Host-microbiota interactions and responses to grass carp reovirus infection in Ctenopharyngodon idellus.

Gut microbiota could facilitate host to defense diseases, but fish-microbiota interactions during viral infection and the underlying mechanism are poorly understood. We examined interactions and responses of gut microbiota to grass carp reovirus (GCRV) infection in Ctenopharyngodon idellus, which is the most important aquaculture fish worldwide. We found that GCRV infection group with serious haemorrhagic symptoms (G7s) showed considerably different gut microbiota, especially with an abnormally high abundance of gram-negative anaerobic Cetobacterium somerae. It also showed the lowest (p < 0.05) alpha-diversity but with much higher ecological process of homogenizing dispersal (28.8%), confirming a dysbiosis of the gut microbiota after viral infection. Interestingly, signaling pathways of NOD-like receptors (NLRs), toll-like receptors (TLRs), and lipopolysaccharide (LPS) stimulation genes were significantly (q-value < 0.01) enriched in G7s, which also significantly (p < 0.01) correlated with the core gut microbial genera of Cetobacterium and Acinetobacter. The results suggested that an expansion of C. somerae initiated by GCRV could aggravate host inflammatory reactions through the LPS-related NLRs and TLRs pathways. This study advances our understanding of the interplay between fish immunity and gut microbiota challenged by viruses; it also sheds new insights for ecological defense of fish diseases with the help of gut microbiota.

First identification of mammalian orthoreovirus type 3 by gut virome analysis in diarrheic child in Brazil.

Diarrhea remains one of the most common causes of deaths in children. Although many studies have investigated the prevalence of enteric pathogens around the globe some diarrheal episodes remain unexplained. It is possible that some yet-unidentified viral agents could be related to these cases of gastroenteritis. By using viral metagenomics techniques, we screened 251 fecal samples of children between 0.5 to 2.5-year-old with acute diarrhea not associated with common pathogens. These children live in rural areas and have different levels of contact with animals such as pigs, cows and bats. Here we report a complete genome of one mammalian orthoreovirus (MRV) type 3, denoted TO-151/BR, detected in a female child in the state of Tocantins (north of Brazil). Brazilian TO-151/BR strain was classified as MRV-3 based on S1 phylogeny and was closely related to porcine Asian strains. Phylogenetic analyses showed that other segments were more similar to MRV-3s of different geographic locations and hosts, including human and bats, highlighting genome reassortment and lack of host-specific barriers. This is the first report of MRV-3 in South America and a hypothesis of a silent long-term circulation of this virus in Brazil has been raised.

Host range of mammalian orthoreovirus type 3 widening to alpine chamois.

Mammalian orthoreoviruses (MRV) type 3 have been recently identified in human and several animal hosts, highlighting the apparent lack of species barriers. Here we report the identification and genetic characterization of MRVs strains in alpine chamois, one of the most abundant wild ungulate in the Alps. Serological survey was also performed by MRV neutralization test in chamois population during five consecutive years (2008-2012). Three novel MRVs were isolated on cell culture from chamois lung tissues. No respiratory or other clinical symptoms neither lung macroscopic lesions were observed in the chamois population. MRV strains were classified as MRV-3 within the lineage III, based on S1 phylogeny, and were closely related to Italian strains identified in dog, bat and diarrheic pig. The full genome sequence was obtained by next-generation sequencing and phylogenetic analyses showed that other segments were more similar to MRVs of different geographic locations, serotypes and hosts, including human, highlighting genome reassortment and lack of host specific barriers. By using serum neutralization test, a high prevalence of MRV-3 antibodies was observed in chamois population throughout the monitored period, showing an endemic level of infection and suggesting a self-maintenance of MRV and/or a continuous spill-over of infection from other animal species.

Efficiency of Reovirus Concentration from Water with Positively Charged Filters.

This study examined the efficacy of reovirus concentration from large volumes of water using two positively charged filters: Zeta Plus 1MDS and NanoCeram. The results indicated that an average of 61 and 81% of input reoviruses were effectively recovered, respectively, from recycled water and tap water using NanoCeram filtration.

First identification of mammalian orthoreovirus type 3 in diarrheic pigs in Europe.

