An update on Toscana virus distribution,genetics, medical and diagnostic aspects

BackgroundToscana virus is an arbovirus transmitted by sand flies within the Mediterranean area where it can cause febrile illness and neuroinvasive infections during the seasonal circulation period of the vector. Although it is an important cause of meningitis and encephalitis, it remains a neglected virus with limited published data, as demonstrated by <250 peer-reviewed articles since the 1970s.ObjectiveThe last review article on Toscana virus was published in 2012. The aim was to compile peer-reviewed articles to provide an updated review highlighting recent findings to complement previous review articles.SourcesPubMed database was searched using the ‘Toscana virus’ keyword from 2010 to present. A total of 152 articles were retrieved and identified studies were assessed for novel information on virus genetics, and geographic and medical aspects compared with existing knowledge reported in previous review articles.ContentStudies addressing medical, veterinary and entomological aspects have provided evidence that Toscana virus is present in North Africa, in the Balkan Peninsula, and in most of the Mediterranean islands. Besides the two previously recognized genetic lineages, a novel evolutionary lineage has been identified in the Balkan Peninsula. Co-circulation of two genetic lineages has been demonstrated in France, in Turkey and in Croatia. In addition to meningitis and meningo-encephalitis, which have been reported for 40 years, various neuroinvasive forms have been recently reported such as Guillain–Barré syndrome, hydrocephalus, myositis, fasciitis, polymyeloradiculopathy, deafness and facial paralysis.ImplicationBecause it is endemic in countries bordering the Mediterranean, physicians should include Toscana virus in the differential diagnosis of patients presenting with febrile illness and/or neurological manifestations.     

Rôle du virus Toscana dans les infections neuroméningées en Tunisie

Objectives

To detect the presence of Toscana virus (TOSV) circulation in Tunisia and to study its role in viral meningo-encephalitis.

Patients and methods

A total of 315 (167 sera and 178 cerobrospinal fluid [CSF]) samples was investigated. These samples are colleted from Tunisian patients with neurological diseases during the period between January 2003 and December 2009. All samples were tested negative for enterovirus, Herpes Simplex virus and West Nile virus. Detection for IgM and IgG specific to TOSV was done by ELISA tests.

Results

Specific IgM for TOSV were detected in 10 % of patients with neurological diseases (31 cases). These recent infections were distributed throughout the study period and predominated during summer and automn. Patients were originated, in the majority from the coastal region. IgG were isolated in 22 cases (7 %) corresponding to previous infection.

Conclusion

This is the first report of TOSV circulating in Tunisia and its frequent implication in neurological diseases. These results incited to include TOSV as one of the viral etiologies to target in the diagnosis of viral meningitis and encephalitis in the country.     

Heartland Virus Neutralizing Antibodies in Vertebrate Wildlife,United States, 2009–2014

Since its discovery in 2009, the tickborne Heartland virus (HRTV) has caused human illness in Missouri, Oklahoma, and Tennessee USA. To better assess the geographic distribution of HRTV, we used wildlife serology as an indicator. This retrospective evaluation determined that HRTV is widespread within the central and eastern United States.     

Severe Fever with Thrombocytopenia Syndrome Virus in Ticks Collected from Humans,South Korea, 2013

We investigated the infection rate for severe fever with thrombocytopenia syndrome virus (SFTSV) among ticks collected from humans during May–October 2013 in South Korea. Haemaphysalis longicornis ticks have been considered the SFTSV vector. However, we detected the virus in H. longicornis, Amblyomma testudinarium, and Ixodes nipponensis ticks, indicating additional potential SFTSV vectors.     

Taxonomy of Phleboviruses,Emphasizing Those That Are Sandfly-Borne

Sandfly-borne phleboviruses (phylum Negarnavaricota, realm Riboviria, kingdom Orthornavirae, genus Phlebovirus) comprise three genome segments of ribonucleic acid (RNA) and which encode an RNA-dependent RNA polymerase, which they use to transcribe the viral RNA genome into messenger RNA and to replicate the genome. At least some of these viruses cause mild 3-day fevers in humans but some also have been associated with more severe illnesses in humans. The 67 recognized phleboviruses are listed here in a table composed by the authors from International Committee on Taxonomy of Viruses reports as well as the scientific literature.     

