and sequencing was performed using the ABI 3100 automated DNA sequencer. The sequences obtained were curated using KODON software for both the reads from both the ends. The curated sequences were aligned using program Clustal W and phylogenetic tree was constructed using neighbour joining algorithm with 500-bootstrap replicates as implemented in Mega v 4.0 software [16].

The tick pools were first tested by qRT-PCR. Homogenates of CCHFV-positive tick pools were inoculated into Swiss albino mice via intracerebral and intraperitoneal routes and into Vero E6 cells for virus isolation. Virus isolation was attempted from the CCHF positive human blood, serum, and urine samples.

Two CCHF IgM ELISA kits were used; a) commercial kit, b) indigenously developed test for detection of IgM antibodies in the human serum samples.

The inactivated animal serum samples were tested for evidence of anti-CCHFV IgG using an ELISA kit provided by CDC, Atlanta. The protocol followed was, ELISA plates were coated with anti-CCHFV hyper immune mouse ascetic fluid (HMAF) (dilution 1∶1000) in phosphate buffer saline pH 7.4 overnight at 4°C. BPL inactivated CCHFV infected Vero E6 cell lysate antigen (1∶20 diluted, 100 µl/well) was added as a positive antigen, normal Vero E6 cell lysate was used as negative antigen and incubated for one hr at 37°C. One hundred µl of 1∶100 diluted serum samples were added and incubated for one hr at 37°C. These wells were washed and anti-sheep IgG HRP conjugate (1∶4000 diluted, 100 µl) was added and incubated for one hr at 37°C. ABTS substrate was added and incubated at 37°C for 30 minutes. The reaction was stopped by adding 1% SDS and plates were read at 414 nm. The plates were washed five times using 10 mM PBS pH 7.4 with 0.1% Tween-20 (Sigma, USA) at the end of each step. Appropriate controls were included in the test. During investigation, ELISA was also performed to detect CCHFV specific IgG antibodies in the animals from the residential area and surrounding villages of index case (case A).

In December 2010 and January 2011, a cluster of viral hemorrhagic fever (VHF) cases was identified in Ahmadabad, Gujarat, India, which was declared as a nosocomial outbreak of Crimean-Congo hemorrhagic fever [13]. The initial case identified was a 25 year old nurse (case C) who worked in a hospital in Ahmadabad, and presented on January 13th, 2011 with a three days history of an acute febrile illness characterized by fever, chills, vomiting and headache followed by hemorrhagic symptoms [13]. Her condition rapidly deteriorated with onset of delirium, multiple hemorrhagic symptoms (palatal petechia, coughing up of blood, bleeding from lips, vaginal bleeding, hematuria, hematemesis, and pulmonary hemorrhage). She was placed in isolation on January 16th and given oral ribavirin based on suspicion of VHF. Despite treatment, she died on January 18th from multi-organ failure and disseminated intravascular coagulation (Table 1). Based on the suspicion of VHF, CCHFV was added to the diagnostic testing and the patient sera tested positive by qRT-PCR and RT-PCR in urine and serum [13].

Epidemiologic investigation revealed that case C had earlier provided care to a patient (case A) with similar VHF symptoms. Probable case A was a 32 year old housewife from Kolat village, approximately 20 km outside Ahmadabad. She had been admitted to the hospital on December 27^th, 2010 and had died on December 31st with VHF-like symptoms (Table 1). While no specimens remained from case A to allow confirmatory testing for the presence of evidence of CCHF infection, case A was strongly suspected to be the source of nosocomial infection of the attending nurse (case C) given the similarity in clinical and laboratory findings (Table 2). Further investigation revealed a similar VHF-like illness in a 42 year old physician (probable case B), who had treated probable case A and had subsequently presented with symptoms on January 6th 2011 (Table 1) and died on January 13th. Virus transmission to two further contacts was documented. The husband (case D) of probable probable case A was admitted to the hospital on January 16th, displayed similar VHF-like symptoms, was treated with oral ribavirin, and recovered and was released on January 26th. Laboratory testing confirmed evidence of CCHF virus infection. The last identified case was a 25 year old doctor (case E) who had contact in the hospital with probable case B and case D. Given his onset of illness on January 26th, case E was likely exposed to case D, who had been admitted on January 16th and released on the 26th (and case B had died on January 13th). Despite oral ribavirin treatment, case E progressed to multi-organ failure and died on January 31st [13].

