Activation of neurotoxic A1-reactive astrocytes by SFTS virus infection accelerates fatal brain damage in IFNAR1-/- mice.

Severe fever with thrombocytopenia syndrome (SFTS) has a high mortality rate compared to other infectious diseases. SFTS is particularly associated with a high risk of mortality in immunocompromised individuals, while most patients who die of SFTS exhibit symptoms of severe encephalitis before death. However, the region of brain damage and mechanisms by which the SFTS virus (SFTSV) causes encephalitis remains unknown. Here, we revealed that SFTSV infects the brainstem and spinal cord, which are regions of the brain associated with respiratory function, and motor nerves in IFNAR1-/- mice. Further, we show that A1-reactive astrocytes are activated, causing nerve cell death, in infected mice. Primary astrocytes of SFTSV-infected IFNAR1-/- mice also induced neuronal cell death through the activation of A1-reactive astrocytes. Herein, we showed that SFTSV induces fatal neuroinflammation in the brain regions important for respiratory function and motor nerve, which may underlie mortality in SFTS patients. This study provides new insights for the treatment of SFTS, for which there is currently no therapeutic approach.

Replication of Akabane virus and related orthobunyaviruses in a fetal-bovine-brain-derived cell line.

Akabane virus (AKAV), Aino virus, Peaton virus, Sathuperi virus, and Shamonda virus are arthropod-borne viruses belonging to the order Elliovirales, family Peribunyaviridae, genus Orthobunyavirus. These viruses cause or may cause congenital malformations in ruminants, including hydranencephaly, poliomyelitis, and arthrogryposis, although their pathogenicity may vary among field cases. AKAV may cause relatively severe congenital lesions such as hydranencephaly in calves. Furthermore, strains of AKAV genogroups I and II exhibit different disease courses. Genogroup I strains predominantly cause postnatal viral encephalomyelitis, while genogroup II strains are primarily detected in cases of congenital malformation. However, the biological properties of AKAV and other orthobunyaviruses are insufficiently investigated in hosts in the field and in vitro. Here, we used an immortalized bovine brain cell line (FBBC-1) to investigate viral replication efficiency, cytopathogenicity, and host innate immune responses. AKAV genogroup II and Shamonda virus replicated to higher titers in FBBC-1 cells compared with the other viruses, and only AKAV caused cytopathic effects. These results may be associated with the severe congenital lesions in the brain caused by AKAV genogroup II. AKAV genogroup II strains replicated to higher titers in FBBC-1 cells than AKAV genogroup I strains, suggesting that genogroup II strains replicated more efficiently in fetal brain cells, accounting for the detection of the latter strains mainly in fetal infection cases. Therefore, FBBC-1 cells may serve as a valuable tool for investigating the virulence and tropism of the orthobunyaviruses for bovine neonatal brain tissues in vitro.

The Severe Fever with Thrombocytopenia Syndrome Virus NSs Protein Interacts with CDK1 To Induce G2 Cell Cycle Arrest and Positively Regulate Viral Replication.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is a newly identified phlebovirus associated with severe hemorrhagic fever in humans. While many viruses subvert the host cell cycle to promote viral growth, it is unknown whether this is a strategy employed by SFTSV. In this study, we investigated how SFTSV manipulates the cell cycle and the effect of the host cell cycle on SFTSV replication. Our results suggest that cells arrest at the G2/M transition following infection with SFTSV. The accumulation of cells at the G2/M transition did not affect virus adsorption and entry but did facilitate viral replication. In addition, we found that SFTSV NSs, a nonstructural protein that forms viroplasm-like structures in the cytoplasm of infected cells and promotes virulence by modulating the interferon response, induces a large number of cells to arrest at the G2/M transition by interacting with CDK1. The interaction between NSs and CDK1, which is inclusion body dependent, inhibits formation and nuclear import of the cyclin B1-CDK1 complex, thereby leading to cell cycle arrest. Expression of a CDK1 loss-of-function mutant reversed the inhibitive effect of NSs on the cell cycle, suggesting that this protein is a potential antiviral target. Our study provides new insight into the role of a specific viral protein in SFTSV replication, indicating that NSs induces G2/M arrest of SFTSV-infected cells, which promotes viral replication.IMPORTANCE Severe fever with thrombocytopenia syndrome virus (SFTSV) is a tick-borne pathogen that causes severe hemorrhagic fever. Although SFTSV poses a serious threat to public health and was recently isolated, its pathogenesis remains unclear. In particular, the relationship between SFTSV infection and the host cell cycle has not been described. Here, we show for the first time that both asynchronized and synchronized SFTSV-susceptible cells arrest at the G2/M checkpoint following SFTSV infection and that the accumulation of cells at this checkpoint facilitates viral replication. We also identify a key mechanism underlying SFTSV-induced G2/M arrest, in which SFTSV NSs interacts with CDK1 to inhibit formation and nuclear import of the cyclin B1-CDK1 complex, thus preventing it from regulating cell cycle progression. Our study highlights the key role that NSs plays in SFTSV-induced G2/M arrest.

