Evaluating the mosquito vector range for two orthobunyaviruses: Oya virus and Ebinur Lake virus.

BACKGROUND: Mosquito-borne viruses cause various infectious diseases in humans and animals. Oya virus (OYAV) and Ebinur Lake virus (EBIV), belonging to the genus Orthobunyavirus within the family Peribunyaviridae, are recognized as neglected viruses with the potential to pose threats to animal or public health. The evaluation of vector competence is essential for predicting the arbovirus transmission risk. METHODS: To investigate the range of mosquito vectors for OYAV (strain SZC50) and EBIV (strain Cu20-XJ), the susceptibility of four mosquito species (Culex pipiens pallens, Cx. quinquefasciatus, Aedes albopictus, and Ae. aegypti) was measured through artificial oral infection. Then, mosquito species with a high infection rate (IR) were chosen to further evaluate the dissemination rate (DR), transmission rate (TR), and transmission efficiency. The viral RNA in each mosquito sample was determined by RT-qPCR. RESULTS: The results revealed that for OYAV, Cx. pipiens pallens had the highest IR (up to 40.0%) among the four species, but the DR and TR were 4.8% and 0.0%, respectively. For EBIV, Cx. pipiens pallens and Cx. quinquefasciatus had higher IR compared to Ae. albopictus (1.7%). However, the EBIV RNA and infectious virus were detected in Cx. pipiens pallens, with a TR of up to 15.4% and a transmission efficiency of 3.3%. CONCLUSIONS: The findings indicate that Cx. pipiens pallens was susceptible to OYAV but had an extremely low risk of transmitting the virus. Culex pipiens pallens and Cx. quinquefasciatus were susceptible to EBIV, and Cx. pipiens pallens had a higher transmission risk to EBIV than Cx. quinquefasciatus.

Pigs in southern Italy are exposed to three ruminant pathogens: an analysis of seroprevalence and risk factors analysis study.

BACKGROUND: Pigs are susceptible to several ruminant pathogens, including Coxiella burnetti, Schmallenberg virus (SBV) and bovine viral diarrhea virus (BVDV). These pathogens have already been described in the pig population, although the dynamics of the infection and the impact on pig farms are currently unclear. The aim of this work was to evaluate the presence of these infections in the pig population of the Campania region, southern Italy, and to evaluate the risk factors associated with a greater risk of exposure. RESULTS: A total of 414 serum samples belonging to 32 herds were tested for the presence of antibodies against SBV, Coxiella, and BVD using commercial multispecies ELISA kits. SBV (5.3%) was the most prevalent pathogen, followed by Coxiella (4.1%) and BVD (3%). The risk factors included in the study (age, sex, province, farming system, ruminant density and major ruminant species) had no influence on the probability of being exposed to BVD and Coxiella, except for the location, in fact more pigs seropositive to Coxiella were found in the province of Caserta. However, the univariate analysis highlighted the influence of age, location, and sex on exposure to SBV. The subsequent multivariate analysis statistically confirmed the importance of these factors. The presence of neutralizing antibodies for SBV and BVDV, or antibodies directed towards a specific phase of infection for Coxiella was further confirmed with virus-neutralization assays and phase-specific ELISAs in a large proportion of positive samples. The presence of high neutralizing antibody titers (especially for SBV) could indicate recent exposures. Twelve of the 17 positive samples tested positive for antibodies against Coxiella phase I or II antigens, indicating the presence of both acute and chronic infections (one animal tested positive for both phases antibodies). CONCLUSIONS: Our study indicates a non-negligible exposure of pigs from southern Italy to the above pathogens. Further studies are necessary to fully understand the dynamics of these infections in pigs, the impact on productivity, and the public health consequences in the case of Coxiella.

Innate immune response against vector-borne bunyavirus infection and viral countermeasures.

Bunyaviruses are a large group of important viral pathogens that cause significant diseases in humans and animals worldwide. Bunyaviruses are enveloped, single-stranded, negative-sense RNA viruses that infect a wide range of hosts. Upon entry into host cells, the components of viruses are recognized by host innate immune system, leading to the activation of downstream signaling cascades to induce interferons (IFNs) and other proinflammatory cytokines. IFNs bind to their receptors and upregulate the expression of hundreds of interferon-stimulated genes (ISGs). Many ISGs have antiviral activities and confer an antiviral state to host cells. For efficient replication and spread, viruses have evolved different strategies to antagonize IFN-mediated restriction. Here, we discuss recent advances in our understanding of the interactions between bunyaviruses and host innate immune response.

Pathogenesis and virulence of Heartland virus.

Heartland virus (HRTV), an emerging tick-borne pathogenic bunyavirus, has been a concern since 2012, with an increasing incidence, expanding geographical distribution, and high pathogenicity in the United States. Infection from HRTV results in fever, thrombocytopenia, and leucopenia in humans, and in some cases, symptoms can progress to severe outcomes, including haemorrhagic disease, multi-organ failure, and even death. Currently, no vaccines or antiviral drugs are available for treatment of the HRTV disease. Moreover, little is known about HRTV-host interactions, viral replication mechanisms, pathogenesis and virulence, further hampering the development of vaccines and antiviral interventions. Here, we aimed to provide a brief review of HRTV epidemiology, molecular biology, pathogenesis and virulence on the basis of published article data to better understand this virus and provide clues for further study.