In total, 3/5 of these hospitalized individuals were confirmed by qRT-PCR, nested RT-PCR & IgM ELISA as having been infected with CCHFV. The patients initially reported fever, headache, myalgia and vomiting. Death occurred within 5–9 days POD in 4 of the 5 patients. Analysis showed that all the probable cases presented with similar laboratory data as laboratory confirmed CCHF cases, including leukopenia, thrombocytopenia, increased SGOT and SGPT and serum LDH, and increased ferritin and prothrombin time (Table 2). None of blood samples were found positive by qRT-PCR from the family and hospital contacts.

With the finding of CCHFV associated with acute hemorrhagic diseases in Gujarat, retrospective analysis was carried out on some samples collected in February, 2010 from patients that showed similar case descriptions and laboratory findings as reported here for these Ahmadabad cases. Acute blood samples from two patients from Rajkot, a town approximately 200 km west of Ahmadabad, were found positive for CCHFV by qRT-PCR and RT-PCR, retrospectively confirming them to be CCHF cases.

Out of 86 samples one hospital and one family contact (both asymptomatic) was positive for presence of IgM antibodies against CCHFV. Case C and E had a very high titter of IgM in the serum samples while case D was negative in the early sample which was positive for qRT-PCR. The 2nd sample of case D was positive for CCHFV specific IgM and also qRT-PCR. The indigenously developed ELISA was compared with commercial Vector-Best assay. The sensitivity and specificity of indigenous ELISA was 75.0% and 97.5% respectively using Vector-Best assay as gold standard (Table 3). The serum samples of case C, D, and E were found positive for IgM antibodies while screening with both the kits. As positive samples were only four, further standardization of indigenously developed assay using more human serum samples is required.

The blood and urine samples collected from the laboratory confirmed CCHF cases (case C, D and E) were inoculated into Vero E6 cells and Swiss albino mice [intra cerebral. and intra peritonial. routes]. Sickness and death was observed in mice on 9^th post infection day (PID). Cytopathic effect was observed in Vero E 6 cells at 3^rd PID and virus was harvested on 7^th PID. CCHFV was isolated from the blood samples in both mice and Vero E6 cells and sequence analysis confirmed that the PCR products were derived from CCHFV RNA. No virus isolate was obtained from the urine samples; the Ct values (34) of the qRT-PCR also suggested low viral titres in the urine.

The initial virus source for this person to person chain of transmission appeared to be probable case A. She and her husband (case D) were reside in the rural village of Kolat, made a living by farming and have daily exposure to livestock and potentially ticks on these animals (Figure 1). In November and December 2010, just prior to this outbreak, a serosurvey was performed to examine livestock for evidence of CCHFV using blood samples collected from slaughterhouses in the northern adjoining state of Rajasthan and some more distant areas from Maharashtra and West Bengal states. The serum samples from buffalo, goat and sheep from Sirohi district, in southern Rajasthan were positive for IgG antibodies against CCHFV. Serum samples from northern West Bengal or Pune area of Maharashtra were negative (Table 4). The area from which positivity was reported was approximately 200 kilometres north of Ahmadabad (Figure 1).

Evidence of CCHFV infection (IgG positive) was also found in a follow-up study of livestock [including buffalo, cattle, goat, and sheep] from Kolat (residence of case “A” and case “D”) and the surrounding villages of Changodar, Jivanpara and Navapura (Table 5). Overall IgG antibodies positivity in the small sample sizes varied between villages, (10–43%) (Table 5). qRT-PCR analysis showed evidence of CCHFV infection in the blood samples from three animals [two cattle and a goat] indicating circulation of the virus in these animal populations (data not shown) which was further confirmed by CCHFV specific nested RT-PCR.

Rodent samples from Rattus rattus rufescens (72), Suncus murinus (6), Mus booduga (3), Mus musculus (8) and Rattus norvegicus (1) were tested by qRT-PCR for detection of CCHFV. All the 90 blood samples were negative for the presence CCHFV.

Out of eight tick pools collected from the livestock around the Kolat village two pools, both Hyalomma anatolicum were positive by qRT-PCR and RT-PCR specific to CCHFV (Table 6). These two Hyalomma tick pools also showed CPE in Vero E-6 cells and propagated in Swiss albino mice also.

CCHFV specific nested RT-PCR products were generated from the blood samples from CCHF cases C, D and E of this outbreak and two retrospective serum samples of year 2010. Along with this, sequences of CCHFV positive two tick pools, and four livestock samples collected in Kolat village were also included for the analysis. Sequencing analysis of the PCR products generated a 226 nt partial fragment of the virus S gene. Comparison of this virus sequence fragment with that of other CCHFVs showed that the strain detected in Ahmadabad had maximum nucleotide identity (98.0%) with Tajikistan strain (TAJ/H08966) of CCHFV which belongs in the Asian/middle east genetic lineage IV (Figure 2).