Mammalian Orthoreoviruses 3 (MRV3) have been described in diarrheic pigs from USA and Asia. We firstly detected MRV3 in Europe (Italy) in piglets showing severe diarrhea associated with Porcine Epidemic Diarrhea. The virus was phylogenetically related to European reoviruses of human and bat origin and to US and Chinese pig MRV3.

A novel pathogenic Mammalian orthoreovirus from diarrheic pigs and Swine blood meal in the United States.

UNLABELLED: Since May 2013, outbreaks of porcine epidemic diarrhea have devastated the U.S. swine industry, causing immense economic losses. Two different swine enteric coronaviruses (porcine epidemic diarrhea virus and Delta coronavirus) have been isolated from the affected swine population. The disease has been reported from at least 32 states of the United States and other countries, including Mexico, Peru, Dominican Republic, Canada, Columbia, Ecuador, and Ukraine, with repeated outbreaks in previously infected herds. Here we report the isolation and characterization of a novel mammalian orthoreovirus 3 (MRV3) from diarrheic feces of piglets from these outbreaks in three states and ring-dried swine blood meal from multiple sources. MRV3 could not be isolated from healthy or pigs that had recovered from epidemic diarrhea from four states. Several MRV3 isolates were obtained from chloroform-extracted pig feces or blood meal in cell cultures or developing chicken embryos. Biological characterization of two representative isolates revealed trypsin resistance and thermostability at 90°C. NextGen sequencing of ultrapurified viruses indicated a strong homology of the S1 segment to mammalian and bat MRV3. Neonatal piglets experimentally infected with these viruses or a chloroform extract of swine blood meal developed severe diarrhea and acute gastroenteritis with 100% mortality within 3 days postinfection. Therefore, the novel porcine MRV3 may contribute to enteric disease along with other swine enteric viruses. The role of MRV3 in the current outbreaks of porcine epidemic diarrhea in the United States remains to be determined, but the pathogenic nature of the virus warrants further investigations on its epidemiology and prevalence. IMPORTANCE: Porcine orthoreoviruses causing diarrhea have been reported in China and Korea but not in the United States. We have isolated and characterized two pathogenic reassortant MRV3 isolates from swine fecal samples from porcine epidemic diarrhea outbreaks and ring-dried swine blood meal in the United States. These fecal and blood meal isolates or a chloroform extract of blood meal induced severe diarrhea and mortality in experimentally infected neonatal pigs. Genetic and phylogenetic analyses of two MRV3 isolates revealed that they are identical but differed significantly from nonpathogenic mammalian orthoreoviruses circulating in the United States. The present study provides a platform for immediate development of suitable vaccines and diagnostics to prevent and control porcine orthoreovirus diarrhea.

Utilization of sialylated glycans as coreceptors enhances the neurovirulence of serotype 3 reovirus.

Mammalian reoviruses display serotype-specific patterns of tropism and disease in the murine central nervous system (CNS) attributable to polymorphisms in viral attachment protein σ1. While all reovirus serotypes use junctional adhesion molecule-A as a cellular receptor, they differ in their utilization of carbohydrate coreceptors. This observation raises the possibility that carbohydrate binding by σ1 influences reovirus pathology in the CNS. In this study, we sought to define the function of carbohydrate binding in reovirus neuropathogenesis. Newborn mice were inoculated intramuscularly with wild-type strain type 3 Dearing (T3D) and T3D-σ1R202W, a point mutant T3D derivative that does not bind sialic acid (SA). Infected mice were monitored for survival, and viral loads at the sites of primary and secondary replication were quantified. Fewer mice inoculated with the wild-type virus survived in comparison to those inoculated with the mutant virus. The wild-type virus also produced higher titers in the spinal cord and brain at late times postinoculation but lower titers in the liver in comparison to those produced by the mutant virus. In addition, the wild-type virus was more virulent and produced higher titers in the brain than the mutant following intracranial inoculation. These animal infectivity studies suggest that T3D-σ1R202W harbors a defect in neural growth. Concordantly, compared with the wild-type virus, the mutant virus displayed a decreased capacity to infect and replicate in primary cultures of cortical neurons, a property dependent on cell surface SA. These results suggest that SA binding enhances the kinetics of reovirus replication in neural tissues and highlight a functional role for sialylated glycans as reovirus coreceptors in the CNS.

