Different tertiary interactions create the same important 3D features in a distinct flavivirus xrRNA.

During infection by a flavivirus (FV), cells accumulate noncoding subgenomic flavivirus RNAs (sfRNAs) that interfere with several antiviral pathways. These sfRNAs are formed by structured RNA elements in the 3' untranslated region (UTR) of the viral genomic RNA, which block the progression of host cell exoribonucleases that have targeted the viral RNA. Previous work on these exoribonuclease-resistant RNAs (xrRNAs) from mosquito-borne FVs revealed a specific three-dimensional fold with a unique topology in which a ring-like structure protectively encircles the 5' end of the xrRNA. Conserved nucleotides make specific tertiary interactions that support this fold. Examination of more divergent FVs reveals differences in their 3' UTR sequences, raising the question of whether they contain xrRNAs and if so, how they fold. To answer this, we demonstrated the presence of an authentic xrRNA in the 3' UTR of the Tamana bat virus (TABV) and solved its structure by X-ray crystallography. The structure reveals conserved features from previously characterized xrRNAs, but in the TABV version these features are created through a novel set of tertiary interactions not previously seen in xrRNAs. This includes two important A-C interactions, four distinct backbone kinks, several ordered Mg2+ ions, and a C+-G-C base triple. The discovery that the same overall architecture can be achieved by very different sequences and interactions in distantly related flaviviruses provides insight into the diversity of this type of RNA and will inform searches for undiscovered xrRNAs in viruses and beyond.

Isolation and Characterisation of Alongshan Virus in Russia.

In recent decades, many new flavi-like viruses have been discovered predominantly in different invertebrates and, as was recently shown, some of them may cause disease in humans. The Jingmenvirus (JMV) group holds a special place among flaviviruses and flavi-like viruses because they have a segmented ssRNA(+) genome. We detected Alongshan virus (ALSV), which is a representative of the JMV group, in ten pools of adult Ixodes persulcatus ticks collected in two geographically-separated Russian regions. Three of the ten strains were isolated in the tick cell line IRE/CTVM19. One of the strains persisted in the IRE/CTVM19 cells without cytopathic effect for three years. Most ALSV virions purified from tick cells were spherical with a diameter of approximately 40.5 nm. In addition, we found smaller particles of approximately 13.1 nm in diameter. We obtained full genome sequences of all four segments of two of the isolated ALSV strains, and partial sequences of one segment from the third strain. Phylogenetic analysis on genome segment 2 of the JMV group clustered our novel strains with other ALSV strains. We found evidence for the existence of a novel upstream open reading frame in the glycoprotein-coding segment of ALSV and other members of the JMV group.

A New Segmented Virus Associated with Human Febrile Illness in China.

BACKGROUND: In 2017, surveillance for tickborne diseases in China led to the identification of a patient who presented to a hospital in Inner Mongolia with a febrile illness that had an unknown cause. The clinical manifestation of the illness was similar to that of tickborne encephalitis virus (TBEV) infection, but neither TBEV RNA nor antibodies against the virus were detected. METHODS: We obtained a blood specimen from the index patient and attempted to isolate and identify a causative pathogen, using genome sequence analysis and electron microscopy. We also initiated a heightened surveillance program in the same hospital to screen for other patients who presented with fever, headache, and a history of tick bites. We used reverse-transcriptase-polymerase-chain-reaction (RT-PCR) and cell-culture assays to detect the pathogen and immunofluorescence and neutralization assays to determine the levels of virus-specific antibodies in serum specimens from the patients. RESULTS: We found that the index patient was infected with a previously unknown segmented RNA virus, which we designated Alongshan virus (ALSV) and which belongs to the jingmenvirus group of the family Flaviviridae. ALSV infection was confirmed by RT-PCR assay in 86 patients from Inner Mongolia and Heilongjiang who presented with fever, headache, and a history of tick bites. Serologic assays showed that seroconversion had occurred in all 19 patients for whom specimens were available from the acute phase and the convalescent phase of the illness. CONCLUSIONS: A newly discovered segmented virus was found to be associated with a febrile illness in northeastern China. (Funded by the National Key Research and Development Program of China and the National Natural Science Foundation of China.).

ICTV Virus Taxonomy Profile: Flaviviridae.

The Flaviviridae is a family of small enveloped viruses with RNA genomes of 9000-13 000 bases. Most infect mammals and birds. Many flaviviruses are host-specific and pathogenic, such as hepatitis C virus in the genus Hepacivirus. The majority of known members in the genus Flavivirus are arthropod borne, and many are important human and veterinary pathogens (e.g. yellow fever virus, dengue virus). This is a summary of the current International Committee on Taxonomy of Viruses (ICTV) report on the taxonomy of the Flaviviridae, which is available at www.ictv.global/report/flaviviridae.

Hepatitis C-like viruses are produced in cells from rabbit and hare DNA.

