Bats are the natural reservoir of numerous known and novel zoonotic viruses, including rabies, Nipah, Hendra, Marburg, and severe acute respiratory syndrome viruses (1). In Bangladesh, Nipah virus (NiV) outbreaks are seasonal, and cases peak during December-April annually. In 2006, the Institute of Epidemiology, Disease Control and Research, Bangladesh (Dhaka, Bangladesh); icddr,b (Dhaka); and US Centers for Disease Control and Prevention (Atlanta, GA, USA) collaboratively established a hospital-based national sentinel surveillance program to address public health risks posed by NiV (2). During 2006-2022, the program enrolled >22,000 patients with symptoms of NiV infection. We report detection of Pteropine orthoreovirus (PRV) from 5 NiV-negative patients with acute respiratory disease and encephalitis during a 2022-2023 outbreak.
PRV is an emerging batborne orthoreoviruses previously linked to acute respiratory infections in humans, especially in Southeast Asia (3-5). PRV is classified under the genus Orthoreovirus genus, family Reoviridae, which includes Nelson Bay virus (NBV), identified in Australia in 1968 (6). Zoonotic potential of NBV was confirmed in 2006, when a human case occurred in Melaka, Malaysia (7).
PRVs are nonenveloped, fusogenic viruses with double-stranded RNA genomes composed of 10 segments (S1, S2, S3, S4, M1, M2, M3, L1, L2, and L3). The S1 segment is tricistronic, encoding 3 proteins: cell-attachment protein, fusion-associated small transmembrane protein, and nonstructural protein p17 of unknown function (8).
The Bangladesh surveillance program uses quantitative PCR on throat swab samples to test for NiV RNA and on serum for NiV IgG or IgM. During December 2022-March 2023, five patients with presumptive NiV infection diagnoses were admitted to hospitals in Bangladesh but tested NiV-negative (Table). Three patients were admitted to Faridpur Medical College Hospital (MCH) (Faridpur, Bangladesh), and 1 patient each was admitted to Rajshahi MCH (Rajshahi, Bangladesh) and Khulna MCH (Khulna, Bangladesh). All patients had clinical signs and symptoms, including fever, disorientation, altered mental status, abnormal gait, and difficulty breathing. Four patients had a primary diagnosis of encephalitis, and 1 pediatric case had mild symptoms and a primary diagnosis of febrile convulsions (Table). All patients reported consuming raw date palm sap within 2 weeks of symptoms developing.
Patients originated from different geographic regions of Bangladesh. Case-patients BDB047, BDB051, and BDB052 were from Faridpur and Rajbari, within a 30-mile radius of central Bangladesh, near the Padma River Basin (Figure 1). Those 3 patients and pediatric case-patient BDB113 from Khulna (≈180 km south of Faridpur and Rajbari) were hospitalized within the same 2-week period in late December 2022 and early January 2023 (Table). Case-patient BDB040 was admitted in Sirajganj (≈150 km north of Faridpur and Rajbari) during March-April 2023. That patient had a history of chronic mental illness and also consumed raw date palm sap while hospitalized.
All patients were discharged after 2-3 weeks. During telehealth follow-up >15 months after discharge, case-patients BDB047 and BDB052 reported persistent fatigue, disorientation, and breathing and walking difficulties. Case-patients BDB051 and BDB113 fully recovered, but case-patient BDB040 died in August 2024, following deteriorating health and unexplained neurologic issues (Table).
We conducted viral discovery by using a capture-based agnostic viral sequencing method, VirCapSeq-VERT (VCS) (9), on total nucleic acid extracted from archived throat swab samples collected in viral-transport media. We perfomed VCS on NextSeq 2000 (Illumina, https://www.illumina.com), as previously described (9). We further used Megablast (MEGA, https://www.megasoftware.net) to compare retrieved sequences to those in GenBank nucleotide databases (Appendix). VCS analysis revealed PRV reads in all patients. We did not identify any other viral or bacterial pathogens in high-throughput sequencing.
We quantified viral load by using an in-house L2-based quantitative PCR (Appendix Table 1). Case-patient BDB051 had the highest viral load, likely due to the short (≈2-day) interval between raw date palm sap consumption and sample collection (Table). Case-patients BDB047 and BDB052 had higher viral loads than did BDB113 and BDB040.
For phylogenetic analysis, we amplified the partial S1 segment encoding the p10 protein (96 aa) by using a consensus PCR (Figure 2; Appendix Table 2). The Bangladesh PRVs clustered at 99.3%-100.0% average nucleotide identity (ANI). Those PRVs showed ≈96% ANI with the Indonesia/2010 detected from a large flying fox (Pteropus vampyrus) in Indonesia, ≈85% ANI with the Nachunsulwe-57 detected from an Egyptian fruit bat (Rousettus aegyptiacus) in Zambia, and ≈77% ANI with the Kasama strain detected from an Angolan soft-furred fruit bat (Lissonycteris angolensis) in Uganda (10,11). The Bangladesh PRVs also had >77.0% ANI with Xi River virus from a fulvous fruit bat (R. leschenaultii) from China, Garut-69 virus from a large flying fox from Indonesia, and the 1968 prototypic NBV from a grey-headed flying fox (P. policephalus) in Australia.
To test whether molecular detection (VCS, quantitative PCR, and partial S1 amplicon sequencing) corresponded to infectious virus presence, we inoculated throat swab samples into MDCK cells and examined for cytopathic effects. After 2 MDCK passages, we passaged PRVs once in Vero cells. We successfully cultured virus from 3 swab samples (case-patients BDB047, BDB051, and BDB113) and sequenced on the MiSeq (Illumina) platform. We mapped reads to PRV genomes by using Geneious Prime (https://www.geneious.com) software.
Complete coding sequences of all 10 Bangladesh PRV segments (GenBank accession nos. PP803379-408) showed 91.1%-100% ANI among themselves (Appendix Table 3). S1 segments showed 96.7%-99.9% ANI with each other and clustered with Indonesia/2010 strain, NBV-Australia, NBV-Nachunsulwe-57, and Kasama virus (Figure 3, panel A). Phylogeny of S2 and S3 segments were partially consistent with S1 segments (Figure 3, panels B, C). S4 segments clustered with Kampar and Melaka NBV strains (Figure 3, panel D), previously linked to mild respiratory illness in humans and reported human-to-human transmission (7,12).
L1, L2, L3, M1, M2, and M3 segments clustered with different PRVs isolated from fruit bats and occasionally from humans in Indonesia and Malaysia (Appendix Figure). That finding suggests unique evolution of each segment from reassortment events among strains circulating in Southeast Asia and long flight ranges of fruit bats. Reassortment is common for segmented RNA virus evolution and enhances risk for zoonotic potential (13). All Banglesh PRV segments showed >76% ANI with NBV-Australia, exceeding the Internationl Committee for Taxonomy of Viruses 2022 species demarcation criteria of <75% ANI (14). Thus, the detected PRVs belong to NBV species but are distinct from other mammalian and avian reoviruses.