Human antibody response to Toscana virus glycoproteins expressed by recombinant baculovirus

The arthropod-borne Toscana virus has been associated with acute neurological disease in humans. In this study, the viral envelope glycoproteins were expressed in soluble form in a baculovirus system. The recombinant sGN and sGC proteins were used as viral antigens in a Western blot assay to analyze the specific immune response in sera from patients with recognized virus-associated aseptic meningitis. The anti-glycoprotein and the anti-nucleoprotein N IgG responses were compared by an immunoassay based on the recombinant proteins. In this system, all the sera showed a high reactivity to the N protein, but they differed in the response to the glycoproteins.     

Rift Valley fever virus lacking NSm proteins retains high virulence in vivo and may provide a model of human delayed onset neurologic disease

Rift Valley fever virus is a significant human and veterinary pathogen responsible for explosive outbreaks throughout Africa and the Arabian Peninsula. Severe acute disease in humans includes rapid onset hepatic disease and hemorrhagic fever or delayed onset encephalitis. A highly efficient reverse genetics system was developed which allowed generation of recombinant RVF viruses to assess the role of NSm protein in virulence in a rat model in which wild-type RVF virus strain ZH501 (wt-ZH501) results in 100% lethal hepatic disease 2-3 days post infection. While extensive genomic analysis indicates conservation of the NSm coding capability of diverse RVF viruses, and viruses deficient in NSs proteins are completely attenuated in vivo, comparison of wt-ZH501, a reverse genetics generated wt-ZH501 virus (R-ZH501), and R-ZH501 virus lacking the NSm proteins (R-DeltaNSm-ZH501) demonstrated that the NSm proteins were nonessential for in vivo virulence and lethality. Surprisingly, while 44% of R-DeltaNSm-ZH501 infected animals quickly developed lethal hepatic disease similar to wt- and R-ZH501, 17% developed delayed onset neurologic disease (lethargy, head tremors, and ataxia) at 13 days post infection. Such infections may provide the basis for study of both RVF acute hepatic disease and delayed onset encephalitic disease in humans.     

Incubation Period for Neuroinvasive Toscana Virus Infections

Toscana virus (TOSV) is an emerging pathogen in the Mediterranean area and is neuroinvasive in its most severe form. Basic knowledge on TOSV biology is limited. We conducted a systematic review on travel-related infections to estimate the TOSV incubation period. We estimated the incubation period at 12.1 days.     

Potent neutralization of Rift Valley fever virus by human monoclonal antibodies through fusion inhibition

Rift Valley fever virus (RVFV), an emerging arboviral and zoonotic bunyavirus, causes severe disease in livestock and humans. Here, we report the isolation of a panel of monoclonal antibodies (mAbs) from the B cells of immune individuals following natural infection in Kenya or immunization with MP-12 vaccine. The B cell responses of individuals who were vaccinated or naturally infected recognized similar epitopes on both Gc and Gn proteins. The Gn-specific mAbs and two mAbs that do not recognize either monomeric Gc or Gn alone but recognized the hetero-oligomer glycoprotein complex (Gc+Gn) when Gc and Gn were coexpressed exhibited potent neutralizing activities in vitro, while Gc-specific mAbs exhibited relatively lower neutralizing capacity. The two Gc+Gn–specific mAbs and the Gn domain A-specific mAbs inhibited RVFV fusion to cells, suggesting that mAbs can inhibit the exposure of the fusion loop in Gc, a class II fusion protein, and thus prevent fusion by an indirect mechanism without direct fusion loop contact. Competition-binding analysis with coexpressed Gc/Gn and mutagenesis library screening indicated that these mAbs recognize four major antigenic sites, with two sites of vulnerability for neutralization on Gn. In experimental models of infection in mice, representative mAbs recognizing three of the antigenic sites reduced morbidity and mortality when used at a low dose in both prophylactic and therapeutic settings. This study identifies multiple candidate mAbs that may be suitable for use in humans against RVFV infection and highlights fusion inhibition against bunyaviruses as a potential contributor to potent antibody-mediated neutralization.

