Pteropine Orthoreovirus Research Articles

Antiviral effects of micafungin against pteropine orthoreovirus, an emerging zoonotic virus carried by bats

Bat-borne emerging zoonotic viruses cause major outbreaks, such as the Ebola virus, Nipah virus, and/or beta coronavirus. Pteropine orthoreovirus (PRV), whose spillover event occurred from fruits bats to humans, causes respiratory syndrome in humans widely in South East Asia. Repurposing approved drugs against PRV is an effective tool to confront future PRV pandemics. We screened 2,943 compounds in an FDA-approved drug library and identified eight hit compounds that reduce viral cytopathic effects on cultured Vero cells. Real-time quantitative PCR analysis revealed that six of eight hit compounds significantly inhibited PRV replication. Among them, micafungin used clinically as an antifungal drug, displayed a prominent antiviral effect on PRV. Secondly, the antiviral effects of micafungin on PRV infected human cell lines (HEK293T and A549), and their transcriptome changes by PRV infection were investigated, compared to four different bat-derived cell lines (FBKT1 (Ryukyu flying fox), DEMKT1 (Leschenault's rousette), BKT1 (Greater horseshoe bat), YUBFKT1 (Eastern bent-wing bats)). In two human cell lines, unlike bat cells that induce an IFN-γ response pathway, an endoplasmic reticulum stress response pathway was commonly activated. Additionally, micafungin inhibits viral release rather than suppressing PRV genome replication in human cells, although it was disturbed in Vero cells. The target of micafungin's action may vary depending on the animal species, but it must be useful for human purposes as a first choice of medical care.

Human cytokeratin 1 plays a role in the interaction of Pteropine orthoreovirus with Hek293 cells but not HeLa cells

Pteropine orthoreovirus (PRV) causes respiratory tract infections in humans. Despite its emergence as a zoonotic and respiratory virus, little is known about its cell tropism, which hampers progress in fully understanding its pathogenesis in humans. Hek293 cells are most susceptible to PRV infection, while HeLa cells are the least. Human cytokeratin 1 (CK1) was identified as the protein that interacts with PRV. The immunofluorescence assay and qPCR results revealed prior treatment with anti-CK1 may provide Hek293 cells protection against PRV. The KRT1-knockout Hek293 cells were less susceptible to PRV infection. Further study into the pathogenesis of PRV in humans is needed.

Pteropine Orthoreovirus (PRV7S) does not Establish Persistent Infection in NP460 Cells but with Inflammatory Response Triggered

Pteropine Orthoreovirus (PRV7S) does not Establish Persistent Infection in NP460 Cells but with Inflammatory Response Triggered

Nelson Bay Reovirus Isolated from Bats and Blood-Sucking Arthropods Collected in Yunnan Province, China.

Nelson Bay reovirus (NBV) is an emerging zoonotic virus that can cause acute respiratory disease in humans. These viruses are mainly discovered in Oceania, Africa, and Asia, and bats have been identified as their main animal reservoir. However, despite recent expansion of diversity for NBVs, the transmission dynamics and evolutionary history of NBVs are still unclear. This study successfully isolated two NBV strains (MLBC1302 and MLBC1313) from blood-sucking bat fly specimens (Eucampsipoda sundaica) and one (WDBP1716) from the spleen specimen of a fruit bat (Rousettus leschenaultii), which were collected at the China-Myanmar border area of Yunnan Province. Syncytia cytopathic effects (CPE) were observed in BHK-21 and Vero E6 cells infected with the three strains at 48 h postinfection. Electron micrographs of ultrathin sections showed numerous spherical virions with a diameter of approximately 70 nm in the cytoplasm of infected cells. The complete genome nucleotide sequence of the viruses was determined by metatranscriptomic sequencing of infected cells. Phylogenetic analysis demonstrated that the novel strains were closely related to Cangyuan orthoreovirus, Melaka orthoreovirus, and human-infecting Pteropine orthoreovirus HK23629/07. Simplot analysis revealed the strains originated from complex genomic reassortment among different NBVs, suggesting the viruses experienced a high reassortment rate. In addition, strains successfully isolated from bat flies also implied that blood-sucking arthropods might serve as potential transmission vectors. IMPORTANCE Bats are the reservoir of many viral pathogens with strong pathogenicity, including NBVs. Nevertheless, it is unclear whether arthropod vectors are involved in transmitting NBVs. In this study, we successfully isolated two NBV strains from bat flies collected from the body surface of bats, which implies that they may be vectors for virus transmission between bats. While the potential threat to humans remains to be determined, evolutionary analyses involving different segments revealed that the novel strains had complex reassortment histories, with S1, S2, and M1 segments highly similar to human pathogens. Further experiments are required to determine whether more NBVs are vectored by bat flies, their potential threat to humans, and transmission dynamics.

