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PLoS Pathogens Sep 2022Reoviridae virus family members, such as mammalian orthoreovirus (reovirus), encounter a unique challenge during replication. To hide the dsRNA from host recognition,...
Reoviridae virus family members, such as mammalian orthoreovirus (reovirus), encounter a unique challenge during replication. To hide the dsRNA from host recognition, the genome remains encapsidated in transcriptionally active proteinaceous core capsids that transcribe and release +RNA. De novo +RNAs and core proteins must repeatedly assemble into new progeny cores in order to logarithmically amplify replication. Reoviruses also produce outercapsid (OC) proteins μ1, σ3 and σ1 that assemble onto cores to create highly stable infectious full virions. Current models of reovirus replication position amplification of transcriptionally-active cores and assembly of infectious virions in shared factories, but we hypothesized that since assembly of OC proteins would halt core amplification, OC assembly is somehow regulated. Kinetic analysis of virus +RNA production, core versus OC protein expression, and core particles versus whole virus particle accumulation, indicated that assembly of OC proteins onto core particles was temporally delayed. All viral RNAs and proteins were made simultaneously, eliminating the possibility that delayed OC RNAs or proteins account for delayed OC assembly. High resolution fluorescence and electron microscopy revealed that core amplification occurred early during infection at peripheral core-only factories, while all OC proteins associated with lipid droplets (LDs) that coalesced near the nucleus in a μ1-dependent manner. Core-only factories transitioned towards the nucleus despite cycloheximide-mediated halting of new protein expression, while new core-only factories developed in the periphery. As infection progressed, OC assembly occurred at LD-and nuclear-proximal factories. Silencing of OC μ1 expression with siRNAs led to large factories that remained further from the nucleus, implicating μ1 in the transition to perinuclear factories. Moreover, late during infection, +RNA pools largely contributed to the production of de-novo viral proteins and fully-assembled infectious viruses. Altogether the results suggest an advanced model of reovirus replication with spatiotemporal segregation of core amplification, OC complexes and fully assembled virions.
Topics: Animals; Capsid Proteins; Cell Line; Cycloheximide; Kinetics; Mammals; RNA, Viral; Reoviridae; Viral Proteins; Virus Assembly
PubMed: 36099325
DOI: 10.1371/journal.ppat.1010641 -
Viruses May 2022Aquareovirus, which is a member of the Reoviridae family, was isolated from aquatic animals. A close molecular evolutionary relationship between aquareoviruses and...
Aquareovirus, which is a member of the Reoviridae family, was isolated from aquatic animals. A close molecular evolutionary relationship between aquareoviruses and mammalian orthoreoviruses was revealed. However, the functions of the aquareovirus genome-encoded proteins are poorly understood. We investigated the molecular characteristics of the outer capsid proteins, namely, VP5 and VP7, of grass carp reovirus (GCRV). The peptides VP5 and VP7 were determined using in-gel tryptic digestion and mass spectrometry. Recovered peptides represented 76% and 66% of the full-length VP5 and VP7 sequences, respectively. Significantly, two-lysine acetylation, as well as two-serine and two-threonine phosphorylation modifications, were first revealed in VP5. We found that the initial amino acid in VP5 was Pro43, suggesting that a lower amount of VP5 remained uncleaved in virions at the autocleavage site (Asn42-Pro43). Further biochemical evidence showed that the cleaved VP5N/VP5C conformation was the major constituent of the particles. Moreover, early cleavage fragments of VP7 and enhanced infectivity were detected after limited tryptic digestion of GCRV, indicating that stepwise VP7 cleavage is essential for VP5 conformational rearrangement. Our results provide insights into the roles of posttranslational modifications in VP5 and its association with VP7 in the viral life cycle.
Topics: Animals; Antibodies, Viral; Capsid Proteins; Carps; Mammals; Orthoreovirus; Reoviridae; Virion
PubMed: 35632773
DOI: 10.3390/v14051032 -
Scientific Reports Apr 2021Bats are known to be reservoirs of several highly pathogenic viruses. Hence, the interest in bat virus discovery has been increasing rapidly over the last decade. So...
Bats are known to be reservoirs of several highly pathogenic viruses. Hence, the interest in bat virus discovery has been increasing rapidly over the last decade. So far, most studies have focused on a single type of virus detection method, either PCR, virus isolation or virome sequencing. Here we present a comprehensive approach in virus discovery, using all three discovery methods on samples from the same bats. By family-specific PCR screening we found sequences of paramyxoviruses, adenoviruses, herpesviruses and one coronavirus. By cell culture we isolated a novel bat adenovirus and bat orthoreovirus. Virome sequencing revealed viral sequences of ten different virus families and orders: three bat nairoviruses, three phenuiviruses, one orbivirus, one rotavirus, one orthoreovirus, one mononegavirus, five parvoviruses, seven picornaviruses, three retroviruses, one totivirus and two thymoviruses were discovered. Of all viruses identified by family-specific PCR in the original samples, none was found by metagenomic sequencing. Vice versa, none of the viruses found by the metagenomic virome approach was detected by family-specific PCRs targeting the same family. The discrepancy of detected viruses by different detection approaches suggests that a combined approach using different detection methods is necessary for virus discovery studies.
