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.

Detection of mammalian orthoreovirus type-3 (Reo-3) infections in mice based on serotype-specific hemagglutination protein sigma-1.

BACKGROUND: Reovirus type-3 infections cause severe pathologies in young mice and thus influence animal experiments in many ways. Therefore, the Federation of Laboratory Animal Science Associations (FELASA) recommends an annual screening in laboratory mice as part of a thorough health monitoring program. Based on the high protein sequence homology among the different reovirus serotypes, immunofluorescence antibody assay and other indirect methods relying on the whole virus are presumably cross-reactive to antibodies triggered by mammalian orthoreovirus infections independent of the serotype. METHODS: The serotype-specific protein σ-1 was expressed in Escherichia coli with an N-terminal Strep-tag and a C-terminal His-tag. The purified Strep-rσ-1-His-construct was used to develop an indirect ELISA by testing defined positive and negative sera obtained by experimental infection of mice as well as field sera. RESULTS: The Strep-rσ-1-His-ELISA provided high sensitivity and specificity during validation. Notably, a high selectivity was also observed for sera positively tested for other relevant FELASA-listed pathogens. Screening of field samples indicated that a commercial reovirus type-3-based ELISA might be cross-reactive to other murine reovirus serotypes and thus produces false-positive results. CONCLUSIONS: The prevalence of reovirus type-3 might be overestimated in German animal facilities and most likely in other countries as well. The occurrence of other reovirus serotypes, however, raises the question if murine health monitoring programs should be extended to these pathogens.

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 natural reassortant and mutant serotype 3 reovirus from mink in China.

Mammalian orthoreoviruses (MRVs) are widespread and infect virtually all mammals. We report here the first case of a natural mutant and reassortant serotype 3 reovirus from mink in China, known as MRV3 SD-14. Whole-genome sequence analysis showed that the MRV3 SD-14 may have resulted from a reassortment involving MRVs that infected swine, humans and mink. Interestingly, the S1 segment, which encodes the viral attachment protein σ1, which influences viral virulence and cell tropism in the host, had a stop codon mutation at amino acid 246. Surveillance of the virulence and evolution of MRVs in humans and other animals deserves more attention.

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.

Serotype-specific differences in inhibition of reovirus infectivity by human-milk glycans are determined by viral attachment protein σ1.

Human milk contains many bioactive components, including secretory IgA, oligosaccharides, and milk-associated proteins. We assessed the antiviral effects of several components of milk against mammalian reoviruses. We found that glucocerebroside (GCB) inhibited the infectivity of reovirus strain type 1 Lang (T1L), whereas gangliosides GD3 and GM3 and 3'-sialyllactose (3SL) inhibited the infectivity of reovirus strain type 3 Dearing (T3D). Agglutination of erythrocytes mediated by T1L and T3D was inhibited by GD3, GM3, and bovine lactoferrin. Additionally, α-sialic acid, 3SL, 6'-sialyllactose, sialic acid, human lactoferrin, osteopontin, and α-lactalbumin inhibited hemagglutination mediated by T3D. Using single-gene reassortant viruses, we found that serotype-specific differences segregate with the gene encoding the viral attachment protein. Furthermore, GD3, GM3, and 3SL inhibit T3D infectivity by blocking binding to host cells, whereas GCB inhibits T1L infectivity post-attachment. These results enhance an understanding of reovirus cell attachment and define a mechanism for the antimicrobial activity of human milk.

Features of the mammalian orthoreovirus 3 Dearing l1 single-stranded RNA that direct packaging and serotype restriction.

