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Infection, Genetics and Evolution :... Dec 2015Mammalian reoviruses (MRVs) are associated with pulmonary infections and have been isolated from humans and various animals experiencing respiratory illness. We report...
Mammalian reoviruses (MRVs) are associated with pulmonary infections and have been isolated from humans and various animals experiencing respiratory illness. We report here the first case of an MRV detected in the masked palm civet, which showed the highest similarity to the serotype 3 MRV. Reovirus particles were identified by electron microscopic examination of both negative-stain and thin-section. Genomic pattern analysis on SDS-PAGE showed that MPC/04 had 10-segmented double-strand RNA genome. Intranasal infection of four-week-old female BALB/c mice resulted in fatal respiratory distress but not other routes. Infections caused tissue damage and inflammation. MPC/04 grew to higher titers in the lungs than in other tissues. This research strongly suggests a need for additional experimentation to understand the pathogenic mechanisms of mammalian orthoreoviruses in infected animals and humans.
Topics: Animals; Cats; Chlorocebus aethiops; Female; Genome, Viral; Mice; Orthoreovirus, Mammalian; Phylogeny; Reoviridae Infections; Sequence Analysis, DNA; Vero Cells; Viral Load
PubMed: 26325682
DOI: 10.1016/j.meegid.2015.08.037 -
Pharmaceutical Nanotechnology 2020A key challenge in the process of virus amplification is the need for a simple and convenient method for measuring virus titers. (Comparative Study)
Comparative Study
BACKGROUND
A key challenge in the process of virus amplification is the need for a simple and convenient method for measuring virus titers.
OBJECTIVE
Real-time unlabeled cell analysis (RTCA) was used to establish a standard curve of correlation between half-cell index time (CIT) and virus titer. At the same time, the virus titer from tunable resistance pulse detection (TRPS) technology was compared with the traditional median tissue culture infectious dose (TCID) method to evaluate the feasibility and application value of the RTCA technique and TRPS technology.
METHODS
Cell index (CI) values for L929 cells under different culture conditions were detected, and the appropriate initial cell inoculation density was screened. The half-cell index (CI) values of reovirus infected L929 cells with TCID titers were analyzed by RTCA, the CI-TCID standard curve was created, and a regression equation was developed. RTCA, TCID50, and TRPS methods were used to detect the reovirus titer obtained by the amplification, and the sensitivity and feasibility of the CIT-TCID standard curve method were analyzed. The virus titer was detected by TRPS technology and the TCID method.
RESULTS
L929 cells were best propagated at an initial density of 6 × 103 cells/well. After infecting L929 cells with different titers of reference reovirus, the linear correlation of CIT50 and TCID50 was y = -2.1806x + 71.023 (R2 = 0.9742). The titer resulting from the RTCA assay was 7×109.6821 pfu/mL, from the TRPS assay was 4.52×1010 pfu/mL, and from the TCID50 assay was 7×109.467 pfu/mL.
CONCLUSION
The CIT-TCID standard curve method established by the RTCA technique can be used to quantitatively detect reovirus titer with L929 cells. Compared with the TCID method, it takes a relatively short time and has high sensitivity and accuracy. The TRPS technology requires even less time to quantify the virus, but its precision is lower than that of the TCID method and RTCA technology. This study provides new technical methods for assessing the virulence of infectious live reovirus particles. Lay Summary: After amplification of the virus, we need to detect the virus titers (the virulence of the virus). The traditional method is to use the virus to infect cells, and then the virus titers can be calculated by 50% of the cells infected. However, this traditional method is time consuming. The ways of RTCA (a real-time cell analysis technique) and TRPS (a nano-bioparticle analysis technique) help us to detect viral titers. The consistency of these three methods determines their feasibility and accuracy. If they are feasible, then these two simple technologies will provide new ideas for detecting viral titers.
Topics: Animals; Cell Line; Cytopathogenic Effect, Viral; Fibroblasts; Mice; Orthoreovirus; Reproducibility of Results; Time Factors; Viral Load; Virulence; Virus Replication
PubMed: 32851967
DOI: 10.2174/2211738508666200826103322 -
Viruses Nov 2020Serpentoviruses are an emerging group of nidoviruses known to cause respiratory disease in snakes and have been associated with disease in other non-avian reptile...