Topics: Animals; Chiroptera; Chlorocebus aethiops; Genome, Viral; Germany; High-Throughput Nucleotide Sequencing; Nairovirus; Orbivirus; Phylogeny; Polymerase Chain Reaction; Rotavirus; Vero Cells; Virome; Viruses
PubMed: 33795699
DOI: 10.1038/s41598-021-86435-4 -
Viruses Jun 2020Mammalian orthoreovirus (reovirus) is a dsRNA virus, which has long been used as a model system to study host-virus interactions. One of the earliest interactions during... (Review)
Review
Mammalian orthoreovirus (reovirus) is a dsRNA virus, which has long been used as a model system to study host-virus interactions. One of the earliest interactions during virus infection is the detection of the viral genomic material, and the consequent induction of an interferon (IFN) based antiviral response. Similar to the replication of related dsRNA viruses, the genomic material of reovirus is thought to remain protected by viral structural proteins throughout infection. Thus, how innate immune sensor proteins gain access to the viral genomic material, is incompletely understood. This review summarizes currently known information about the innate immune recognition of the reovirus genomic material. Using this information, we propose hypotheses about host detection of reovirus.
Topics: Genome, Viral; Host-Pathogen Interactions; Humans; Immunity, Innate; Interferons; RNA, Viral; Reoviridae
PubMed: 32575691
DOI: 10.3390/v12060667 -
Journal of Virological Methods Jun 2020Reassortment of segmented viruses can be an important source of genetic diversity underlying viral evolution and emergence. Methods for the quantification of...
Reassortment of segmented viruses can be an important source of genetic diversity underlying viral evolution and emergence. Methods for the quantification of reassortment have been described but are often cumbersome and best suited for the analysis of reassortment between highly divergent parental strains. While it is useful to understand the potential of divergent parents to reassort, outcomes of such heterologous reassortment are driven by differential selection acting on the progeny and are typically strain specific. To quantify reassortment robustly, a system free of differential selection is needed. We have generated such a system for influenza A virus and for mammalian orthoreovirus by constructing well-matched parental viruses carrying small genetic tags. The method utilizes high-resolution melt technology for the identification of reassortant viruses. Ease of sample preparation and data analysis enables streamlined genotyping of a large number of virus clones. The method described here thereby allows quantification of the efficiency of reassortment and can be applied to diverse segmented viruses.
Topics: Animals; Cell Line; Flow Cytometry; Genetic Markers; Genetic Variation; Genome, Viral; Genotype; High-Throughput Nucleotide Sequencing; Humans; Influenza A virus; Mutation; Orthoreovirus, Mammalian; Reassortant Viruses; Recombination, Genetic; Sequence Analysis, RNA
PubMed: 32353455
DOI: 10.1016/j.jviromet.2020.113878 -
The Journal of Veterinary Medical... Feb 2022Fowl adenoviruses (FAdVs) and avian reoviruses (ARVs) are ubiquitous in poultry farms and most of them are not pathogenic, yet often cause damage to chicks. A total of...
Fowl adenoviruses (FAdVs) and avian reoviruses (ARVs) are ubiquitous in poultry farms and most of them are not pathogenic, yet often cause damage to chicks. A total of 104 chicken fecal samples were collected from 7 farms of breeder chickens (layers and broilers) in Japan from 2019 to 2021, and yielded 26 FAdV plus 14 ARV isolates. By sequencing, FAdV isolates were classified as FAdV-1, 5 and 8b. ARV isolates were classified as genotype II, IV and V. These results suggest that FAdVs and ARVs are resident in the breeder chicken farms in Japan.
Topics: Adenoviridae Infections; Animals; Aviadenovirus; Chickens; Japan; Orthoreovirus, Avian; Phylogeny; Poultry Diseases
PubMed: 34980758
DOI: 10.1292/jvms.21-0616 -
PLoS Pathogens Mar 2022Cholesterol homeostasis is required for the replication of many viruses, including Ebola virus, hepatitis C virus, and human immunodeficiency virus-1. Niemann-Pick C1...