A series of recombinant mammalian orthoreoviruses (mammalian orthoreovirus 3 Dearing, MRV-3De) were generated that express an MRV-3De lambda3-CAT fusion protein. Individual viruses contain L1CAT double-stranded (ds) RNAs that range in length from a minimum of 1020 bp to 4616 bp. The engineered dsRNAs were generated from in vitro-transcribed single-stranded (ss) RNAs and incorporated into infectious virus particles by using reverse genetics. In addition to defining the sequences required for these ssRNAs to be 'identified' as l1 ssRNAs, the individual nucleotides in these regions that 'mark' each ssRNA as originating from mammalian orthoreovirus 1 Lang (MRV-1La), mammalian orthoreovirus 2 D5/Jones (MRV-2Jo) or MRV-3De have been identified. A C at position 81 in the MRV-1La 5' 129 nt sequence was able to be replaced with a U, as normally present in MRV-3De; this toggled the activity of the MRV-1La ssRNA to that of an MRV-3De 5' l1. RNA secondary-structure predictions for the 5' 129 nt of both the biologically active MRV-3De l1 ssRNA and the U81-MRV-3De-restored MRV-1La 5' ssRNA predicted a common structure.

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.

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.

Identification of signals required for the insertion of heterologous genome segments into the reovirus genome.

In cells simultaneously infected with any two of the three reovirus serotypes ST1, ST2, and ST3, up to 15% of the yields are intertypic reassortants that contain all possible combinations of parental genome segments. We have now found that not all genome segments in reassortants are wild type. In reassortants that possess more ST1 than ST3 genome segments, all ST1 genome segments appear to be wild type, but the incoming ST3 genome segments possess mutations that make them more similar to the ST1 genome segments that they replace. In reassortants resulting from crosses of the more distantly related ST3 and ST2 viruses that possess a majority of ST3 genome segments, all incoming ST2 genome segments are wild type, but the ST3 S4 genome segment possesses two mutations, G74 to A and G624 to A, that function as acceptance signals. Recognition of these signals has far-reaching implications for the construction of reoviruses with novel properties and functions.

A sigma 1 region important for hemagglutination by serotype 3 reovirus strains.

Hemagglutination (HA) by the mammalian reoviruses is mediated by interactions between the viral sigma 1 protein and sialoglycoproteins on the erythrocyte surface. Three serotype 3 (T3) reovirus strains were identified that do not agglutinate either bovine or type O human erythrocytes (HA negative): T3 clone 43 (T3C43), T3 clone 44 (T3C44), and T3 clone 84 (T3C84). These three strains also showed a diminished capacity to bind the major erythrocyte sialoglycoprotein, glycophorin, in an enzyme-linked immunosorbent assay. To determine the molecular basis for these findings, we examined the deduced sigma 1 amino acid sequences of the three HA-negative T3 strains and four HA-positive T3 strains. The limited number of sequence differences in the sigma 1 proteins of these seven strains allowed us to identify single unique amino acid residues in each of the HA-negative strains (aspartate 198 in T3C43, leucine 204 in T3C44, and tryptophan 202 in T3C84) that cluster within a discrete region of the sigma 1 tail. The identification of sigma 1 residues important for HA and glycophorin binding suggests that tail-forming sequences are exposed on the virion surface, where they interact with carbohydrate residues on the surface of cells.

Isolation and characteristics of an equine reovirus type 3 and an antibody prevalence survey to reoviruses in horses located in New York State.

Reoviruses have been isolated from a number of species including human, bovine, feline, canine and equine. In most species they seem to produce mild to inapparent disease. We have isolated a reovirus type 3 from a foal with diarrhea. The virus designated the Ralph strain has been propagated in both the MA-104 and A-72 cell lines. The strain produced cytoplasmic inclusion bodies in these cell cultures. Tissue-cultured virus fixed complement in the presence of reovirus antibodies, but failed to do so in the presence of rotavirus antiserum. By electron microscopy the viral particle measured +/- 65 nm. The virus hemagglutinated pig erythrocytes, but not human O, human A, calf, cow, chicken or guinea pig erythrocytes. In the hemagglutination test there was complete reciprocal crossing between the Ralph strain and the NIH reovirus type 3, but there was no crossing with the NIH reovirus types 1 and 2. A limited serological survey was completed on serum samples from New York State horses collected in 1976-1977 and 1981 using the hemagglutination-inhibition test. The percentage with antibodies to reovirus types 1, 2 and 3 for 1976-1977 was 24.5, 42.2 and 3.9% and in 1981, 8.8, 9.8 and 3.9%, respectively.