Serpentoviruses are an emerging group of nidoviruses known to cause respiratory disease in snakes and have been associated with disease in other non-avian reptile species (lizards and turtles). This study describes multiple episodes of respiratory disease-associated mortalities in a collection of juvenile veiled chameleons (). Histopathologic lesions included rhinitis and interstitial pneumonia with epithelial proliferation and abundant mucus. Metagenomic sequencing detected coinfection with two novel serpentoviruses and a novel orthoreovirus. Veiled chameleon serpentoviruses are most closely related to serpentoviruses identified in snakes, lizards, and turtles (approximately 40-50% nucleotide and amino acid identity of ORF1b). Veiled chameleon orthoreovirus is most closely related to reptilian orthoreoviruses identified in snakes (approximately 80-90% nucleotide and amino acid identity of the RNA-dependent RNA polymerase). A high prevalence of serpentovirus infection (>80%) was found in clinically healthy subadult and adult veiled chameleons, suggesting the potential for chronic subclinical carriers. Juvenile veiled chameleons typically exhibited a more rapid progression compared to subadults and adults, indicating a possible age association with morbidity and mortality. This is the first description of a serpentovirus infection in any chameleon species. A causal relationship between serpentovirus infection and respiratory disease in chameleons is suspected. The significance of orthoreovirus coinfection remains unknown.
Topics: Animals; Animals, Zoo; Coinfection; Disease Outbreaks; Female; Lizards; Lung Diseases, Interstitial; Male; Metagenomics; Nidovirales; Orthoreovirus; Prevalence; Reoviridae Infections
PubMed: 33228135
DOI: 10.3390/v12111329 -
Virology Jun 2022Bats have recently been identified as potential reservoir hosts for mammalian orthoreoviruses (MRVs) throughout Europe and China. Here we present the first evolutionary...
Bats have recently been identified as potential reservoir hosts for mammalian orthoreoviruses (MRVs) throughout Europe and China. Here we present the first evolutionary and biological characterization of bat-borne MRVs in North America, including phylogenomic analysis, in vitro relative infectivity in bat and other mammalian cell cultures, host cell receptor specificity, and epifluorescence microscopy of viral factory formation. Through genetic and phylogenetic comparisons, we show that two divergent MRV serotype 2 (T2) strains - isolated from a silver-haired bat (Lasionycteris noctivagans) and a big brown bat (Eptesicus fuscus) from Pennsylvania, USA - provide an evolutionary link to an MRV strain (T2W) recovered from an 8-week-old infant who died in Winnipeg, Manitoba, Canada in 1997. Although these findings suggest North American bats may represent a previously unrecognized source for the cross-species transmission of MRVs to other animals, including humans, the ecology and epidemiology of MRVs in wildlife remain enigmatic.
Topics: Animals; Animals, Wild; Chiroptera; Host Specificity; Humans; Orthoreovirus, Mammalian; Phylogeny
PubMed: 35421704
DOI: 10.1016/j.virol.2022.03.012 -
Frontiers in Veterinary Science 2023Reovirus infections in reptiles are frequently detected and associated with various clinical diseases; yet, our knowledge about their genetic diversity and evolutionary...
Reovirus infections in reptiles are frequently detected and associated with various clinical diseases; yet, our knowledge about their genetic diversity and evolutionary relationships remains limited. In this study, we characterize at the genomic level five reptile origin orthoreovirus strains isolated from exotic snakes and lizards in Hungary and Germany. The genomic organization of the study strains was similar to that of the representative strains of reptile origin reoviruses belonging to species and . Additionally, all five study strains clustered with the bush viper origin reference strain, 47/02. The nucleotide sequence divergence among strains fell from 56.64 to 99.36%. Based on genome segment constellations two well separated groups were observed, which may represent two genetic lineages of reptilian orthoreoviruses we tentatively referred here as genogroups, classifying two squamata origin strains with available whole genome sequences into genogroup I (GGI) and four strains into genogroup II (GGII). The representative GGI and GGII strains are characterized by moderate-to-high nucleotide and amino acid similarities within genogroups (range, 69.45 to 99.36% and 74.64 to 100.00%), whereas lower nucleotide and amino acid similarities (range, 56.64 to 77.24% and 54.53 to 93.85%) and different structures of the bicistronic S1 segment were found between genogroups. Further studies are needed to explore the genomic diversity among reptilian reoviruses of squamata origin; this would be critical to establish a robust classification system for these viruses and to see if interaction among members of distinct lineages may result in viable progenies with novel genetic features.