Incidence of hepatotropic viruses in biliary atresia.

Biliary atresia (BA) is the most frequent indication for paediatric liver transplantation. We tested the hypothesis of a viral aetiology of this disease by screening liver samples of a large number of BA patients for the common human hepatotropic viruses. Moreover, we correlated our findings to the expression of Mx protein, which has been shown to be significantly up-regulated during viral infections. Seventy-four liver biopsies (taken during Kasai portoenterostomy) were tested by polymerase chain reaction (PCR) for DNA viruses (herpes simplex virus [HSV], Epstein-Barr virus [EBV], varicella zoster virus [VZV], cytomegalovirus [CMV], adenovirus, parvovirus B19 and polyoma BK) and RNA viruses (enteroviruses, rotavirus and reovirus 3). Mx protein expression was assessed by immunohistochemistry. Virus DNA/RNA was found in less than half of the biopsies (8/74 CMV, 1/74 adenovirus; 21/64 reovirus, 1/64 enterovirus). A limited number presented with double infection. Patients that had detectable viral RNA/DNA in their liver biopsies were significantly older than virus-free patients (P = 0.037). The majority (54/59) of the liver biopsies showed expression of Mx proteins in hepatocytes, bile ducts and epithelium. Our data suggest that the known hepatotropic viruses do not play a major role in the aetiology and progression of BA. Their incidence appears to be, rather, a secondary phenomenon. Nonetheless, the inflammatory response in the livers of BA patients mimics that observed during viral infections.

Rapid and reliable quantification of reovirus type 3 by high performance liquid chromatography during manufacturing of Reolysin.

Reolysin, a human reovirus type 3, is being evaluated in the clinic as an oncolytic therapy for various types of cancer. To facilitate the optimization and scale-up of the current process, a high performance liquid chromatography (HPLC) method has been developed that is rapid, specific and reliable for the quantification of reovirus type 3 particles. Using an anion-exchange column, the intact virus eluted from the contaminants in 9.78 min at 350 mM NaCl in 50mM HEPES, pH 7.10 in a total analysis time of 25 min. The virus demonstrated a homogenous peak with no co-elution of other compounds as analyzed by photodiode array analysis. The HPLC method facilitated the optimization of the purification process which resulted in the improvement of both total and infectious particle recovery and contributed to the successful scale-up of the process at the 20 L, 40 L and 100 L production scale. The method is suitable for the analysis of crude virus supernatants, crude lysates, semi-purified and purified preparations and therefore is an ideal monitoring tool during process development and scale-up.

Validation of a high-performance liquid chromatographic assay for the quantification of Reovirus particles type 3.

An anion exchange high-performance liquid chromatography (HPLC) method for the quantification of human Reovirus type 3 particles was validated according to the performance criteria of precision, specificity, linearity of calibration and working range, limits of detection and quantification, accuracy and recovery. Samples taken at various stages of Reovirus purification were used for the validation of the method. The method was specific for Reovirus which eluted around 9.8min without interference from any other component in the sample. Reovirus can be detected between 0.32E+12 and 2.10E12VP/mL by the proposed method that has the correlation coefficient of linearity equal to 0.9974 and the slope of linearity equal to 5.74E-07 area units/(VPmL).

Efficacy and safety evaluation of human reovirus type 3 in immunocompetent animals: racine and nonhuman primates.