Rift Valley fever virus (RVFV) is an emerging arbovirus and zoonotic threat to human and animal health with pandemic potential because of the global presence of its vectors and hosts (1). First identified in 1931 (2), RVFV causes ongoing infections in Africa, with occasional rises in incidence related to the propagation of mosquitos due to alternating weather patterns (3). These outbreak episodes are characterized by mass livestock die-off events, particularly in young animals and abortions in pregnant females. Spillover events to humans occur by infected mosquito exposures or contact with the blood and/or organs of infected animals (4). Human-to-human transmission of RVFV has not been documented, but concern is growing that RVFV can be transmitted from mother to fetus in utero (5, 6). In humans, disease presentation ranges from a mild influenza-like illness to a potentially lethal hemorrhagic fever syndrome. The World Health Organization (W.H.O.) has reported more than 4,600 cases and 957 deaths from 2000 to 2016 due to RVFV infections, with a case fatality rate of more than 20% (7). RVFV outbreaks typically occur in sub-Saharan and North Africa, but in 2000, the first reported cases outside the African continent occurred in the Arabian Peninsula (8). Given the increasing observed mortality rate, spread to new regions, and potential use as a bioterrorist agent (9), the W.H.O. and the US National Institute of Allergy and Infectious Diseases have designated RVFV as a priority pathogen for urgent research and therapeutic development (10, 11).RVFV, a member of the Phlebovirus genus in the Phenuiviridae family of the Bunyavirales order (12) has a tripartite RNA genome containing large (L), medium (M), and small (S) gene segments. The sequences in the M segment encode for the viral envelope glycoproteins (Gc and Gn). These two glycoproteins allow for viral attachment, entry, fusion, and assembly when oriented as a pentamer of heterodimers (13–16). Data from animal and human vaccine trials indicate that high-serum neutralizing titers and protection (in animals) are associated with responses to Gn, Gc, or entire virion particles (17–23). Protection against experimental RVFV challenge is observed in vaccinated animals with high-serum titers of neutralizing antibodies (22, 23).On the viral surface, Gn and Gc organize into a pentamer or hexamers of Gc-Gn heterodimers with icosahedral symmetry (13, 14). Receptor engagement of DC-SIGN or possibly other receptors like heparan sulfate (24) by Gn allows for cellular attachment and initiates caveolae-mediated endocytosis (25, 26). Premature fusion and extension of the fusion loop is prevented by Gn, which provides a shield for the fusion loop in Gc (27). Upon exposure to acidic conditions in the late endosome, Gn repositions, and the class II fusion protein, Gc extends so that its fusion loop interacts with the host membrane (27–29). The extended Gc conformation subsequently rearranges so the membranes organize in close proximity to fuse (29). Given the broad host, tissue, and cell tropism of RVFV (24), inhibiting RVFV by blocking DC-SIGN engagement may not fully explain the neutralizing and protective capacity of potent mAbs. The fusion process, a common mechanistic feature of RVFV entry facilitating broad tropism, may be a desirable target to enable neutralization. These observations may explain why neutralization is observed in cells that do not express DC-SIGN.Here, we report the isolation of 20 human monoclonal antibodies (mAbs) from the circulating B cells of RVFV MP-12 vaccines or survivors of natural RVFV infection using neutralization as the primary screening method. This panel of antibodies targets diverse antigenic sites on Gc, Gn, and an undefined epitope that may present only in the glycoprotein complex (Gc+Gn) of properly hetero-oligomerized Gc-Gn. Here, we refer to mAbs that bind to coexpressed full-length Gc-Gn proteins but not to monomeric Gc or Gn as “Gc+Gn–specific mAbs.” Four competition groups for binding to viral antigen on the RVFV surface were recognized by neutralizing antibodies, and all groups contained antibodies that cross-neutralized diverse strains of RVFV. Antibodies from both vaccinated and naturally infected individuals primarily neutralized virus by targeting domain A on the Gn protein. Previously, mAbs derived from a single patient in China inhibited receptor engagement by binding to Gn domain A (30). We sought to explore the role of fusion inhibition as a second major mechanism of neutralization, by which mAbs could contribute to the overall potent neutralizing activity observed and prevent infection even in cells and tissues that do not readily express DC-SIGN. Fusion-inhibiting activity of anti-RVFV–specific mAbs has not been reported to date but has been observed for murine mAbs (31). We observed two classes of potently neutralizing antibodies, those that target domain A and cause partial inhibition of fusion and Gc+Gn–specific mAbs that cause complete inhibition of fusion. Studies of a representative mAb from each group indicate the Gn domain A-specific mAb loses fusion inhibition activity when used as Fab fragments, whereas the Gc+Gn–specific mAb retains fusion inhibition activity as a Fab, suggesting different mechanisms of action for these two classes of mAbs. Antibodies that recognize domain A, domain B, and Gc+Gn–specific epitopes neutralized RVFV with varying potencies, and all provided protection against lethal ZH501 wild-type (wt) virus challenge in prophylactic or therapeutic mouse models of experimental infection when used as a monotherapy. These results suggest these mAbs can be investigated as therapies against RVFV infection and that multiple antigenic sites of vulnerability for neutralization by inhibition of viral fusion exist on the virion surface.