Reverse Genetics System for Heartland Bandavirus: NSs Protein Contributes to Heartland Bandavirus Virulence.

Heartland bandavirus (HRTV), which is an emerging tick-borne virus first identified in Missouri in 2009, causes fever, fatigue, decreased appetite, headache, nausea, diarrhea, and muscle or joint pain in humans. HRTV is genetically close to Dabie bandavirus, which is the causative agent of severe fever with thrombocytopenia syndrome (SFTS) in humans and is known as SFTS virus (SFTSV). The generation of infectious HRTV entirely from cloned cDNAs has not yet been reported. The absence of a reverse genetics system for HRTV has delayed efforts to understand its pathogenesis and to generate vaccines and antiviral drugs. Here, we developed a reverse genetics system for HRTV, which employs an RNA polymerase I-mediated expression system. A recombinant nonstructural protein (NSs)-knockout HRTV (rHRTV-NSsKO) was generated. We found that NSs interrupted signaling associated with innate immunity in HRTV-infected cells. The rHRTV-NSsKO was highly attenuated, indicated by the apparent absence of symptoms in a mouse model of HRTV infection. Moreover, mice immunized with rHRTV-NSsKO survived a lethal dose of HRTV. These findings suggest that NSs is a virulence factor of HRTV and that rHRTV-NSsKO could be a vaccine candidate for HRTV. IMPORTANCE Heartland bandavirus (HRTV) is a tick-borne virus identified in the United States in 2009. HRTV causes fever, fatigue, decreased appetite, headache, nausea, diarrhea, and muscle or joint pain in humans. FDA-approved vaccines and antiviral drugs are unavailable. The lack of a reverse genetics system hampers efforts to develop such antiviral therapeutics. Here, we developed a reverse genetics system for HRTV that led to the generation of a recombinant nonstructural protein (NSs)-knockout HRTV (rHRTV-NSsKO). We found that NSs interrupted signaling associated with innate immunity in HRTV-infected cells. Furthermore, rHRTV-NSsKO was highly attenuated and immunogenic in a mouse model. These findings suggest that NSs is a virulence factor of HRTV and that rHRTV-NSsKO could be a vaccine candidate for HRTV.

The Input of Structural Vaccinology in the Search for Vaccines against Bunyaviruses.

A significant increase in the number of viruses causing unexpected illnesses and epidemics among humans, wildlife and livestock has been observed in recent years. These new or re-emerging viruses have often caught the scientific community off-guard, without sufficient knowledge to combat them, as shown by the current coronavirus pandemic. The bunyaviruses, together with the flaviviruses and filoviruses, are the major etiological agents of viral hemorrhagic fever, and several of them have been listed as priority pathogens by the World Health Organization for which insufficient countermeasures exist. Based on new techniques allowing rapid analysis of the repertoire of protective antibodies induced during infection, combined with atomic-level structural information on viral surface proteins, structural vaccinology is now instrumental in the combat against newly emerging threats, as it allows rapid rational design of novel vaccine antigens. Here, we discuss the contribution of structural vaccinology and the current challenges that remain in the search for an efficient vaccine against some of the deadliest bunyaviruses.

Encephalitic Arboviruses of Africa: Emergence, Clinical Presentation and Neuropathogenesis.

Many mosquito-borne viruses (arboviruses) are endemic in Africa, contributing to systemic and neurological infections in various geographical locations on the continent. While most arboviral infections do not lead to neuroinvasive diseases of the central nervous system, neurologic diseases caused by arboviruses include flaccid paralysis, meningitis, encephalitis, myelitis, encephalomyelitis, neuritis, and post-infectious autoimmune or memory disorders. Here we review endemic members of the Flaviviridae and Togaviridae families that cause neurologic infections, their neuropathogenesis and host neuroimmunological responses in Africa. We also discuss the potential for neuroimmune responses to aide in the development of new diagnostics and therapeutics, and current knowledge gaps to be addressed by arbovirus research.

Implicating bites from a leishmaniasis sand fly vector in the loss of tolerance in pemphigus.