Severe Fever with Thrombocytopenia Syndrome Virus NSs Interacts with TRIM21 To Activate the p62-Keap1-Nrf2 Pathway.

Nuclear factor erythroid 2-related factor 2 (Nrf2) dissociates from its inhibitor, Keap1, upon stress signals and subsequently induces an antioxidant response that critically controls the viral life cycle and pathogenesis. Besides intracellular Fc receptor function, tripartite motif 21 (TRIM21) E3 ligase plays an essential role in the p62-Keap1-Nrf2 axis pathway for redox homeostasis. Specifically, TRIM21-mediated p62 ubiquitination abrogates p62 oligomerization and sequestration activity and negatively regulates the Keap1-Nrf2-mediated antioxidant response. A number of viruses target the Nrf2-mediated antioxidant response to generate an optimal environment for their life cycle. Here we report that a nonstructural protein (NSs) of severe fever with thrombocytopenia syndrome virus (SFTSV) interacts with and inhibits TRIM21 to activate the Nrf2 antioxidant signal pathway. Mass spectrometry identified TRIM21 to be a binding protein for NSs. NSs bound to the carboxyl-terminal SPRY subdomain of TRIM21, enhancing p62 stability and oligomerization. This facilitated p62-mediated Keap1 sequestration and ultimately increased Nrf2-mediated transcriptional activation of antioxidant genes, including those for heme oxygenase 1, NAD(P)H quinone oxidoreductase 1, and CD36. Mutational analysis found that the NSs-A46 mutant, which no longer interacted with TRIM21, was unable to increase Nrf2-mediated transcriptional activation. Functionally, the NS wild type (WT), but not the NSs-A46 mutant, increased the surface expression of the CD36 scavenger receptor, resulting in an increase in phagocytosis and lipid uptake. A combination of reverse genetics and assays with Ifnar-/- mouse models revealed that while the SFTSV-A46 mutant replicated similarly to wild-type SFTSV (SFTSV-WT), it showed weaker pathogenic activity than SFTSV-WT. These data suggest that the activation of the p62-Keap1-Nrf2 antioxidant response induced by the NSs-TRIM21 interaction contributes to the development of an optimal environment for the SFTSV life cycle and efficient pathogenesis.IMPORTANCE Tick-borne diseases have become a growing threat to public health. SFTSV, listed by the World Health Organization as a prioritized pathogen, is an emerging phlebovirus, and fatality rates among those infected with this virus are high. Infected Haemaphysalis longicornis ticks are the major source of human SFTSV infection. In particular, the recent spread of this tick to over 12 states in the United States has increased the potential for outbreaks of this disease beyond Far East Asia. Due to the lack of therapies and vaccines against SFTSV infection, there is a pressing need to understand SFTSV pathogenesis. As the Nrf2-mediated antioxidant response affects viral life cycles, a number of viruses deregulate Nrf2 pathways. Here we demonstrate that the SFTSV NSs inhibits the TRIM21 function to upregulate the p62-Keap1-Nrf2 antioxidant pathway for efficient viral pathogenesis. This study not only demonstrates the critical role of SFTSV NSs in viral pathogenesis but also suggests potential future therapeutic approaches to treat SFTSV-infected patients.

