Crimean-Congo Hemorrhagic Fever Virus in Humans and Livestock, Pakistan, 2015-2017.

We detected Crimean-Congo hemorrhagic fever virus infections in 4 provinces of Pakistan during 2017-2018. Overall, seroprevalence was 2.7% in humans and 36.2% in domestic livestock. Antibody prevalence in humans was highest in rural areas, where increased contact with animals is likely.


Animal and Tick Samples
For animal sampling, we selected 14 districts from Punjab, 3 from Khyber Pakhtunkhwa, 7 from Balochistan, and 5 from Sindh. We chose sampling sites on the basis of animal populations, ease of sampling, and the presence of veterinary clinics. We sampled a total of 311 buffaloes, 480 camels, 183 cattle, 440 goats, and 424 sheep; from each animal, we drew ≈4 mL blood aseptically directly into the gel-clot activator containing vacutainer (Improvacuter). We centrifuged samples, and then harvested serum samples and stored them at −40°C. The animal sampling period spanned 30 months during 2015-2018. In addition, Punjab Livestock and Dairy Development Department provided 98 plasma samples (24 from goats, 28 from buffaloes, and 46 from cows) from the household livestock of the CCHF-suspected human case-patients.
We collected a total of 509 Hyalomma spp., 134 Rhipicephalus spp., 77 Haemaphysalis spp., and 54 Rhipicephalus spp. ticks from livestock from Punjab province of Pakistan. The ticks were collected at the time of field sampling. Animals randomly selected for serum collection were also carefully examined for the presence of ticks; if infested, we collected the ticks by removing them with forceps from the animals. We stored ticks at 4°C in the field and sent them to University of Agriculture Faisalabad, Pakistan, where they were stored at −40°C.

Serologic Testing
For human serum samples, we used a 2-step approach. In the first step, we screened all human serum samples using a commercial ELISA (Vector-Best, https://vector-best.ru) according to the manufacturer's instructions. In the second step, all ELISA-positive serum samples were confirmed by indirect immunofluorescence assay. The cells were fixed with phosphate-buffered saline (PBS, pH = 7.5) containing 4% paraformaldehyde, permeablised with 0.2% TritonX-100, and blocked with 5% bovine serum albumin at 37°C for 1 hour. We added serum samples diluted in 1% bovine serum albumin and incubated plates overnight at 4°C. We washed the culture plates and added FITC-tagged anti-human IgG at a dilution of 1:1,000 (Sigma, USA). We incubated plates for 1 hour, then washed them and added 100 μL of 1× PBS solution. We observed fluorescence using a fluorescence microscope and acquired images. We first tested serum samples at a dilution of 1:100; negative samples were further tested at a dilution of 1:20.
We used anti-CCHFV NP monoclonal antibody (43E5) as the positive control.
For animal serum samples, we conducted screening of anti-CCHFV IgGs by ELISA (ID Vet, https://www.id-vet.com). This double antigen multi-species ELISA can detect IgG antibodies against NP protein of CCHFV in caprine, ovine, bovine, and other susceptible species' serum samples.
We used animal plasma samples and ticks to screen CCHFV antigen by VectoCrimea-CHF-antigen (Vector-Best, https://vector-best.ru) ELISA kit according to the manufacturer's instructions.

Whole-Genome Sequencing and Analysis
We prepared libraries for next-generation sequencing using Truseq mRNA kit (TruSeq Stranded mRNA Library Prep Kit, Cat # RS-122-2101; Illumina) following the manufacturer's instructions. We performed sequencing on a HiSeq 3000 sequencer (). We analyzed the data in Metavisitor (a suite of galaxy tools) as described previously (3). We then used the BLAST guided scaffold to reference align the reads in Geneious R11. We set up RT-PCR reaction to fill the gap in L segment by using 3508 F(CCHF-L): GGCCAGCTTATCACTCATGGA and 3974 R(CCHF-L): CATTCTGCTGCCACCTCCTT primers.

Statistical Analysis
We conducted statistical analysis using R version 3.5.1 (https://www.r-project.org). We