Cholesterol homeostasis is required for the replication of many viruses, including Ebola virus, hepatitis C virus, and human immunodeficiency virus-1. Niemann-Pick C1 (NPC1) is an endosomal-lysosomal membrane protein involved in cholesterol trafficking from late endosomes and lysosomes to the endoplasmic reticulum. We identified NPC1 in CRISPR and RNA interference screens as a putative host factor for infection by mammalian orthoreovirus (reovirus). Following internalization via clathrin-mediated endocytosis, the reovirus outer capsid is proteolytically removed, the endosomal membrane is disrupted, and the viral core is released into the cytoplasm where viral transcription, genome replication, and assembly take place. We found that reovirus infection is significantly impaired in cells lacking NPC1, but infection is restored by treatment of cells with hydroxypropyl-β-cyclodextrin, which binds and solubilizes cholesterol. Absence of NPC1 did not dampen infection by infectious subvirion particles, which are reovirus disassembly intermediates that bypass the endocytic pathway for infection of target cells. NPC1 is not required for reovirus attachment to the plasma membrane, internalization into cells, or uncoating within endosomes. Instead, NPC1 is required for delivery of transcriptionally active reovirus core particles from endosomes into the cytoplasm. These findings suggest that cholesterol homeostasis, ensured by NPC1 transport activity, is required for reovirus penetration into the cytoplasm, pointing to a new function for NPC1 and cholesterol homeostasis in viral infection.
Topics: Animals; Cholesterol; Endosomes; Homeostasis; Humans; Mammals; Niemann-Pick C1 Protein; Reoviridae; Reoviridae Infections
PubMed: 35263388
DOI: 10.1371/journal.ppat.1010322 -
Frontiers in Immunology 2022Piscine orthoreovirus (PRV) is a virus in the genus Orthoreovirus of the Reoviridae family, first described in 2010 associated with Heart and Skeletal Muscle... (Review)
Review
Piscine orthoreovirus (PRV) is a virus in the genus Orthoreovirus of the Reoviridae family, first described in 2010 associated with Heart and Skeletal Muscle Inflammation (HSMI) in Atlantic salmon (). Three phases of PRV infection have been described, the early entry and dissemination, the acute dissemination phase, and the persistence phase. Depending on the PRV genotype and the host, infection can last for life. Mechanisms of immune response to PRV infection have been just beginning to be studied and the knowledge in this matter is here revised. PRV induces a classical antiviral immune response in experimental infection of salmonid erythrocytes, including transcriptional upregulation of , , , and . In addition, transcript upregulation of has been observed in Atlantic salmon infected with PRV, indicating that PRV elicited a Th1 type response probably as a host defense strategy. The high expression levels of , , and in PRV-infected fish suggest a positive modulatory effect on the CTL-mediated immune response. This is consistent with PRV-dependent upregulation of the genes involved in antigen presentation, including MHC class I, transporters, and proteasome components. We also review the potential immune mechanisms associated with the persistence phenotype of PRV-infected fish and its consequence for the development of a secondary infection. In this scenario, the application of a vaccination strategy is an urgent and challenging task due to the emergence of this viral infection that threatens salmon farming.
Topics: Animals; Fish Diseases; Immunity; Orthoreovirus; Reoviridae Infections
PubMed: 35464421
DOI: 10.3389/fimmu.2022.768621 -
Microbiology Spectrum Aug 2023Nelson 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...
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 () 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. 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.
Topics: Animals; Humans; Arthropods; China; Genome, Viral; Orthoreovirus; Phylogeny
PubMed: 37306586
DOI: 10.1128/spectrum.05122-22 -
Emerging Microbes & Infections Dec 2021Mammalian orthoreovirus (MRV) infects multiple mammalian species including humans. A United States Midwest swine farm with approximately one thousand 3-month-old pigs...
Mammalian orthoreovirus (MRV) infects multiple mammalian species including humans. A United States Midwest swine farm with approximately one thousand 3-month-old pigs experienced an event, in which more than 300 pigs showed neurological signs, like "down and peddling", with approximately 40% mortality. A novel MRV was isolated from the diseased pigs. Sequence and phylogenetic analysis revealed that the isolate was a reassortant virus containing viral gene segments from three MRV serotypes that infect human, bovine and swine. The M2 and S1 segment of the isolate showed 94% and 92% nucleotide similarity to the M2 of the MRV2 D5/Jones and the S1 of the MRV1 C/bovine/Indiana/MRV00304/2014, respectively; the remaining eight segments displayed 93%-95% nucleotide similarity to those of the MRV3 FS-03/Porcine/USA/2014. Pig studies showed that both MRV-infected and native contact pigs displayed fever, diarrhoea and nasal discharge. MRV RNA was detected in different intestinal locations of both infected and contact pigs, indicating that the MRV isolate is pathogenic and transmissible in pigs. Seroconversion was also observed in experimentally infected pigs. A prevalence study on more than 180 swine serum samples collected from two states without disease revealed 40%-52% positive to MRV. All results warrant the necessity to monitor MRV epidemiology and reassortment as the MRV could be an important pathogen for the swine industry and a novel MRV might emerge to threaten animal and public health.
Topics: Animals; Cattle; Dogs; High-Throughput Nucleotide Sequencing; Humans; Madin Darby Canine Kidney Cells; Orthoreovirus, Mammalian; Phylogeny; RNA, Viral; Reassortant Viruses; Reoviridae Infections; Sequence Analysis, RNA; Swine; United States
PubMed: 34018466
DOI: 10.1080/22221751.2021.1933608