A possible etiological link between the onset of endemic pemphigus in Tunisia and bites of Phlebotomus papatasi, the vector of zoonotic cutaneous leishmaniasis, has been previously suggested. We hypothesized that the immunodominant P. papatasi salivary protein PpSP32 binds to desmogleins 1 and 3 (Dsg1 and Dsg3), triggering loss of tolerance to these pemphigus target autoantigens. Here, we show using far-Western blot that the recombinant PpSP32 protein (rPpSP32) binds to epidermal proteins with a MW of approximately 170 kDa. Coimmunoprecipitation revealed the interaction of rPpSP32 with either Dsg1 or Dsg3. A specific interaction between PpSP32 and Dsg1 and Dsg3 was further demonstrated by ELISA assays. Finally, mice immunized with rPpSP32 twice per week exhibited significantly increased levels of anti-Dsg1 and -Dsg3 antibodies from day 75 to 120. Such antibodies were specific for Dsg1 and Dsg3 and were not the result of cross-reactivity to PpSP32. In this study, we demonstrated for the first time to our knowledge a specific binding between PpSP32 and Dsg1 and Dsg3, which might underlie the triggering of anti-Dsg antibodies in patients exposed to sand fly bites. We also confirmed the development of specific anti-Dsg1 and -Dsg3 antibodies in vivo after PpSP32 immunization in mice. Collectively, our results provide evidence that environmental factors, such as the exposure to P. papatasi bites, can trigger the development of autoimmune antibodies.

The Nonstructural Protein of Guertu Virus Disrupts Host Defenses by Blocking Antiviral Interferon Induction and Action.

Guertu virus (GTV) is a potentially highly pathogenic bunyavirus newly isolated in China, which is genetically related to the severe fever with thrombocytopenia syndrome virus (SFTSV) and Heartland virus (HRTV), two other emerging life-threatening bunyaviruses. Previous studies suggested that SFTSV and HRTV antagonize the interferon (IFN) system by targeting antiviral signaling proteins in different ways. However, whether and how GTV counteracts the host innate immunity are unclear. Here, we found that GTV strongly inhibits both IFN induction and action through its nonstructural protein (NSs). Different from the NSs of SFTSV and HRTV, GTV NSs (G-NSs) induced the formation of two distinctive cytoplasmic structures, compact inclusion bodies (IBs) and extended filamentous structures (FSs). Protein interaction and colocalization analyses demonstrated that G-NSs interacts with TBK1 (TANK binding kinase-1, the pivotal kinase for IFN induction) and STAT2 (signal transducer and activator of transcription 2, the essential transcription factor for IFN action) and irreversibly sequesters the host proteins into the viral IBs and FSs. Consistently, G-NSs thus inhibited phosphorylation/activation and nuclear translocation of IFN-regulatory factor 3 (IRF3, the substrate of TBK1), diminishing the IFN induction. Furthermore, G-NSs sequestration of STAT2 blocked phosphorylation/activation and nuclear translocation of STAT2, disabling IFN action and host antiviral state establishment. Collectively, this study shows the robust subversion of the two phases of the IFN antiviral system by GTV and unravels the respective molecular mechanisms, exhibiting some notable differences from those employed by SFTSV and HRTV, providing insights into the virus-host interactions and pathogenesis, and probably also benefiting the prevention and treatment of the related infectious diseases in the future.

Structural and functional similarities in bunyaviruses: Perspectives for pan-bunya antivirals.

The order of Bunyavirales includes numerous (re)emerging viruses that collectively have a major impact on human and animal health worldwide. There are no vaccines for human use or antiviral drugs available to prevent or treat infections with any of these viruses. The development of efficacious and safe drugs and vaccines is a pressing matter. Ideally, such antivirals possess pan-bunyavirus antiviral activity, allowing the containment of every bunya-related threat. The fact that many bunyaviruses need to be handled in laboratories with biosafety level 3 or 4, the great variety of species and the frequent emergence of novel species complicate such efforts. We here examined the potential druggable targets of bunyaviruses, together with the level of conservation of their biological functions, structure, and genetic similarity by means of heatmap analysis. In the light of this, we revised the available models and tools currently available, pointing out directions for antiviral drug discovery.

Serologic Survey of Snowshoe Hares (Lepus americanus) in the Greater Yellowstone Area for Brucellosis, Tularemia, and Snowshoe Hare Virus.