PURPOSE: Human reovirus type 3 has been proposed to kill cancer cells with an activated Ras signaling pathway. The purpose of this study was to investigate the efficacy of reovirus in immunocompetent glioma animal models and safety/toxicity in immunocompetent animals, including nonhuman primates. EXPERIMENTAL DESIGN: Racine glioma cells 9L and RG2 were implanted s.c. or intracranially in Fisher 344 rats with or without reovirus antibodies, followed by treatment of reovirus. To study whether reovirus kills contralateral tumors in the brain and to determine viral distribution, we established an in situ dual tumor model followed by reovirus intratumoral inoculation only into the ipsilateral tumor. To evaluate neurotoxicity/safety of reovirus, Cynomolgus monkeys and immunocompetent rats were given intracranially with reovirus, and pathological examination and/or behavioral studies were done. Viral shedding and clinical biochemistry were systematically studied in monkeys. RESULTS: Intratumorally given reovirus significantly suppressed the growth of both s.c. and intracranially tumors and significantly prolonged survival. The presence of reovirus-neutralizing antibodies did not abort the reovirus' antitumor effect. Reovirus inhibited glioma growth intracranially in the ipsilateral but not the contralateral tumors; viral load in ipsilateral tumors was 15 to 330-fold higher than the contralateral tumors. No encephalitis or behavioral abnormalities were found in monkeys and rats given reovirus intracranially. No treatment-related clinical biochemistry changes or diffuse histopathological abnormality were found in monkeys inoculated intracranially with Good Manufacturing Practice prepared reovirus. Microscopic changes were confined to the region of viral inoculation and were dose related, suggesting reovirus intracranially was well tolerated in nonhuman primates. CONCLUSIONS: These data show the efficacy and safety of reovirus when it is used in the treatment of gliomas in immunocompetent hosts. Inoculation of reovirus into the brain of nonhuman primates did not produce significant toxicities.

Isolation and molecular characterization of a novel type 3 reovirus from a child with meningitis.

Mammalian reoviruses are non-enveloped viruses that contain a segmented, double-stranded RNA genome. Reoviruses infect most mammalian species, although infection with these viruses in humans is usually asymptomatic. We report the isolation of a novel reovirus strain from a 6.5-week-old child with meningitis. Hemagglutination and neutralization assays indicated that the isolate is a serotype 3 strain, leading to the designation T3/Human/Colorado/1996 (T3C/96). Sequence analysis of the T3C/96 S1 gene segment, which encodes the viral attachment protein, sigma 1, confirmed the serotype assignment for this strain and indicated that T3C/96 is a novel reovirus isolate. T3C/96 is capable of systemic spread in newborn mice after peroral inoculation and produces lethal encephalitis. These results suggest that serotype 3 reoviruses can cause meningitis in humans.

Optimization of reovirus production from mouse L-929 cells in suspension culture.

Reovirus serotype 3 Dearing (T3D) has shown potential as a novel cancer therapy. To support the increasing demand for reovirus, a two-stage perfusion mode scheme is proposed for cell growth and reovirus production. Mouse L-929 cells were used as the host for reovirus infection due to their ability to grow well in suspension culture. Several L-929 cell growth and reovirus infection characteristics were investigated and optimized in spinner flask batch cultures. For the growth of L-929 cells, a balanced nutrient-fortification of SMEM medium increased the maximum cell density by 30%, compared to normal SMEM; however, ammonia and lactate accumulations were found to inhibit further cell growth. For the production of reovirus, approximately 90% increase in viral yield resulted when the infection temperature was reduced from 37 to 33 degrees C. Infectious reovirus particles were shown to be stable in conditioned medium at 37 and 33 degrees C. The final virus titer was dependent on the multiplicity of infection (MOI) and the host cell density at the time of infection. A combination of an MOI of 0.1 pfu/cell and an initial host cell density of 1.0 x 10(6) cells/mL in fortified medium resulted in a maximum virus titer of (4.59 +/- 0.16) x 10(9) pfu/mL and a specific yield of (2.34 +/- 0.08) x 10(3) pfu/cell. At an optimal harvest time of the infection process, 99% of the virus was associated with the cellular debris. Finally, the presence of 5.0 mM ammonia in the culture medium was shown to seriously inhibit the reovirus yield, whereas lactate concentrations up to 20 mM had no effect.

Detection of Reovirus type 3 by use of fluorogenic nuclease reverse transcriptase polymerase chain reaction.

