Henipavirus-related Sequences in Fruit Bat Bushmeat, Republic of Congo

Animals obtained for this study were euthanized under anesthesia with xylazine and ketamine by cardiac exsanguination. Tissue samples were extracted by using the NucleoSpin RNA II Kit (Macherey-Nagel, Düren, Germany); throat swab specimens were dissolved in 500 μL of phosphate-buffered saline and extracted by using the QIAamp Viral RNA Mini Kit (QIAGEN, Hilden, Germany). The same kit was utilized for RNA extraction from urine. Fecal samples were extracted with the Stool DNA Extraction Kit (ROBOKLON, Berlin, Germany) in the presence of carrier RNA. RNA was reverse transcribed with random hexamer primers. The 339 samples, from 42 bats that were tested, included major organs of all animals (kidney, spleen, lung, small intestine). Liver samples were only available from 41, blood samples from 39, salivary gland of 1, throat swab specimens from 28, fecal samples from 31, and urine samples from 21 individual animals. Fifteen samples that tested positive with respirovirus, morbillivirus, and henipavirus (RES-MOR-HEN) PCR included 10 spleens, 3 kidneys, 1 liver, and 1 urine sample. Only 1 organ from each animal tested positive, except for 1 animal (RC09_Eid_239) which had viral sequences found in kidney, liver, and spleen. In 1 animal (RC09_Eid_236), spleen and urine samples tested positive. Five sequence-specific real-time PCRs were designed and used to re-screen spleen, kidney, and urine samples. Two spleen samples and 1 urine sample displayed additional positive results with very low copy numbers in 1 of 2 independent experiments. Because of low copy numbers and limitation of material, no sequences were derived for these samples. Paramyxovirinae (PAR) sequences were obtained for the positive liver and urine samples and for 2 positive spleen samples.