Polyamine Depletion Inhibits Bunyavirus Infection via Generation of Noninfectious Interfering Virions.

Several host and viral processes contribute to forming infectious virions. Polyamines are small host molecules that play diverse roles in viral replication. We previously demonstrated that polyamines are crucial for RNA viruses; however, the mechanisms by which polyamines function remain unknown. Here, we investigated the role of polyamines in the replication of the bunyaviruses Rift Valley fever virus (vaccine strain MP-12) and La Crosse virus (LACV). We found that polyamine depletion did not impact viral RNA or protein accumulation, despite significant decreases in titer. Viral particles demonstrated no change in morphology, size, or density. Thus, polyamine depletion promotes the formation of noninfectious particles. These particles interfere with virus replication and stimulate innate immune responses. We extended this phenotype to Zika virus; however, coxsackievirus did not similarly produce noninfectious particles. In sum, polyamine depletion results in the accumulation of noninfectious particles that interfere with replication and stimulate immune signaling, with important implications for targeting polyamines therapeutically, as well as for vaccine strategies.IMPORTANCE Bunyaviruses are emerging viral pathogens that cause encephalitis, hemorrhagic fevers, and meningitis. We have uncovered that diverse bunyaviruses require polyamines for productive infection. Polyamines are small, positively charged host-derived molecules that play diverse roles in human cells and in infection. In polyamine-depleted cells, bunyaviruses produce an overabundance of noninfectious particles that are indistinguishable from infectious particles. However, these particles interfere with productive infection and stimulate antiviral signaling pathways. We further find that additional enveloped viruses are similarly sensitive to polyamine depletion but that a nonenveloped enterovirus is not. We posit that polyamines are required to maintain bunyavirus infectivity and that polyamine depletion results in the accumulation of interfering noninfectious particles that limit infectivity. These results highlight a novel means by which bunyaviruses use polyamines for replication and suggest promising means to target host polyamines to reduce virus replication.

Keystone Virus Isolated From a Florida Teenager With Rash and Subjective Fever: Another Endemic Arbovirus in the Southeastern United States?

Keystone virus, a California-serogroup orthobunyavirus, was first isolated in 1964 from mosquitoes in Keystone, Florida. There were no prior reports of isolation from humans, despite studies suggesting that ~20% of persons living in the region are seropositive. We report virus isolation from a Florida teenager with a rash and fever.

Experimental infection of golden hamsters with Guama virus (Peribunyaviridae, Orthobunyavirus).

The Guama virus (GMAV) is a member of Peribunyaviridae family, Orthobunyavirus genus. Several strains of the virus were isolated in South and Central Americas from several hosts, such as humans, wild animals, including nonhuman primates, wild rodents and mosquitoes as well as mice used as sentinels. The virus is able to cause febrile disease in humans. Here we describe for the first time pathologic and biochemical findings in golden hamsters (Mesocricetus auratus) infected with the prototype GMAV. Blood and organs of infected and control animals were collected every 24 h after infection from the 1st to the 7th day post infection (dpi) and at 21 dpi when experiment was ended. The tissues were processed for histopathology and immunohistochemistry. The blood and serum were used to determine viremia and biochemical markers plus to detect anti-GMAV antibodies. The viremia was early detected already on the 1st dpi and it was no longer detected on the 3rd dpi. Total anti-GMAV antibodies were detected from the 6th dpi. Hepatic markers as ALT of infected animals were increased and showed statistically significant difference in comparison with control animals, indicating damage of the liver; indeed the liver was the most affected organ, but other organs presented lesions and positive GMAV immunostaining as brain, lung, liver, spleen, and kidney. Our findings indicate that golden hamsters are a good animal model for experimental infection of the GMAV.

Multiple organ involvement in severe fever with thrombocytopenia syndrome: an immunohistochemical finding in a fatal case.