Pteropine orthoreoviruses use cell surface heparan sulphate as an attachment receptor

ABSTRACT Pteropine orthoreoviruses (PRVs) are an emerging group of fusogenic, bat-borne viruses from the Orthoreovirus genus. Since the isolation of PRV from a patient with acute respiratory tract infections in 2006, the zoonotic potential of PRV has been further highlighted following subsequent isolation of PRV species from patients in Malaysia, Hong Kong and Indonesia. However, the entry mechanism of PRV is currently unknown. In this study, we investigated the role of previously identified mammalian orthoreovirus (MRV) receptors, sialic acid and junctional adhesion molecule-1 for PRV infection. However, none of these receptors played a significant role in PRV infection, suggesting PRV uses a distinct entry receptor from MRV. Given its broad tissue tropism, we hypothesized that PRV may use a receptor that is widely expressed in all cell types, heparan sulphate (HS). Enzymatic removal of cell surface HS by heparinase treatment and genetic ablation of HS biosynthesis genes, SLC35B2, exostosin-1, N-deacetylase/N-sulfotransferase I and beta-1,3-glucuronyltransferase 3, significantly reduced infection with multiple genetically distinct PRV species. Replication kinetic of PRV3M in HS knockout cells revealed that HS plays a crucial role in the early phase of PRV infection. Mechanistic studies demonstrated that HS is an essential host-factor for PRV attachment and internalization into cells. To our knowledge, this is the first report on the use of HS as an attachment receptor by PRVs.

Surveillance, isolation and genomic characterization of Pteropine orthoreovirus of probable bat origin among patients with acute respiratory infection in Malaysia.

Pteropine orthoreovirus (PRV), an emerging bat-borne virus, has been linked to cases of acute respiratory infections (ARI) in humans. The prevalence, epidemiology and genomic diversity of PRV among ARI of unknown origin were studied. Among 632 urban outpatients tested negative for all known respiratory viruses, 2.2% were PRV-positive. Patients mainly presented with moderate to severe forms of cough, sore throat and muscle ache, but rarely with fever. Phylogenetic analysis revealed that over 90% of patients infected with the Melaka virus (MelV)-like PRV, while one patient infected with the Pulau virus previously found only in fruit bats. Human oral keratinocytes and nasopharyngeal epithelial cells were susceptible to clinical isolates of PRV, including the newly isolated MelV-like 12MYKLU1034. Whole genome sequence of 12MYKLU1034 using Nanopore technique revealed a novel reassortant strain. Evolutionary analysis of the global PRV strains suggests the continuous evolution of PRV through genetic reassortment among PRV strains circulating in human, bats and non-human primate hosts, creating a spectrum of reassortant lineages with complex evolutionary characteristics. In summary, the role of PRV as a common etiologic agent of ARI is evident. Continuous monitoring of PRV prevalence, pathogenicity and diversity among human and animal hosts is important to trace the emergence of novel reassortants.

Laying the foundation for single-cell studies in bats.

Bats serve as hosts of viruses that can cause disease in humans. In this issue of Immunity, Gamage etal. characterize the immune cell repertoire in Eonycteris spelaea bat lung tissue using single-cell transcriptomics, providing insight into the invivo immune response to infection with a Pteropine orthoreovirus (PRV3M) and establishing a paradigm for future comparative immunology studies.