Hepatitis C virus (HCV), a major causative agent of acute and chronic liver disease, belongs to the Flaviviridæ family and contains a single-strand positive-sense RNA genome, which upon virus entry and uncoating, functions as mRNAs and thus can be directly translated into proteins by host cell machinery. To date the HCV origin remains unclear and HCV life cycle and pathogenesis are not enlightened processes due to the absence of HCV efficient cell cultures systems or animals models. Here we show that rabbit and hare HCV-like viruses, RHCV and HHCV respectively, are formed after the inoculation of genomic DNA in Madin-Darby bovine kidney cell line cultures. RHCV is closely related to the HCV-1a/HCV-1b genotypes and HHCV is more closely related to the HCV-1b genotype. These findings could contribute to the understanding of HCV origin as well as clarify the virus life cycle, pathogenesis, evolution and diversity.

Membranous replication factories induced by plus-strand RNA viruses.

In this review, we summarize the current knowledge about the membranous replication factories of members of plus-strand (+) RNA viruses. We discuss primarily the architecture of these complex membrane rearrangements, because this topic emerged in the last few years as electron tomography has become more widely available. A general denominator is that two "morphotypes" of membrane alterations can be found that are exemplified by flaviviruses and hepaciviruses: membrane invaginations towards the lumen of the endoplasmatic reticulum (ER) and double membrane vesicles, representing extrusions also originating from the ER, respectively. We hypothesize that either morphotype might reflect common pathways and principles that are used by these viruses to form their membranous replication compartments.

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.

Cell culture and electron microscopy for identifying viruses in diseases of unknown cause.

During outbreaks of infectious diseases or in cases of severely ill patients, it is imperative to identify the causative agent. This report describes several events in which virus isolation and identification by electron microscopy were critical to initial recognition of the etiologic agent, which was further analyzed by additional laboratory diagnostic assays. Examples include severe acute respiratory syndrome coronavirus, and Nipah, lymphocytic choriomeningitis, West Nile, Cache Valley, and Heartland viruses. These cases illustrate the importance of the techniques of cell culture and electron microscopy in pathogen identification and recognition of emerging diseases.

[Virus isolated in the human blood leukocyte culture and its interaction with hepatitis C and G viruses]. / Virus, vydelennyi na kul'ture leikotsitov krovi cheloveka, i ego vzaimootnoshenie s virusami gepatitov C i G.

Electrone-microscopic investigations are indicative of that the cultures of healthy donors, stimulated by phytohemagglutinin (PHA), can be successfully used to study the etiology of parenterally transmitted hepatitis. An electronic-microscopic study of a virus, isolated from the blood serum of a patient with hepatitis on the basis of the PHA-stimulated human leukocyte cultures and named a hepatitis leukocytic virus (HLV), enabled, by using the negative contrasting method, to detect viral particles of the hexagonal shape, sized 50-65 nm, with a coating divided by a 4-5 nm light space. Therefore, the HLV was described as belonging to the Flaviviridae family. RNA of the C hepatitis virus was detected in the K HLV strain stored, for 24 years, at the Museum of the Viruses Research Institute, Russian Academy of Medical Sciences, in a lyophilizated bed at -5 degrees C, however, an attempt to genotype the RNA failed. No RNA donor leukocytes were found in the materials of further passing of HLV by using the PHA-stimulated cultures, which can be explained by an inactivation of HLV at storage. No RNA of the C hepatitis virus was found in the above materials either, however, in 1999, DNA of the TT virus was detected at passing the strain, which indicates that the virus is widely spread in the population of healthy donors, whose lymphocytes are used preparing the blood leukocyte cultures.

[Kama, a new virus (Flaviviridae, Flavivirus, Tiulenii antigenic group), isolated from Ixodes lividus ticks]. / Novyi virus kama (Flaviviridae, Flavivirus, antigennaia gruppa tiulenii), izolirovannyi iz kleshchei Ixodes lividus.

Three identical strains of a new virus Kama (Flaviviridae, Flavivirus, Tyuleny antigenic group) were isolated from Ixodes lividus Roch, obligate parasites of Riparia riparia L. The ticks were collected in June, 1990 in Tatarstan on the islands in the basin of the Kama river. The strains were examined under electron microscope and by serological tests (neutralization, complement fixation, hemagglutination inhibition, and indirect immunofluorescence). The virus is antigenically related but not identical to Tyuleny virus. Hence, the Tyuleny antigenic group at present includes viruses Tyuleny, Meaban, Gadgest Gally, Saumares Reef, and Kama. All these viruses are associated with an ecosystem including ixodide ticks, obligate parasites of colonial birds.

Human disease caused by an arbovirus closely related to Ilheus virus: report of five cases.

We report five cases of human disease caused by arbovirus in 5 patients from the State of São Paulo, Brazil, residing in the municipalities of Osasco, Atibaia, Guarujá, and the capital São Paulo, respectively. One of the patients resides in São Luis, capital of the State of Maranhão. The sites of infection probably were the states of Paraná and Goiás, both in cave regions, the State of Amazonas, and Rondônia in two cases. Laboratory tests for malaria were negative and 1 patient showed a positive serum reaction for leptospirosis. Serum samples from the acute and convalescent phases were tested by hemagglutination inhibition, complement fixation, and neutralization in mice. Acute phase samples were inoculated into suckling mice by the intracerebral route. A close antigenic relationship was observed between the five agents isolated and the flavivirus Ilheus. Serologic tests demonstrated the absence of antibodies in all samples from the 5 patients during convalescence and even for more than 1 year after infection in 1 of them.