BACKGROUND: Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease caused by SFTS bunyavirus (SFTSV), a tick borne bunyavirus. However, Immunohistochemistry of SFTS patients are not well studied. METHODS: We obtained multiple of tissues from a fatal case with SFTS, including blood, lungs, kidneys, heart, and spleen. The blood samples were used to isolate the causative agent for detection of viral RNA and further expression of recombinant viral protein as primary antibody. Immunohistochemistry of the heart, lungs, spleen and kidneys was used to characterize the viral antigen in tissue sections. RESULTS: A 79-year-old man, together with his wife, was admitted because of fever. Both patients were diagnosed with SFTS by the positive SFTSV RNA in the blood. The gentleman died of multiple organ failure 8 days after hospitalization. However, his wife recovered and was discharged. Immunohistochemistry indicated that SFTSV antigens were present in all studied organs including the heart, kidney, lung and spleen, of which the spleen presented with the highest amount of SFTSV antigens. The kidney was next while the heart and lungs showed lower amount of SFTSV antigens. CONCLUSIONS: SFTSV can direct infect multiple organs, resulting in multiple organ failure and ultimately in an unfavorable outcome.

Pathophysiology of severe fever with thrombocytopenia syndrome and development of specific antiviral therapy.

Severe fever with thrombocytopenia syndrome (SFTS) caused by SFTS virus (SFTSV), a novel phlebovirus, was reported to be endemic to central and northeastern PR China and was also to be endemic to South Korea and western Japan. SFTS is an emerging viral infection, which should be categorized as a viral hemorrhagic fever disease as Crimean-Congo hemorrhagic fever (CCHF) is caused by CCHF virus. SFTS is a tick-borne viral infection. SFTSV is maintained between several species of ticks and wild and domestic animals in nature. Patients with SFTS show symptoms of fever, general fatigue, and gastrointestinal symptoms such as bloody diarrhea. The severely ill SFTS patients usually show gastrointestinal hemorrhage and deteriorated consciousness. The case fatality rate of SFTS ranges from 5 to 40%. Pathological studies on SFTS have revealed that the mechanisms behind the high case fatality rate are virus infection-related hemophagocytic syndrome associated with cytokine storm, coagulopathy due to disseminated intravascular coagulation causing bleeding tendency, and multi-organ failure. Favipiravir was reported to show efficacy in the prevention and treatment of SFTSV infections in an animal model. A clinical study to evaluate the efficacy of favipiravir in the treatment of SFTS patients has been initiated in Japan. SFTSV is circulating in nature in PR China, Korea, and Japan, indicating that we cannot escape from the risk being infected with SFTSV. The development of specific therapy and preventive measures is a pressing issue requiring resolution to reduce the morbidity and mortality of SFTS patients.

Novel Clinical and Pathologic Findings in a Heartland Virus-Associated Death.

We describe an investigation into a Heartland virus (HRTV)-associated death in Tennessee with novel clinical and pathologic findings. HRTV can cause rapidly fatal, widely disseminated infection with multisystem organ failure in patients without substantial comorbidities. We identified viral antigen in multiple organ tissues where it was not detected previously.

Congenital Malformations of Calves Infected with Shamonda Virus, Southern Japan.

In 2015 and 2016, we observed 15 malformed calves that were exposed to intrauterine infection with Shamonda virus, a Simbu serogroup orthobunyavirus, in Japan. Characteristic manifestations were arthrogryposis and gross lesions in the central nervous system. Our results indicate that this arbovirus should be considered a teratogenic virus in ruminants.

Correlation of cytokine level with the severity of severe fever with thrombocytopenia syndrome.

BACKGROUND: Severe fever with thrombocytopenia syndrome (SFTS) was an emerging hemorrhagic fever that was caused by a tick-borne bunyavirus, SFTSV. Although SFTSV nonstructural protein can inhibit type I interferon (IFN-I) production Ex Vivo and IFN-I played key role in resistance SFTSV infection in animal model, the role of IFN-I in patients is not investigated. METHODS: We have assayed the concentration of IFN-α, a subtype of IFN-I as well as other cytokines in the sera of SFTS patients and the healthy population with CBA (Cytometric bead array) assay. RESULTS: The results showed that IFN-α, tumor necrosis factor (TNF-α), granulocyte colony-stimulating factor (G-CSF), interferon-γ (IFN-γ), macrophage inflammatory protein (MIP-1α), interleukin-6 (IL-6), IL-10, interferon-inducible protein (IP-10), monocyte chemoattractant protein (MCP-1) were significantly higher in SFTS patients than in healthy persons (p < 0.05); the concentrations of IFN-α, IFN-γ, G-CSF, MIP-1α, IL-6, and IP-10 were significant higher in severe SFTS patients than in mild SFTS patients (p < 0.05). CONCLUSION: The concentration of IFN-α as well as other cytokines (IFN-γ, G-CSF, MIP-1α, IL-6, and IP-10) is correlated with the severity of SFTS, suggesting that type I interferon may not be significant in resistance SFTSV infection in humans and it may play an import role in cytokine storm.