We examined sera from snowshoe hares (Lepus americanus) livetrapped in the northern Greater Yellowstone Area (GYA), US, for antibodies to Brucella abortus, Francisella tularensis, and snowshoe hare virus (SSHV). Zero of 90, 0 of 67, and 40 of 100 samples were antibody positive for B. abortus, F. tularensis, and SSHV, respectively. Hares were trapped from 2009 to 2012, and of the six animals that were captured twice with at least 1 yr between captures, four developed antibody to SSHV, indicating active exposure to the agent. These findings suggest snowshoe hares in the GYA do not play a significant role as a reservoir of B. abortus, but do maintain the zoonotic, encephalitic SSHV in the population.

Seasonal dynamics of thrips (Thrips tabaci) (Thysanoptera: Thripidae) transmitters of iris yellow spot virus: a serious viral pathogen of onion bulb and seed crops.

Thrips-transmitted Iris yellow spot virus (IYSV) is an important economic constraint to the production of bulb and seed onion crops in the United States and many other parts of the world. Because the virus is exclusively spread by thrips, the ability to rapidly detect the virus in thrips vectors would facilitate studies on the role of thrips in virus epidemiology, and thus formulation of better vector management strategies. Using a polyclonal antiserum produced against the recombinant, Escherichia coli-expressed nonstructural protein coded by the small (S) RNA of IYSV, an enzyme linked immunosorbent assay was developed for detecting IYSV in individual as well as groups of adult thrips. The approach enabled estimating the proportion of potential thrips transmitters in a large number of field-collected thrips collected from field-grown onion plants. Availability of a practical and inexpensive test to identify viruliferous thrips would be useful in epidemiological studies to better understand the role of thrips vectors in outbreaks of this economically important virus of onion.

Human antibody neutralizes severe Fever with thrombocytopenia syndrome virus, an emerging hemorrhagic Fever virus.

Severe fever with thrombocytopenia syndrome virus (SFTSV), a newly discovered member of the Bunyaviridae family, is the causative agent of an emerging hemorrhagic fever, SFTS, in China. Currently, there are no vaccines or effective therapies against SFTS. In this study, a combinatorial human antibody library was constructed from the peripheral lymphocytes of 5 patients who had recovered from SFTS. The library was screened against purified virions for the production of single-chain variable-region fragments (ScFv). Of the 6 positive clones, one clone (monoclonal antibody [MAb] 4-5) showed neutralizing activity against SFTSV infection in Vero cells. MAb 4-5 was found to effectively neutralize all of the clinical isolates of SFTSV tested, which were isolated from patients in China from 2010 to 2012. MAb 4-5 was found to bind a linear epitope in the ectodomain of glycoprotein Gn. Its neutralizing activity is attributed to blockage of the interactions between the Gn protein and the cellular receptor, indicating that inhibition of virus-cell attachment is its main mechanism. These data suggest that MAb 4-5 can be used as a promising candidate molecule for immunotherapy against SFTSV infection.

Infection with a novel virus causes hemorrhagic fever in China.

BACKGROUND: Patients have been identified with hemorrhagic fever (HF) caused by Huaiyangshan virus (HYSV) infection since 2009. This study aimed to investigate the characteristics of clinical symptoms, laboratory examinations, epidemiological factors, and therapeutic responses in patients with Huaiyangshan hemorrhagic fever (HYSHF). METHODS: A total of 57 patients with a suspected HF were admitted to the Department of Infectious Diseases, the Affiliated Union Hospital of Tongji Medical College between June 2009 and October 2010. A potential infection with HYSV was determined by indirect immunofluorescent assay and reverse-transcription (RT)-PCR. The clinical symptoms, epidemiological characteristics, laboratory examinations, and therapeutic responses of these patients were evaluated. RESULTS: Forty-eight out of 57 patients were diagnosed with HYSHF. They displayed diverse clinical symptoms, such as an acute febrile flu-like illness, and progressed to proteinuria, hemorrhagic manifestations, and encephalopathy. Some patients exhibited progressive leukopenia, thrombocytopenia, liver and kidney dysfunction, and systemic cell injury. Following symptom-specific treatment, 35 patients recovered completely and 13 patients died from severe complications, including central nervous system manifestations. CONCLUSIONS: Patients with HYSHF displayed diverse clinical symptoms and laboratory findings. Such patients should be treated immediately and closely monitored to prevent severe complications.

Identification of a novel subtype of South River virus (family Bunyaviridae).