PubMed: 36816198
DOI: 10.3389/fvets.2023.1058133 -
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 -
Journal of Virology Oct 2020Triple-negative breast cancer (TNBC) constitutes 10 to 15% of all breast cancer and is associated with worse prognosis than other subtypes of breast cancer. Current...
Triple-negative breast cancer (TNBC) constitutes 10 to 15% of all breast cancer and is associated with worse prognosis than other subtypes of breast cancer. Current therapies are limited to cytotoxic chemotherapy, radiation, and surgery, leaving a need for targeted therapeutics to improve outcomes for TNBC patients. Mammalian orthoreovirus (reovirus) is a nonenveloped, segmented, double-stranded RNA virus in the family. Reovirus preferentially kills transformed cells and is in clinical trials to assess its efficacy against several types of cancer. We previously engineered a reassortant reovirus, r2Reovirus, that infects TNBC cells more efficiently and induces cell death with faster kinetics than parental reoviruses. In this study, we sought to understand the mechanisms by which r2Reovirus induces cell death in TNBC cells. We show that r2Reovirus infection of TNBC cells of a mesenchymal stem-like (MSL) lineage downregulates the mitogen-activated protein kinase/extracellular signal-related kinase pathway and induces nonconventional cell death that is caspase-dependent but caspase 3-independent. Infection of different MSL lineage TNBC cells with r2Reovirus results in caspase 3-dependent cell death. We map the enhanced oncolytic properties of r2Reovirus in TNBC to epistatic interactions between the type 3 Dearing M2 gene segment and type 1 Lang genes. These findings suggest that the genetic composition of the host cell impacts the mechanism of reovirus-induced cell death in TNBC. Together, our data show that understanding host and virus determinants of cell death can identify novel properties and interactions between host and viral gene products that can be exploited for the development of improved viral oncolytics. TNBC is unresponsive to hormone therapies, leaving patients afflicted with this disease with limited treatment options. We previously engineered an oncolytic reovirus (r2Reovirus) with enhanced infective and cytotoxic properties in TNBC cells. However, how r2Reovirus promotes TNBC cell death is not known. In this study, we show that reassortant r2Reovirus can promote nonconventional caspase-dependent but caspase 3-independent cell death and that the mechanism of cell death depends on the genetic composition of the host cell. We also map the enhanced oncolytic properties of r2Reovirus in TNBC to interactions between a type 3 M2 gene segment and type 1 genes. Our data show that understanding the interplay between the host cell environment and the genetic composition of oncolytic viruses is crucial for the development of efficacious viral oncolytics.
Topics: Antineoplastic Agents; Apoptosis; Breast Neoplasms; Caspase 3; Cell Death; Cell Line; Cell Survival; Humans; Mitochondria; Oncolytic Virotherapy; Oncolytic Viruses; Orthoreovirus, Mammalian; Reoviridae; Viral Proteins
PubMed: 32847857
DOI: 10.1128/JVI.01613-20 -
Proceedings of the National Academy of... May 2023Mammalian orthoreoviruses (reoviruses) serve as potential triggers of celiac disease and have oncolytic properties, making these viruses potential cancer therapeutics....