Oropouche virus infection and pathogenesis are restricted by MAVS, IRF-3, IRF-7, and type I interferon signaling pathways in nonmyeloid cells.

UNLABELLED: Oropouche virus (OROV) is a member of the Orthobunyavirus genus in the Bunyaviridae family and a prominent cause of insect-transmitted viral disease in Central and South America. Despite its clinical relevance, little is known about OROV pathogenesis. To define the host defense pathways that control OROV infection and disease, we evaluated OROV pathogenesis and immune responses in primary cells and mice that were deficient in the RIG-I-like receptor signaling pathway (MDA5, RIG-I, or MAVS), downstream regulatory transcription factors (IRF-3 or IRF-7), beta interferon (IFN-ß), or the receptor for type I IFN signaling (IFNAR). OROV replicated to higher levels in primary fibroblasts and dendritic cells lacking MAVS signaling, the transcription factors IRF-3 and IRF-7, or IFNAR than in wild-type (WT) cells. In mice, deletion of IFNAR, MAVS, or IRF-3 and IRF-7 resulted in uncontrolled OROV replication, hypercytokinemia, extensive liver damage, and death, whereas WT congenic animals failed to develop disease. Unexpectedly, mice with a selective deletion of IFNAR on myeloid cells (CD11c Cre(+) Ifnar(f/f) or LysM Cre(+) Ifnar(f/f)) did not sustain enhanced disease with OROV or a selective (flox/flox) deletion La Crosse virus, a closely related encephalitic orthobunyavirus. In bone marrow chimera studies, recipient irradiated Ifnar(-/-) mice reconstituted with WT hematopoietic cells sustained high levels of OROV replication and liver damage, whereas WT mice reconstituted with Ifnar(-/-) bone marrow were resistant to disease. Collectively, these results establish a dominant protective role for MAVS, IRF-3 and IRF-7, and IFNAR in restricting OROV infection and tissue injury and suggest that IFN signaling in nonmyeloid cells contributes to the host defense against orthobunyaviruses. IMPORTANCE: Oropouche virus (OROV) is an emerging arthropod-transmitted orthobunyavirus that causes episodic outbreaks of a debilitating febrile illness in humans in countries of South and Central America. The continued expansion of the range and number of its arthropod vectors increases the likelihood that OROV will spread into new regions. At present, the pathogenesis of OROV in humans or other vertebrate animals remains poorly understood. To define cellular mechanisms of control of OROV infection, we performed infection studies in a series of primary cells and mice that were deficient in key innate immune genes involved in pathogen recognition and control. Our results establish that a MAVS-dependent type I IFN signaling pathway has a dominant role in restricting OROV infection and pathogenesis in vivo.

Generation of a Recombinant Akabane Virus Expressing Enhanced Green Fluorescent Protein.