Single-cell transcriptome analysis of the invivo response to viral infection in the cave nectar bat Eonycteris spelaea.

Bats are reservoir hosts of many zoonotic viruses with pandemic potential. We utilized single-cell transcriptome sequencing (scRNA-seq) to analyze the immune response in bat lungs upon invivo infection with a double-stranded RNA virus, Pteropine orthoreovirus PRV3M. Bat neutrophils were distinguished by high basal IDO1 expression. NK cells and Tcells were the most abundant immune cells in lung tissue. Three distinct CD8+ effector Tcell populations could be delineated by differential expression of KLRB1, GFRA2, and DPP4. Select NK and T clusters increased expression of genes involved in Tcell activation and effector function early after viral infection. Alveolar macrophages and classical monocytes drove antiviral interferon signaling. Infection expanded a CSF1R+ population expressing collagen-like genes, which became the predominant myeloid cell type post-infection. This work uncovers features relevant to viral disease tolerance in bats, lays a foundation for future experimental work, and serves as a resource for comparative immunology studies.

Seroprevalence of Pteropine orthoreovirus (PRV) Infection among Outpatients in a Clinic in Rembau, Malaysia

Seroprevalence of Pteropine orthoreovirus (PRV) Infection among Outpatients in a Clinic in Rembau, Malaysia

Pteropine Orthoreovirus, PRV7S (Sikamat Virus) Demonstrates Oncolysis in Nasopharyngeal Carcinoma Cell Lines.

Oncolytic properties had been demonstrated in Mammalian Orthoreovirus (MRV) and Avian Orthorevirus (ARV). Besides MRV and ARV, Pteropine Orthoreovirus (PRV) is also categorized under the genus Orthoreovirus. PRV7S (Sikamat virus) is an orthoreovirus isolated in Malaysia. Present study aims to investigate the oncolytic effects of PRV7S on ranges of nasopharyngeal carcinoma (NPC) cells through apoptosis in comparison to MRV3. Non-cancerous nasopharyngeal (NCNP) and NPC cells were infected by PRV7S and MRV3. The effects of PRV7S on the proliferation inhibition and apoptotic activity of NPC cells was examined using MTT assay and flow cytometry. Additionally, western blot assay was performed to analyze the expression of RAS and apoptotic protein. Lastly, qPCR assay was performed to demonstrate that PRV7S and MRV3 replicated in infected-NPC and infected-NCNP cells. The proliferation of NPC cells were significantly inhibited after PRV7S infection in a time dependent manner in comparison to infected-NCPC cells. Flow cytometry analysis showed that PRV7S infection was able to induce apoptosis on NPC cells at 48 hpi. Western blot results showed that upon PRV7S infection, N/H/K RAS protein expression was reduced, whereas caspase-3 protein expression increased in NPC cells. qPCR assay showed higher viral load of PRV7S found in infected-NPC compared to infected-NCNP cells. PRV7S inhibits the proliferation and induces apoptosis of NPC cells similar to MRV3. Therefore, PRV7S is a potential oncolytic virus.

Pteropine orthoreovirus of Bat Origin: An Emerging Pathogen Associated with Cases of Acute Respiratory Infections

Pteropine orthoreovirus of Bat Origin: An Emerging Pathogen Associated with Cases of Acute Respiratory Infections

Seroprevalence of Pteropine orthoreovirus in humans remain similar after nearly two decades (2001-2002 vs. 2017) in Tioman Island, Malaysia.