Mammalian orthoreoviruses (reoviruses) serve as potential triggers of celiac disease and have oncolytic properties, making these viruses potential cancer therapeutics. Primary attachment of reovirus to host cells is mainly mediated by the trimeric viral protein, σ1, which engages cell-surface glycans, followed by high-affinity binding to junctional adhesion molecule-A (JAM-A). This multistep process is thought to be accompanied by major conformational changes in σ1, but direct evidence is lacking. By combining biophysical, molecular, and simulation approaches, we define how viral capsid protein mechanics influence virus-binding capacity and infectivity. Single-virus force spectroscopy experiments corroborated by in silico simulations show that GM2 increases the affinity of σ1 for JAM-A by providing a more stable contact interface. We demonstrate that conformational changes in σ1 that lead to an extended rigid conformation also significantly increase avidity for JAM-A. Although its associated lower flexibility impairs multivalent cell attachment, our findings suggest that diminished σ1 flexibility enhances infectivity, indicating that fine-tuning of σ1 conformational changes is required to successfully initiate infection. Understanding properties underlying the nanomechanics of viral attachment proteins offers perspectives in the development of antiviral drugs and improved oncolytic vectors.
Topics: Animals; Capsid Proteins; Reoviridae; Orthoreovirus; Viral Proteins; Virus Attachment; Antibodies, Viral; Mammals
PubMed: 37186838
DOI: 10.1073/pnas.2220741120 -
Viruses Dec 2022Grasshoppers can swarm in the millions and destroy crops over wide areas, posing a major economic threat to agriculture. A wide range of insect-related viruses has...
Grasshoppers can swarm in the millions and destroy crops over wide areas, posing a major economic threat to agriculture. A wide range of insect-related viruses has recently been reported in the metagenomics of grasshoppers. Here, we identified and isolated a novel reovirus from grasshoppers, named Acrididae reovirus (ARV). The complete genome of ARV was composed of nine dsRNA segments. Phylogenetic analysis revealed that ARV formed a monophyletic lineage with unclassified insect-associated reoviruses and was sufficiently distinct from known genera of . ARV could replicate in its host and result in host death. Lower-dose ARV infection affected ovary development and resulted in a significant reduction in fecundity. The identification and characterization of a novel pathogenic reovirus could potentially promote the development of new biological control agents.
Topics: Animals; Grasshoppers; Phylogeny; Reoviridae; Orthoreovirus; Reoviridae Infections
PubMed: 36560814
DOI: 10.3390/v14122810 -
Transboundary and Emerging Diseases Sep 2022A fusogenic virus was isolated from a flock of breeder Pekin ducks in 2019, Hungary. The affected flock experienced a marked decrease in egg production....
A fusogenic virus was isolated from a flock of breeder Pekin ducks in 2019, Hungary. The affected flock experienced a marked decrease in egg production. Histopathological lesions were seen in the oviduct and in the lungs of birds sent for diagnostic investigation. The fusogenic agent was characterized as an orthoreovirus by viral metagenomics. The assembled viral genome was composed of 10 genomic segments and was 23,433 nucleotides (nt) in length. The study strain, designated Reo/HUN/DuckDV/2019, shared low-to-medium gene-wise sequence identity with avian orthoreovirus strains from galliform and anseriform birds (nt, 38.90%-72.33%) as well as with representative strains of neoavian orthoreoviruses (nt, 40.07%-68.23%). On the contrary, the study strain shared 86.48%-95.01% pairwise nt sequence identities with recent German and Chinese reovirus isolates, D2533/6 and Ych, respectively. Phylogenetic analysis clustered all three unusual waterfowl pathogens on a monophyletic branch, indicating a common evolutionary origin of Reo/HUN/DuckDV/2019 with these enigmatic orthoreoviruses described over the past few years. The finding that a candidate new orthoreovirus species, tentatively called Avian orthoreovirus B, was isolated in recent years in Europe and Asia in moribund ducks seems an alarming sign that needs to be better evaluated by extending laboratory diagnosis of viral pathogens in countries where the waterfowl industry is important.
Topics: Animals; Birds; Ducks; Genome, Viral; Nucleotides; Orthoreovirus; Orthoreovirus, Avian; Phylogeny; Reoviridae Infections; Sequence Analysis, DNA
PubMed: 35810357
DOI: 10.1111/tbed.14654