Reovirus type 3 (Reo-3) can infect numerous rodent species and induces the clinical syndrome 'oily skin disease' in neonatal mice, and is a common contaminant of biological materials. The reverse transcriptase polymerase chain reaction (RT-PCR) assay has proven useful for the detection of Reo-3 in rodents and contaminated biological materials. Fluorogenic nuclease reverse transcriptase polymerase chain reaction assays (fnRT-PCR) combine RT-PCR with an internal fluorogenic hybridization probe, thereby potentially enhancing specificity and eliminating post-PCR processing. Therefore, an fnRT-PCR assay specific for Reo-3 was developed by targeting primer and probe sequences to a unique region of the Reo-3 M3 gene. The fnRT-PCR detected both strains of Reo-3 (Dearing and Abney), but did not detect Reovirus types 1 or 2, other viruses in the family Reoviridae, or other RNA viruses that naturally infect rodents. The fnRT-PCR detected less than 1 fg of target template and detected viral RNA in tissues obtained from mice experimentally infected with Reo-3. The assay also displayed comparable sensitivity when compared to the mouse antibody production test commonly used to detect viral contamination of biological materials. In conclusion, this fnRT-PCR assay offers a potentially high-throughput diagnostic assay for detecting Reo-3 RNA in infected mice and contaminated biological materials.

Reovirus as an oncolytic agent against experimental human malignant gliomas.

BACKGROUND: Reovirus is a naturally occurring oncolytic virus that usurps activated Ras-signaling pathways of tumor cells for its replication. Ras pathways are activated in most malignant gliomas via upstream signaling by receptor tyrosine kinases. The purpose of this study was to determine the effectiveness of reovirus as an experimental treatment for malignant gliomas. METHODS: We investigated whether reovirus would infect and lyse human glioma cell lines in vitro. We also tested the effect of injecting live reovirus in vivo on human gliomas grown subcutaneously or orthotopically (i.e., intracerebrally) in mice. Finally, reovirus was tested ex vivo against low-passage cell lines derived from human glioma specimens. All P values were two-sided. RESULTS: Reovirus killed 20 (83%) of 24 established malignant glioma cell lines tested. It caused a dramatic and often complete tumor regression in vivo in two subcutaneous (P =.0002 for both U251N and U87) and in two intracerebral (P =.0004 for U251N and P =.0009 for U87) human malignant glioma mouse models. As expected, serious toxic effects were found in these severely immunocompromised hosts. In a less immunocompromised mouse model, a single intratumoral inoculation of live reovirus led to a dramatic prolongation of survival (compared with control mice treated with dead virus; log-rank test, P<.0001 for both U251N and U87 cell lines). The animals treated with live virus also appeared to be healthier and gained body weight (P =.0001). We then tested the ability of reovirus to infect and kill primary cultures of brain tumors removed from patients and found that it killed nine (100%) of nine glioma specimens but none of the cultured meningiomas. CONCLUSIONS: Reovirus has potent activity against human malignant gliomas in vitro, in vivo, and ex vivo. Oncolysis with reovirus may be a potentially useful treatment for a broad range of human cancers.

Detection and identification of mammalian reoviruses in surface water by combined cell culture and reverse transcription-PCR.

Reoviruses are a common class of enteric viruses capable of infecting a broad range of mammalian species, typically with low pathogenicity. Previous studies have shown that reoviruses are common in raw water sources and are often found along with other animal viruses. This suggests that in addition to the commonly monitored enteroviruses, reoviruses might serve as an informative target for monitoring fecal contamination of drinking water sources. Mammalian reoviruses were detected and identified by a combined cell culture-reverse transcription-PCR (RT-PCR) assay with novel primers targeting the L3 gene that encodes the lambda3 major core protein. Five of 26 (19.2%) cytopathic effect-positive cell culture lysates inoculated with surface water were positive for reoviruses by RT-PCR. DNA sequence analysis of RT-PCR products revealed significant sequence diversity among isolates, which is consistent with the sequence diversity among previously characterized mammalian reoviruses. Sequence analysis revealed persistence of a reovirus genotype at a single sampling site, while a sample from another site contained two different reovirus genotypes.

A comparative analysis of freon substitutes in the purification of reovirus and calicivirus.

Freon 113 (Freon) is an essential component used in some viral purification methods to separate virus from infected cell debris. With its environmental and toxic hazards, Freon's availability is limited and more tightly regulated. Several organic solvent substitutes were selected to identify a suitable Freon replacement for the purification of both cultivable reovirus and fastidious calicivirus. Reovirus was extracted from tissue cultured cells with each solvent tested and purified in cesium chloride gradients by standard techniques. Purified virions were analyzed for conservation of physical and biological properties by morphological examination and infectivity studies. The purification of calicivirus nucleic acid from stool samples using selected solvents was also examined. Solvent-extracted calicivirus RNA was reverse transcribed and quantified by polymerase chain reaction amplification of a standard diagnostic 117 bp amplicon. These studies indicated that Vertrel XF (a newly developed environmentally friendly Freon substitute) and a 7:3 mixture of isopentane/1-chlorobutane are suitable replacements. Considerations of flammability and ease of use suggest that Vertrel XF is the preferred choice as a Freon substitute for the purification of these non-enveloped viruses.