We previously reported the isolation of South River virus (SORV) from a pool of mosquitoes collected in the Yucatan Peninsula of Mexico (Farfan-Ale et al. in Vector Borne Zoonotic Dis 10:777-783, 5). The isolate (designated SORV-252) was identified as SORV after a 197-nucleotide region of its small RNA genome segment was sequenced. In the present study, the complete small and medium RNA genome segments and part of the large RNA genome segment of SORV-252 were sequenced and shown to have 92%, 85% and 90% nucleotide sequence identity, respectively, to the homologous regions of the prototype SORV isolate (NJO-94F). To determine the antigenic relationship between SORV-252 and NJO-94F, cross-plaque reduction neutralization tests (PRNTs) were performed using sera from mice inoculated with these viruses. SORV-252 and NJO-94F were distinguishable in the cross-neutralization assays; there was a twofold difference in the PRNT titers in one direction and a fourfold difference in the other direction, suggesting that SORV-252 represents a novel subtype of SORV. Additionally, SORV-252 and NJO-94F have distinct plaque morphologies in African green monkey kidney (Vero) cells. In conclusion, we provide evidence that a novel subtype of SORV is present in the Yucatan Peninsula of Mexico.

Antiviral escape strategies developed by bunyaviruses pathogenic for humans.

New or re-emerging pathogens for humans have emerged outside of their usual endemic range during the last decade originating severe public health concern and economical losses. Climate changes have played a significant role in the emergence or re-emergence of arboviruses. Among these pathogens, several viruses belong to the Bunyaviridae family. This family is composed of RNA viruses grouped into five genera Orthobunyavirus, Hantavirus, Nairovirus, Phlebovirus and Tospovirus characterized by their antigenic, genetic and ecological properties. These viruses use cellular proteins to promote their own replication/transcription and reciprocally the host induces, in response, an important transcriptional reprogramming to activate antiviral defences including the interferon type I pathways. The virulence of the pathogenic bunyaviruses is directly linked to the roles of viral virulence factors and their capacity to counteract the host pathways. This review summarizes the various strategies developed by the different genera of the Bunyaviridae family to overcome and escape the innate immune response and eventually other cellular functions.

Viruses in the Anopheles A, Anopheles B, and Tete serogroups in the Orthobunyavirus genus (family Bunyaviridae) do not encode an NSs protein.

Viruses in the genus Orthobunyavirus, family Bunyaviridae, have a genome comprising three segments (called L, M, and S) of negative-sense RNA. Serological studies have classified the >170 named virus isolates into 18 serogroups, with a few additional as yet ungrouped viruses. Until now, molecular studies and full-length S-segment nucleotide sequences were available for representatives of eight serogroups; in all cases, the S segment encodes two proteins, N (nucleocapsid) and NSs (nonstructural), in overlapping open reading frames (ORFs) that are translated from the same mRNA. The NSs proteins of Bunyamwera virus (BUNV) and California serogroup viruses have been shown to play a role in inhibiting host cell mRNA and protein synthesis, thereby preventing induction of interferon (IFN). We have determined full-length sequences of the S segments of representative viruses in the Anopheles A, Anopheles B, and Tete serogroups, and we report here that these viruses do not show evidence of having an NSs ORF. In addition, these viruses have rather longer N proteins than those in the other serogroups. Most of the naturally occurring viruses that lack the NSs protein behaved like a recombinant BUNV with the NSs gene deleted in that they failed to prevent induction of IFN-beta mRNA. However, Tacaiuma virus (TCMV) in the Anopheles A serogroup inhibited IFN induction in a manner similar to that of wild-type BUNV, suggesting that TCMV has evolved an alternative mechanism, not involving a typical NSs protein, to antagonize the host innate immune response.

[Selected immunological processes in viral haemorrhagic fevers infections]. / Wybrane procesy immunologiczne zwiazane z wirusami goraczek krwotocznych.

The paper reviews the known facts on the immunological response in infections with viral haemorrhagic fevers--dangerous pathogens for life and health of people. Immunological process registered in infections with viruses from Arenaviridae, Bunyaviridae, Filoviridae and Flaviviridae have been described. Moreover, the immunological response in infection with the RHD (rabbit haemorrhagic disease) virus from Caliciviridae family have been shown as a potential model for laboratory research on the duration and pathogenesis of viral haemorrhagic fevers.

Characterization of temperature-sensitive Akabane virus mutants and their roles in attenuation.