Pteropine orthoreovirus (PRV) is an emerging zoonotic respiratory virus that can be transmitted from bats to humans. In Malaysia, aside from PRV2P (Pulau virus) being isolated from Pteropus hypomelanus sampled in Tioman Island, PRV3M (Melaka virus), PRV4K (Kampar virus), and PRV7S (Sikamat virus) were all isolated from samples of patients who reported having a disease spectrum from acute respiratory distress to influenza-like illness and sometimes even with enteric symptoms such as diarrhea and abdominal pain. Screening of sera collected from human volunteers on Tioman Island in 2001-2002 demonstrated that 12.8% (14/109) were positive for PRV2P and PRV3M. Taking all these together, we aim to investigate the serological prevalence of PRV (including PRV4K and PRV7S) among Tioman Island inhabitants again with the assumption that the seroprevalence rate will remain nearly similar to the above reported if human exposure to bats is still happening in the island. Using sera collected from human volunteers on the same island in 2017, we demonstrated seroprevalence of 17.8% (28/157) against PRV2P and PRV3M, respectively. Seropositivity of 11.4% among Tioman Island inhabitants against PRV4K and PRV7S, respectively, was described in this study. In addition, the seroprevalence of 89.5% (17/19), 73.6% (14/19), 63.0% (12/19), and 73.6% (14/19) against PRV2P, PRV3M, PRV4K, and PRV7S, respectively, were observed among pteropid bats in the island. We revealed that the seroprevalence of PRV among island inhabitants remains nearly similar after nearly two decades, suggesting that potential spill-over events in bat-human interface areas in the Tioman Island. We are unclear whether such spillover was directly from bats to humans, as suspected for the PRV3M human cases, or from an intermediate host(s) yet to be identified. There is a high possibility of the viruses circulating among the bats as demonstrated by high seroprevalence against PRV in the bats.

Establishment of Intestinal Organoid from Rousettus leschenaultii and the Susceptibility to Bat-Associated Viruses, SARS-CoV-2 and Pteropine Orthoreovirus.

Various pathogens, such as Ebola virus, Marburg virus, Nipah virus, Hendra virus, Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), Middle East Respiratory Syndrome Coronavirus (MERS-CoV), and SARS-CoV-2, are threatening human health worldwide. The natural hosts of these pathogens are thought to be bats. The rousette bat, a megabat, is thought to be a natural reservoir of filoviruses, including Ebola and Marburg viruses. Additionally, the rousette bat showed a transient infection in the experimental inoculation of SARS-CoV-2. In the current study, we established and characterized intestinal organoids from Leschenault’s rousette, Rousettus leschenaultii. The established organoids successfully recapitulated the characteristics of intestinal epithelial structure and morphology, and the appropriate supplements necessary for long-term stable culture were identified. The organoid showed susceptibility to Pteropine orthoreovirus (PRV) but not to SARS-CoV-2 in experimental inoculation. This is the first report of the establishment of an expandable organoid culture system of the rousette bat intestinal organoid and its sensitivity to bat-associated viruses, PRV and SARS-CoV-2. This organoid is a useful tool for the elucidation of tolerance mechanisms of the emerging rousette bat-associated viruses such as Ebola and Marburg virus.

Attenuated infection by a Pteropine orthoreovirus isolated from an Egyptian fruit bat in Zambia.

BackgroundPteropine orthoreovirus (PRV) is an emerging bat-borne zoonotic virus that causes severe respiratory illness in humans. Although PRVs have been identified in fruit bats and humans in Australia and Asia, little is known about the prevalence of PRV infection in Africa. Therefore, this study performed an PRV surveillance in fruit bats in Zambia.MethodsEgyptian fruit bats (Rousettus aegyptiacus, n = 47) and straw-colored fruit bats (Eidolon helvum, n = 33) captured in Zambia in 2017–2018 were screened for PRV infection using RT-PCR and serum neutralization tests. The complete genome sequence of an isolated PRV strain was determined by next generation sequencing and subjected to BLAST and phylogenetic analyses. Replication capacity and pathogenicity of the strain were investigated using Vero E6 cell cultures and BALB/c mice, respectively.ResultsAn PRV strain, tentatively named Nachunsulwe-57, was isolated from one Egyptian fruit bat. Serological assays demonstrated that 98% of sera (69/70) collected from Egyptian fruit bats (n = 37) and straw-colored fruit bats (n = 33) had neutralizing antibodies against PRV. Genetic analyses revealed that all 10 genome segments of Nachunsulwe-57 were closely related to a bat-derived Kasama strain found in Uganda. Nachunsulwe-57 showed less efficiency in viral growth and lower pathogenicity in mice than another PRV strain, Miyazaki-Bali/2007, isolated from a patient.ConclusionsA high proportion of Egyptian fruit bats and straw-colored fruit bats were found to be seropositive to PRV in Zambia. Importantly, a new PRV strain (Nachunsulwe-57) was isolated from an Egyptian fruit bat in Zambia, which had relatively weak pathogenicity in mice. Taken together, our findings provide new epidemiological insights about PRV infection in bats and indicate the first isolation of an PRV strain that may have low pathogenicity to humans.