Linkage between reovirus-induced apoptosis and inhibition of cellular DNA synthesis: role of the S1 and M2 genes.

The mammalian reoviruses are capable of inhibiting cellular DNA synthesis and inducing apoptosis. Reovirus strains type 3 Abney (T3A) and type 3 Dearing (T3D) inhibit cellular DNA synthesis and induce apoptosis to a substantially greater extent than strain type 1 Lang (T1L). We used T1L x T3A and T1L x T3D reassortant viruses to identify viral genes associated with differences in the capacities of reovirus strains to elicit these cellular responses to viral infection. We found that the S1 and M2 genome segments determine differences in the capacities of both T1L x T3A and T1L x T3D reassortant viruses to inhibit cellular DNA synthesis and to induce apoptosis. These genes encode viral outer-capsid proteins that play important roles in viral attachment and disassembly. To extend these findings, we used field isolate strains of reovirus to determine whether the strain-specific differences in inhibition of cellular DNA synthesis and induction of apoptosis are also associated with viral serotype, a property determined by the S1 gene. In these experiments, type 3 field isolate strains were found to inhibit cellular DNA synthesis and to induce apoptosis to a greater extent than type 1 field isolate strains. Statistical analysis of these data indicate a significant correlation between the capacity of T1L x T3A and T1L x T3D reassortant viruses and field isolate strains to inhibit cellular DNA synthesis and to induce apoptosis. These findings suggest that reovirus-induced inhibition of cellular DNA synthesis and induction of apoptosis are linked and that both phenomena are induced by early steps in the viral replication cycle.

Circulating immunoglobulin G can play a critical role in clearance of intestinal reovirus infection.

Reoviruses are encapsidated double-stranded RNA viruses that cause systemic disease in mice after peroral (p.o.) inoculation and primary replication in the intestine. In this study, we define components of the immune system involved in the clearing of reovirus from the proximal small intestine. The intestines of immunocompetent adult CB17, 129, and C57BL/6 mice were cleared of reovirus serotype 3 clone 9 (T3C9) within 7 days of p.o. inoculation. Antigen-specific lymphocytes were important for the clearance of intestinal infection, since severe combined immunodeficient (SCID) mice failed to clear T3C9 infection. To define specific immune components required for intestinal clearance, reovirus infection of mice with null mutations in the immunoglobulin M (IgM) transmembrane exon (MuMT; B cell and antibody deficient) or beta 2 microglobulin gene (beta 2-/-; CD8 deficient) was evaluated. beta 2-/- mice cleared reovirus infection with normal kinetics, while MuMT mice showed delayed clearance of T3C9 7 to 11 days after p.o. inoculation. Adoptive transfer of splenic lymphocytes from reovirus-immune CB17 mice inhibited growth of T3C9 in CB17 SCID mouse intestine 11 days after p.o. inoculation. The efficiency of viral clearance by adoptively transferred cells was significantly diminished by depletion of B cells prior to adoptive transfer. Results in SCID and MuMT mice demonstrate an important role for B cells or IgG in clearance of reovirus from the intestines. Polyclonal reovirus-immune rabbit serum, protein A-purified immune IgG, and murine monoclonal IgG2a antibody specific for reovirus outer capsid protein sigma 3 administered intraperitoneally all normalized clearance of reovirus from intestinal tissue in MuMT mice. This result demonstrates an IgA-independent role for IgG in the clearance of intestinal virus infection. Polyclonal reovirus-immune serum also significantly decreased reovirus titers in the intestines of SCID mice, demonstrating a T-cell-independent role for antibody in the clearance of intestinal reovirus infection. B cells and circulating IgG play an important role in the clearance of reovirus from intestines, suggesting that IgG may play a more prominent functional role at mucosal sites of primary viral replication than was previously supposed.