Severe Fever with Thrombocytopenia Syndrome, South Korea, 2012

Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging tick-borne pathogen that was first reported in China in 2009. Phylogenetic analysis of the viral genome showed that SFTS virus represents a new lineage within the Phlebovirus genus, distinct from the existing sandfly fever and Uukuniemi virus groups, in the family Bunyaviridae. SFTS disease is characterized by gastrointestinal symptoms, chills, joint pain, myalgia, thrombocytopenia, leukocytopenia, and some hemorrhagic manifestations with a case fatality rate of about 2 to 15%. Here we report the development of reverse genetics systems to study STFSV replication and pathogenesis. We developed and optimized functional T7 polymerase-based M-and S-segment minigenome assays, which revealed errors in the published terminal sequences of the S segment of the Hubei 29 strain of SFTSV. We then generated recombinant viruses from cloned cDNAs prepared to the antigenomic RNAs both of the minimally passaged virus (HB29) and of a cell culture-adapted strain designated HB29pp. The growth properties, pattern of viral protein synthesis, and subcellular localization of viral N and NSs proteins of wild-type HB29pp (wtHB29pp) and recombinant HB29pp viruses were indistinguishable. We also show that the viruses fail to shut off host cell polypeptide production. The robust reverse genetics system described will be a valuable tool for the design of therapeutics and the development of killed and attenuated vaccines against this important emerging pathogen. IMPORTANCESFTSV and related tick-borne phleboviruses such as Heartland virus are emerging viruses shown to cause severe disease in humans in the Far East and the United States, respectively. Study of these novel pathogens would be facilitated by technology to manipulate these viruses in a laboratory setting using reverse genetics. Here, we report the generation of infectious SFTSV from cDNA clones and demonstrate that the behavior of recombinant viruses is similar to that of the wild type. This advance will allow for further dissection of the roles of each of the viral proteins in the context of virus infection, as well as help in the development of antiviral drugs and protective vaccines.T he family Bunyaviridae is one of the largest taxonomic groupings of RNA viruses, containing over 350 named isolates that are classified into five genera: Hantavirus, Nairovirus, Orthobunyavirus, Phlebovirus, and Tospovirus (1). All viruses share a genome structure that comprises three segments of negative-sense or ambisense RNA, named small (S), medium (M), and large (L). The S segment encodes the nucleocapsid (N) protein; the M segment encodes the virion glycoproteins Gn and Gc; and the L segment encodes the L protein, the viral RNA-dependent RNA polymerase. Some viruses also encode nonstructural proteins on the S and M segments. Viruses that impinge on human health, either directly by causing human disease or indirectly by causing economic losses of domestic animals or crop plants, are found in each of the five gener...

Section: Discussionmentioning

confidence: 99%

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confidence: 96%

Reverse Genetics System for Severe Fever with Thrombocytopenia Syndrome Virus

Brennan

Zhang

et al. 2015

“…Four deaths due to SFTSV infection were recently reported in Japan (3), and 14 cases of SFTSV (with 9 deaths) were confirmed in South Korea during 2012 to 2013 (4). Clinical signs of the disease mainly include severe fever, thrombocytopenia, and leukopenia syndrome, frequently with gastrointestinal symptoms such as diarrhea and abdominal pain.…”

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confidence: 99%

Nonmuscle Myosin Heavy Chain IIA Is a Critical Factor Contributing to the Efficiency of Early Infection of Severe Fever with Thrombocytopenia Syndrome Virus

Sun

Liu

et al. 2014

100

106

“…Originally described in the Henan and Hubei provinces in China (17), SFTSV has now been found to be more widespread in China (18) and has also been isolated in Japan and South Korea, suggesting a more widespread distribution (19,20). HRTV isolates have so far been restricted to Missouri and Tennessee in the United States, with 10 cases and 2 fatalities (21-23), but one serological survey suggested a widespread occurrence of antigenically similar viruses in farm animals throughout Minnesota (24).…”

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confidence: 99%

Generation of Mutant Uukuniemi Viruses Lacking the Nonstructural Protein NSs by Reverse Genetics Indicates that NSs Is a Weak Interferon Antagonist

Rezelj

Överby

Elliott

2015

Uukuniemi virus (UUKV) is a tick-borne member of the Phlebovirus genus (family Bunyaviridae) and has been widely used as a safe laboratory model to study aspects of bunyavirus replication. Recently, a number of new tick-borne phleboviruses have been discovered, some of which, like severe fever with thrombocytopenia syndrome virus and Heartland virus, are highly pathogenic in humans. UUKV could now serve as a useful comparator to understand the molecular basis for the different pathogenicities of these related viruses. We established a reverse-genetics system to recover UUKV entirely from cDNA clones. We generated two recombinant viruses, one in which the nonstructural protein NSs open reading frame was deleted from the S segment and one in which the NSs gene was replaced with green fluorescent protein (GFP), allowing convenient visualization of viral infection. We show that the UUKV NSs protein acts as a weak interferon antagonist in human cells but that it is unable to completely counteract the interferon response, which could serve as an explanation for its inability to cause disease in humans. IMPORTANCE Uukuniemi virus (UUKV) is a tick-borne phlebovirus that is apathogenic for humans and has been used as a convenient model to investigate aspects of phlebovirus replication. Recently, new tick-borne phleboviruses have emerged, such as severe fever with thrombocytopenia syndrome virus in China and Heartland virus in the UnitedStates, that are highly pathogenic, and UUKV will now serve as a comparator to aid in the understanding of the molecular basis for the virulence of these new viruses. To help such investigations, we have developed a reverse-genetics system for UUKV that permits manipulation of the viral genome. We generated viruses lacking the nonstructural protein NSs and show that UUKV NSs is a weak interferon antagonist. In addition, we created a virus that expresses GFP and thus allows convenient monitoring of virus replication. These new tools represent a significant advance in the study of tick-borne phleboviruses. The Bunyaviridae are the largest grouping of RNA viruses, comprising Ͼ350 members. The family includes a wide range of enveloped, single-stranded RNA viruses with tripartite genomes of negative-sense or ambisense polarity. The Phlebovirus genus contains 70 viruses divided into two groups: the sandfly fever group, transmitted mainly by phlebotomines and mosquitoes, and the Uukuniemi-like virus group, transmitted by ticks. Uukuniemi virus (UUKV) itself was originally isolated in 1964 from an Ixodes ricinus tick in Finland (1). UUKV has served as a prototype tick-borne phlebovirus for many years and has aided in advances in bunyavirus structural and architectural determination (2-4), bunyavirus glycoprotein studies (5-9), and studies of bunyavirus entry into mammalian cells (10-12).Like other phleboviruses, the three UUKV genome segments, designated small (S), medium (M), and large (L), are found in the form of ribonucleoprotein (RNP) complexes in association with the nucleocapsid (N)...