Distinct interferon response in bat and other mammalian cell lines infected with Pteropine orthoreovirus.

Bats serve as natural hosts of Pteropine orthoreovirus (PRV), an emerging group of bat-borne, zoonotic viruses. Bats appear to possess unique innate immune system responses that can inhibit viral replication, thus reducing clinical symptoms. We examined the innate immune response against PRV and assessed viral replication in cell lines derived from four bat species (Miniopterus fuliginosus, Pteropus dasymallus, Rhinolophus ferrumequinum, and Rousettus leschenaultii), one rodent (Mesocricetous auratus), and human (Homo sapiens). The expression levels of pattern recognition receptors (PRRs) (TLR3, RIG-I, and MDA5) and interferons (IFNB1 and IFNL1) were higher and PRV replication was lower in cell lines derived from M. fuliginosus, R. ferrumequinum, and R. leschenaultii. Reduction of IFNB1 expression by the knockdown of PRRs in the cell line derived from R. ferrumequinum was associated with increased PRV replication. The knockdown of RIG-I led to the most significant reduction in viral replication for all cell lines. These results suggest that RIG-I production is important for antiviral response against PRV in R. ferrumequinum.Supplementary InformationThe online version contains supplementary material available at 10.1007/s11262-021-01865-6.

Pteropine Orthoreovirus in an Angolan Soft-Furred Fruit Bat (Lissonycteris angolensis) in Uganda Dramatically Expands the Global Distribution of an Emerging Bat-Borne Respiratory Virus.

Pteropine orthoreovirus (PRV; Reoviridae: Spinareovirinae) is an emerging bat-borne zoonotic virus that causes influenza-like illness (ILI). PRV has thus far been found only in Australia and Asia, where diverse old-world fruit bats (Pteropodidae) serve as hosts. In this study, we report the discovery of PRV in Africa, in an Angolan soft-furred fruit bat (Lissonycteris angolensis ruwenzorii) from Bundibugyo District, Uganda. Metagenomic characterization of a rectal swab yielded 10 dsRNA genome segments, revealing this virus to cluster within the known diversity of PRV variants detected in bats and humans in Southeast Asia. Phylogeographic analyses revealed a correlation between geographic distance and genetic divergence of PRVs globally, which suggests a geographic continuum of PRV diversity spanning Southeast Asia to sub-Saharan Africa. The discovery of PRV in an African bat dramatically expands the geographic range of this zoonotic virus and warrants further surveillance for PRVs outside of Southeast Asia.

Detection of Recombinant Rousettus Bat Coronavirus GCCDC1 in Lesser Dawn Bats (Eonycteris spelaea) in Singapore.

Rousettus bat coronavirus GCCDC1 (RoBat-CoV GCCDC1) is a cross-family recombinant coronavirus that has previously only been reported in wild-caught bats in Yúnnan, China. We report the persistence of a related strain in a captive colony of lesser dawn bats captured in Singapore. Genomic evidence of the virus was detected using targeted enrichment sequencing, and further investigated using deeper, unbiased high throughput sequencing. RoBat-CoV GCCDC1 Singapore shared 96.52% similarity with RoBat-CoV GCCDC1 356 (NC_030886) at the nucleotide level, and had a high prevalence in the captive bat colony. It was detected at five out of six sampling time points across the course of 18 months. A partial segment 1 from an ancestral Pteropine orthoreovirus, p10, makes up the recombinant portion of the virus, which shares high similarity with previously reported RoBat-CoV GCCDC1 strains that were detected in Yúnnan, China. RoBat-CoV GCCDC1 is an intriguing, cross-family recombinant virus, with a geographical range that expands farther than was previously known. The discovery of RoBat-CoV GCCDC1 in Singapore indicates that this recombinant coronavirus exists in a broad geographical range, and can persist in bat colonies long-term.