UNLABELLED: We generated a recombinant Akabane virus (AKAV) expressing enhanced green fluorescence protein (eGFP-AKAV) by using reverse genetics. We artificially constructed an ambisense AKAV S genome encoding N/NSs on the negative-sense strand, and eGFP on the positive-sense strand with an intergenic region (IGR) derived from the Rift Valley fever virus (RVFV) S genome. The recombinant virus exhibited eGFP fluorescence and had a cytopathic effect in cell cultures, even after several passages. These results indicate that the gene encoding eGFP in the ambisense RNA could be stably maintained. Transcription of N/NSs and eGFP mRNAs of eGFP-AKAV was terminated within the IGR. The mechanism responsible for this appears to be different from that in RVFV, where the termination sites for N and NSs are determined by a defined signal sequence. We inoculated suckling mice intraperitoneally with eGFP-AKAV, which resulted in neurological signs and lethality equivalent to those seen for the parent AKAV. Fluorescence from eGFP in frozen brain slices from the eGFP-AKAV-infected mice was localized to the cerebellum, pons, and medulla oblongata. Our approach to producing a fluorescent virus, using an ambisense genome, helped obtain eGFP-AKAV, a fluorescent bunyavirus whose viral genes are intact and which can be easily visualized. IMPORTANCE: AKAV is the etiological agent of arthrogryposis-hydranencephaly syndrome in ruminants, which causes considerable economic loss to the livestock industry. We successfully generated a recombinant enhanced green fluorescent protein-tagged AKAV containing an artificial ambisense S genome. This virus could become a useful tool for analyzing AKAV pathogenesis in host animals. In addition, our approach of using an ambisense genome to generate an orthobunyavirus stably expressing a foreign gene could contribute to establishing alternative vaccine strategies, such as bivalent vaccine virus constructs, for veterinary use against infectious diseases.

Toscana virus infects dendritic and endothelial cells opening the way for the central nervous system.

Toscana virus (TOSV) is a Phlebovirus responsible for human neurological infections in endemic Mediterranean areas. The main viral target is the central nervous system, with viremia as a way of dissemination throughout the host. This study was aimed at understanding the spread of TOSV in the host by identifying the cell population infected by the virus and the vehicle to the organs. In vivo studies provided evidence that endothelial cells are infected by TOSV, indicating their potential role in the diffusion of the virus following viremic spread. These results were further confirmed in vitro. Human peripheral mononuclear blood cells were infected with TOSV; only monocyte-derived dendritic cells were identified as susceptible to TOSV infection. Productive viral replication was then observed in human monocyte-derived dendritic cells (moDCs) and in human endothelial cells by recovery of the virus from a cell supernatant. Interleukin-6 was produced by both cell types upon TOSV infection, mostly by endothelial cells, while moDCs particularly expressed TNF-α, which is known to induce a long-lasting decrease in endothelial cell barrier function. These cells could therefore be implicated in the spread of the virus in the host and in the infection of tissues that are affected by the disease, such as the central nervous system. The identification of in vitro and in vivo TOSV cell targets is an important tool for understanding the pathogenesis of the infection, providing new insight into virus-cell interaction for improved knowledge and control of this viral disease.

Experimental infection of cows with newly isolated Akabane virus strain (AKAV-7) causing encephalomyelitis.

Akabane virus (AKAV), an arthropod-transmitted bunyavirus, is a major cause of congenital abnormalities and encephalomyelitis in ruminants. In 2010, there was a major outbreak of encephalomyelitis in Korea and fifteen AKAV strains, including AKAV-7, were isolated from cows. To identify the neuropathogenicity of AKAV-7, we performed experimental infection of cows. Six-month-old female Korean Holstein dairy cattle were inoculated with AKAV-7 by various routes, including intracerebral (IC), intrasubarachnoid space (IS), subcutaneous (SC) and intravenous (IV); a separate group was vaccinated before intravenous infection. Five of the six cows in the IC group and two of the six cows in the IS group showed clinical signs such as locomotor ataxia and paralysis of the hind limbs. Three of six cows died after IC infection 9-12 days post infection (dpi). Histopathologic changes such as nonsuppurative encephalomyelitis were confirmed in various parts of the central nervous system in the IC, IS and SC groups. Early onset of neutralizing antibodies in the serum and lower viral mRNA levels in the peripheral blood mononuclear cells (PBMCs) and various tissues in the vaccinated group was noticeable compared to the unvaccinated group (IV group). We suggest that the AKAV vaccine currently used in Korea may be partially effective for protection against AKAV-7 in cows.

[ZOOPATHOGENIC ORTHOBUNIAVIRUSES (ORTHOBUNYAVIRUS, BUNYAVIRIDAE)].