Akabane virus (AKAV) of the genus Orthobunyavirus in the family Bunyaviridae is an important animal pathogen; however, studies on AKAV biology are scarce. Therefore, we generated temperature-sensitive (ts) mutants of AKAV in order to study its pathogenesis. The ts AKAV mutants were generated by incubating the virulent OBE-1 strain with the chemical mutagen 5-fluorouracil. Each ts mutant was inoculated intracerebrally into mice to assess its virulence, and the genomic sequences of the attenuated mutants were also determined. Three of the twelve ts mutants studied showed a mortality rate of less than 10%. Although no mutation was detected in the S RNA segment of these three mutants, amino acid substitutions were observed in both the M and L RNA segments. Three of the mutants and the wild-type virus demonstrated a similar pattern of immunoreactivity in an ELISA with anti-Gc monoclonal antibodies. On the other hand, using a minireplicon system, the level of L protein activity of each ts mutant decreased as the temperature increased. These results suggest that the L RNA segment could be involved in the virulence of AKAV, which increases our understanding of how the viral gene products contribute to pathogenesis.

Arthrogryposis, hydranencephaly and cerebellar hypoplasia syndrome in neonatal calves resulting from intrauterine infection with Aino virus.

To determine the teratogenic potential of Aino virus (AINOV) in cattle, pregnant cows and fetal cattle were infected with a fresh isolate of AINOV. Five pregnant cows were inoculated intravenously with the virus at 122 to 162 days of gestation and allowed to give birth. All of the cows developed neutralizing antibodies to the virus, indicating that the cows had been infected with the virus; however, no clinical abnormalities were seen in their six newborn calves, and no specific antibodies to the virus were detected in the precolostral serum of calves. Five fetuses with fetal ages ranging from 132 to 156 days were inoculated in utero with the virus. One weak newborn and four stillborn calves were delivered at gestation days 256 to 263, i.e., less than the standard gestation term; they had congenital abnormalities including arthrogryposis, hydranencephaly and cerebellar hypoplasia. Antibodies specific to AINOV were detected in their precolostral serum. These results demonstrate that AINOV is a potential etiological agent of congenital malformation of cattle.

Induction of severe disease in hamsters by two sandfly fever group viruses, Punta toro and Gabek Forest (Phlebovirus, Bunyaviridae), similar to that caused by Rift Valley fever virus.

Adult golden hamsters inoculated subcutaneously with either of two sandfly fever group viruses, Punta Toro and Gabek Forest (Phlebovirus, Bunyaviridae), developed a fulminating fatal illness characterized by hepatic and splenic necrosis and interstitial pneumonitis. Most animals died within three days after infection; this was accompanied by high levels of viremia. Necropsy and histopathologic examination of the infected animals revealed pathologic changes involving multiple organs that resembled those described in Rift Valley fever. These two hamster-phlebovirus systems may serve as alternative animal models for Rift Valley fever and should be useful in studying the pathogenesis of severe phlebovirus infection and for testing potential therapeutic agents.

[The use of monoclonal antibodies in studying the causative agents of viral hemorrhagic fevers]. / Ispol'zovanie monoclonal'nykh antitel v issledovanii vozbuditelei virusnykh gemorragicheskikh likhoradok.

The most promising trends of using monoclonal antibodies (Mabs) in virology research of the viral hemorrhagic-fevers' agents are related with studying viral antigen structure and with developing diagnostic preparations for indicating and identifying infectious agents of the mentioned pathologies. The methodological specificity of obtaining the Mabs to viral hemorrhagic-fevers' agents as well as data on its practical use are discussed.

Seroprevalence of three emerging arboviral infections in Kuwaiti nationals.

Diseases caused by dengue, sandfly fever and hanta viruses pose a major health risk in many countries. We determined the threat of these arboviral infections through a serologic using enzyme linked immunosorbent assay (ELISA) based tests. Hantavirus-specific antibodies were also detected using immunofluorescence. Of 499 samples tested for dengue virus IgG antibodies l4% were as positive for dengue positive by all the ELISA tests. Among the 42 showing strong IgG reactivity, only 1 was positive for dengue virus IgM antibodies. All samples tested for IgG antibodies to sandfly fever virus were negative. Hantavirus antibodies were detected in 11% of the 46 samples from high-risk individuals. The low prevalences suggest that at present these infections are not a serious problem in Kuwait.