Detection and Characterization of a Novel Reassortant Mammalian Orthoreovirus in Bats in Europe.

A renewed interest in mammalian orthoreoviruses (MRVs) has emerged since new viruses related to bat MRV type 3, detected in Europe, were identified in humans and pigs with gastroenteritis. This study reports the isolation and characterization of a novel reassortant MRV from the lesser horseshoe bat (Rhinolophus hipposideros). The isolate, here designated BatMRV1-IT2011, was first identified by electron microscopy and confirmed using PCR and virus-neutralization tests. The full genome sequence was obtained by next-generation sequencing. Molecular and antigenic characterizations revealed that BatMRV1-IT2011 belonged to serotype 1, which had not previously been identified in bats. Phylogenetic and recombination detection program analyses suggested that BatMRV1-IT2011 was a reassortant strain containing an S1 genome segment similar to those of MRV T1/bovine/Maryland/Clone23/59 and C/bovine/ Indiana/MRV00304/2014, while other segments were more similar to MRVs of different hosts, origins and serotypes. The presence of neutralizing antibodies against MRVs has also been investigated in animals (dogs, pigs, bovines and horses). Preliminary results suggested that MRVs are widespread in animals and that infections containing multiple serotypes, including MRVs of serotype 1 with an S1 gene similar to BatMRV1-IT2011, are common. This paper extends the current knowledge of MRVs and stresses the importance to continue and improve MRV surveillance in bats and other mammals through the development and standardization of specific diagnostic tools.

A tick-borne segmented RNA virus contains genome segments derived from unsegmented viral ancestors.

Although segmented and unsegmented RNA viruses are commonplace, the evolutionary links between these two very different forms of genome organization are unclear. We report the discovery and characterization of a tick-borne virus--Jingmen tick virus (JMTV)--that reveals an unexpected connection between segmented and unsegmented RNA viruses. The JMTV genome comprises four segments, two of which are related to the nonstructural protein genes of the genus Flavivirus (family Flaviviridae), whereas the remaining segments are unique to this virus, have no known homologs, and contain a number of features indicative of structural protein genes. Remarkably, homology searching revealed that sequences related to JMTV were present in the cDNA library from Toxocara canis (dog roundworm; Nematoda), and that shared strong sequence and structural resemblances. Epidemiological studies showed that JMTV is distributed in tick populations across China, especially Rhipicephalus and Haemaphysalis spp., and experiences frequent host-switching and genomic reassortment. To our knowledge, JMTV is the first example of a segmented RNA virus with a genome derived in part from unsegmented viral ancestors.

Biophysical characterization of rotavirus serotypes G1, G3 and G4.

The stability of attenuated virus vaccines has traditionally been assessed by a plaque assay to measure the virus's loss of replication competency in response to a variety of environmental perturbations. Although this method provides information regarding the impact of the vaccine formulation, it involves an empirical approach to evaluate stability. Biophysical studies on the other hand have the potential to provide insight into the mechanisms of inactivation of a viral vaccine in response to a variety of stressed conditions. Herein, we have employed a variety of spectroscopic techniques (i.e., circular dichroism, fluorescence spectroscopy and dynamic light scattering) for a comprehensive examination of the thermal stability of three live-attenuated human-bovine reassortant rotavirus strains (G1, G3 and G4) in the 5-8 pH range. The spectroscopic methods employed are not specific and response changes reflect an average change over the entire virus structure. The present work, however, suggests the utility of these methods in early formulation of rotaviral vaccines due to their ability to identify regions of marginal stability over which high throughput excipient screening assays can be designed. We have further shown that these methods are sufficiently sensitive to differentiate the stability of the three homologous G-subtypes differing only in the composition of their surface antigenic proteins. The data from these spectroscopic methods are also compared to biological activity using a tissue culture viral infectivity assay. Partial correlation between the structural alterations and losses in activity are observed, further suggesting the utility of biophysical studies in early formulation studies of rotavirus vaccines.

Reverse genetics studies on the filamentous morphology of influenza A virus.

We have investigated the genetic determinants responsible for the filamentous morphology of influenza A viruses, a property characteristic of primary virus isolates. A plasmid-based reverse genetics system was used to transfer the M segment of influenza A/Udorn/72 (H3N2) virus into influenza A/WSN/33 (H1N1) virus. While WSN virions display spherical morphology, recombinant WSN-Mud virus acquired the ability of the parental Udorn strain to form filamentous virus particles. This was determined by immunofluorescence studies in infected MDCK cells and by electron microscopy of purified virus particles. To determine the gene product within the M segment responsible for filamentous virus morphology, we generated four recombinant viruses carrying different sets of M1 and M2 genes from WSN or Udorn strains in a WSN background. These studies revealed that the M1 gene of Udorn, independently of the origin of the M2 gene, conferred filamentous budding properties and filamentous virus morphology to the recombinant viruses. We also constructed two WSN viruses encoding chimeric M1 proteins containing the amino-terminal 1-162 amino acids or the carboxy-terminal 163-252 amino acids of the Udorn M1 protein. Neither of these two viruses acquired filamentous phenotypes, indicating that both amino- and carboxy-terminal domains of the M1 protein contribute to filamentous virus morphology. We next rescued seven mutant WSN-M1ud viruses containing Udorn M1 proteins carrying single amino acid substitutions corresponding to the seven amino acid differences with the M1 protein of WSN virus. Characterization of these recombinant viruses revealed that amino acid residues 95 and 204 are critical in determining filamentous virus particle formation.

A reassortant bunyavirus isolated from acute hemorrhagic fever cases in Kenya and Somalia.

In late 1997 and early 1998, a large outbreak of hemorrhagic fever occurred in East Africa. Clinical samples were collected in Kenya and southern Somalia, and 27 of 115 (23%) hemorrhagic fever patients tested showed evidence of acute infection with Rift Valley fever (RVF) virus as determined by IgM detection, virus isolation, detection of virus RNA by reverse transcription-polymerase chain reaction (RT-PCR), or immunohistochemistry. However, two patients (one from Kenya and the other from Somalia) whose illness met the hemorrhagic fever case definition yielded virus isolates that were not RVF. Electron microscopy suggested these two virus isolates were members of the family Bunyaviridae. RT-PCR primers were designed to detect bunyavirus RNA in these samples. Regions of the S and L segments of the two isolates were successfully amplified, and their nucleotide sequences exhibited nearly complete identity with Bunyamwera virus, a mosquito-borne virus not previously associated with severe human disease. Unexpectedly, the virus M segment appeared to be reassorted, as the sequences detected exhibited 32-33% nucleotide and 28% amino acid differences relative to the corresponding M segment sequence of Bunyamwera virus. The association of this reassortant bunyavirus, proposed name Garissa virus, with severe disease is supported by the detection of the virus RNA in acute-phase sera taken from 12 additional hemorrhagic fever cases in the region.