Replication kinetics and cellular tropism of emerging reoviruses in sheep and swine respiratory ex vivo organ cultures

Ex vivo organ cultures (EVOCs) are extensively used to study the cellular tropism and infectivity of different pathogens. In this study, we used ovine and porcine respiratory EVOCs to investigate the replication kinetics and cellular tropism of selected emerging reoviruses namely Pteropine orthoreovirus, an emerging bat-borne zoonotic respiratory virus, and atypical Bluetongue virus (BTV) serotypes which, unlike classical serotypes, do not cause Bluetongue, a major OIE-listed disease of ruminants. BTV failed to replicate in ovine EVOCs. Instead, PRV showed slight replication in porcine lower respiratory EVOCs and a more sustained replication in all ovine respiratory tissues. By confocal laser scanning microscopy, PRV was demonstrated to infect bronchiolar and type I pneumocytes of ovine tissues. Overall, respiratory EVOCs from different animal species, eventually obtained at slaughterhouse, are a useful tool for testing and preliminarily characterize novel and emerging viruses addressing the essential in vivo animal work. Further experiments are, indeed, warranted in order to characterize the pathogenesis and transmission of these emerging reoviruses.

Cell-cell fusion induced by reovirus FAST proteins enhances replication and pathogenicity of non-enveloped dsRNA viruses.

Fusogenic reoviruses encode fusion-associated small transmembrane (FAST) protein, which induces cell–cell fusion. FAST protein is the only known fusogenic protein in non-enveloped viruses, and its role in virus replication is not yet known. We generated replication-competent, FAST protein-deficient pteropine orthoreovirus and demonstrated that FAST protein was not essential for viral replication, but enhanced viral replication in the early phase of infection. Addition of recombinant FAST protein enhanced replication of FAST-deficient virus and other non-fusogenic viruses in a fusion-dependent and FAST-species-independent manner. In a mouse model, replication and pathogenicity of FAST-deficient virus were severely impaired relative to wild-type virus, indicating that FAST protein is a major determinant of the high pathogenicity of fusogenic reovirus. FAST-deficient virus also conferred effective protection against challenge with lethal homologous virus strains in mice. Our results demonstrate a novel role of a viral fusogenic protein and the existence of a cell–cell fusion-dependent replication system in non-enveloped viruses.

Serological evidence and experimental infection of cynomolgus macaques with pteropine orthoreovirus reveal monkeys as potential hosts for transmission to humans

ABSTRACT Pteropine orthoreoviruses (PRV) are emerging bat-borne viruses with proven zoonotic transmission. We recently demonstrated human exposure to PRV in Singapore, which together with previous reports from Malaysia and Vietnam suggest that human infection of PRV may occur periodically in the region. This raises the question whether bats are the only sources of human infection. In this study, we screened 517 cynomolgus macaques caught in Singapore for evidence of exposure to PRV3M (also known as Melaka virus), which was first isolated from human patients in Melaka, Malaysia. We found that 67 serum samples were PRV3M positive by ELISA and 34 were also positive by virus neutralization assay. To investigate whether monkeys could act as hosts for PRV transmission, we experimentally infected cynomolgus macaques with PRV3M and housed these animals with uninfected monkeys. Although no clinical signs of infection were observed in infected animals, viral RNA was detected in nasal and rectal swabs and all infected macaques seroconverted. Additionally, one of the uninfected animals seroconverted, implying active shedding and transmission of PRV3M. We provide evidence that PRV exposure in the macaque population in Singapore occurs at a relatively high prevalence and this study suggests that cynomolgus macaques may be an intermediate or reservoir host for PRVs.