This work deals with the systematics and taxonomy of orthobunyaviruses, little-studied dangerous and new iruses Akabane, Aino, Schmallenberg, Cache Valley diseases, Oropouche fever. The significance of the reassortment mechanism of their origin and diversification is discussed.

The pathogenesis of severe fever with thrombocytopenia syndrome virus infection in alpha/beta interferon knockout mice: insights into the pathologic mechanisms of a new viral hemorrhagic fever.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is a newly discovered Phlebovirus causing an emerging hemorrhagic fever in East Asia, with reported case fatality rates up to 30%. Despite the high case fatality rate and large number of persons at risk of infection, the pathobiology of the disease is unknown, and no effective animal model has been available for investigating its pathogenesis. We have studied mice and hamsters as potential small-animal models of SFTSV infection following subcutaneous, intraperitoneal, or intracerebral inoculation. Animal tissues were processed for viral load determination, histopathology, immunohistochemistry, and confocal microscopic studies. We found that immunocompetent adult mice and hamsters did not become ill after SFTSV infection. However, alpha/beta interferon receptor knockout (IFNAR(-/-)) mice were highly susceptible to SFTSV infection, and all mice died within 3 to 4 days after subcutaneous inoculation of 10(6) focus-forming units of SFTSV. Histologic examination of tissues of IFNAR(-/-) mice infected with SFTSV showed no detectable lesions. In contrast, by immunohistochemistry virus antigen was found in liver, intestine, kidney, spleen, lymphoid tissue, and brain, but not in the lungs. Mesenteric lymph nodes and spleen were the most heavily infected tissues. Quantitative reverse transcription-PCR (RT-PCR) confirmed the presence of virus in these tissues. Confocal microscopy showed that SFTSV colocalized with reticular cells but did not colocalize with dendritic cells, monocytes/macrophages, neutrophils, or endothelium. Our results indicate that SFTSV multiplied in all organs except for lungs and that mesenteric lymph nodes and spleen were the most heavily infected tissues. The major target cells of SFTSV appear to be reticular cells in lymphoid tissues of intestine and spleen.

Schmallenberg virus pathogenesis, tropism and interaction with the innate immune system of the host.

Schmallenberg virus (SBV) is an emerging orthobunyavirus of ruminants associated with outbreaks of congenital malformations in aborted and stillborn animals. Since its discovery in November 2011, SBV has spread very rapidly to many European countries. Here, we developed molecular and serological tools, and an experimental in vivo model as a platform to study SBV pathogenesis, tropism and virus-host cell interactions. Using a synthetic biology approach, we developed a reverse genetics system for the rapid rescue and genetic manipulation of SBV. We showed that SBV has a wide tropism in cell culture and "synthetic" SBV replicates in vitro as efficiently as wild type virus. We developed an experimental mouse model to study SBV infection and showed that this virus replicates abundantly in neurons where it causes cerebral malacia and vacuolation of the cerebral cortex. These virus-induced acute lesions are useful in understanding the progression from vacuolation to porencephaly and extensive tissue destruction, often observed in aborted lambs and calves in naturally occurring Schmallenberg cases. Indeed, we detected high levels of SBV antigens in the neurons of the gray matter of brain and spinal cord of naturally affected lambs and calves, suggesting that muscular hypoplasia observed in SBV-infected lambs is mostly secondary to central nervous system damage. Finally, we investigated the molecular determinants of SBV virulence. Interestingly, we found a biological SBV clone that after passage in cell culture displays increased virulence in mice. We also found that a SBV deletion mutant of the non-structural NSs protein (SBVΔNSs) is less virulent in mice than wild type SBV. Attenuation of SBV virulence depends on the inability of SBVΔNSs to block IFN synthesis in virus infected cells. In conclusion, this work provides a useful experimental framework to study the biology and pathogenesis of SBV.

First molecular detection and characterization of Akabane virus in small ruminants in Turkey.

Abortion outbreaks associated with congenital malformations in two distinct small-ruminant flocks were reported in Turkey in 2013-2014. This paper describes the first molecular characterization of Turkish Akabane virus strains in small-ruminant flocks using partial sequence analysis of the S segment and pathological findings.