First case of Oropouche fever detected in the international border region of the Colombian Amazon: clinical characteristics and molecular diagnosis.

OBJECTIVES: We report the first case of Oropouche fever detected in the border region of Colombia. METHODS: Using a multiplex real-time polymerase chain reaction (PCR), genetic sequencing and clinical characteristics during the dengue epidemic in 2019, a total of 175 samples were analysed, from cases notified to the system epidemiological surveillance such as dengue. FINDINGS: The Oropouche virus (OROV) isolate from Leticia belongs to lineage 2 according to both M and S genome segments maximum likelihood (ML) analysis, shares a common ancestor with samples obtained in Esmeraldas, Ecuador and Turbaco, Colombia. The patient: a woman resident in the border neighbourhood of the municipality of Leticia had the following symptoms: fever, headache, retro-orbital pain and myalgias. MAIN CONCLUSION: This cross-border surveillance can be useful to give an alert about the entry or exit of arboviruses circulation in the region, which are often underreported in public health surveillance systems.

Evaluation of the vector competence for Batai virus of native Culex and exotic Aedes species in Central Europe.

BACKGROUND: Batai virus (BATV) is a zoonotic arbovirus of veterinary importance. A high seroprevalence in cows, sheep and goats and infection in different mosquito species has been observed in Central Europe. Therefore, we studied indigenous as well as exotic species of the genera Culex and Aedes for BATV vector competence at different fluctuating temperature profiles. METHODS: Field caught Culex pipiens biotype pipiens, Culex torrentium, Aedes albopictus and Aedes japonicus japonicus from Germany and Aedes aegypti laboratory colony were infected with BATV strain 53.3 using artificial blood meals. Engorged mosquitoes were kept under four (Culex species) or three (Aedes species) fluctuating temperature profiles (18 ± 5 °C, 21 ± 5 °C, 24 ± 5 °C, 27 ± 5 °C) at a humidity of 70% and a dark/light rhythm of 12:12 for 14 days. Transmission was measured by testing the saliva obtained by forced salivation assay for viable BATV particles. Infection rates were analysed by testing whole mosquitoes for BATV RNA by quantitative reverse transcription PCR. RESULTS: No transmission was detected for Ae. aegypti, Ae. albopictus or Ae. japonicus japonicus. Infection was observed for Cx. p. pipiens, but only in the three conditions with the highest temperatures (21 ± 5 °C, 24 ± 5 °C, 27 ± 5 °C). In Cx. torrentium infection was measured at all tested temperatures with higher infection rates compared with Cx. p. pipiens. Transmission was only detected for Cx. torrentium exclusively at the highest temperature of 27 ± 5 °C. CONCLUSIONS: Within the tested mosquito species, only Cx. torrentium seems to be able to transmit BATV if the climatic conditions are feasible.

Development of monoclonal antibodies against oropouche virus and its applicability to immunohistochemical diagnosis.

Orthobunyavirus oropouche ense virus (OROV), the causative agent of Oropouche fever, is widely dispersed in Brazil and South America, causing sporadic outbreaks. Due to the similarity of initial clinical symptoms caused by OROV with other arboviruses found in overlapping geographical areas, differential diagnosis is challenging. As for most neglected tropical diseases, there is a shortage of reagents for diagnosing and studying OROV pathogenesis. We therefore developed and characterized mouse monoclonal antibodies and, one of them recognizes the OROV nucleocapsid in indirect immunofluorescent (IFA) and immunohistochemistry (IHC) assays. Considering that it is the first monoclonal antibody produced for detecting OROV infections, we believe that it will be useful not only for diagnostic purposes but also for performing serological surveys and epidemiological surveillance on the dispersion and prevalence of OROV in Brazil and South America.

Modeling Heartland virus disease in mice and therapeutic intervention with 4'-fluorouridine.

Heartland virus (HRTV) is an emerging tick-borne bandavirus that causes a febrile illness of varying severity in humans, with cases reported in eastern and midwestern regions of the United States. No vaccines or approved therapies are available to prevent or treat HRTV disease. Here, we describe the genetic changes, natural history of disease, and pathogenesis of a mouse-adapted HRTV (MA-HRTV) that is uniformly lethal in 7- to 8-week-old AG129 mice at low challenge doses. We used this model to assess the efficacy of the ribonucleoside analog, 4'-fluorouridine (EIDD-2749), and showed that once-daily oral treatment with 3 mg/kg of drug, initiated after the onset of disease, protects mice against lethal MA-HRTV challenge and reduces viral loads in blood and tissues. Our findings provide insights into HRTV virulence and pathogenesis and support further development of EIDD-2749 as a therapeutic intervention for HRTV disease. IMPORTANCE: More than 60 cases of HRTV disease spanning 14 states have been reported to the United States Centers for Disease Control and Prevention. The expanding range of the Lone Star tick that transmits HRTV, the growing population of at-risk persons living in geographic areas where the tick is abundant, and the lack of antiviral treatments or vaccines raise significant public health concerns. Here, we report the development of a new small-animal model of lethal HRTV disease to gain insight into HRTV pathogenesis and the application of this model for the preclinical development of a promising new antiviral drug candidate, EIDD-2749. Our findings shed light on how the virus causes disease and support the continued development of EIDD-2749 as a therapeutic for severe cases of HRTV infection.

Difference in Intraspecies Transmissibility of Severe Fever with Thrombocytopenia Syndrome Virus Depending on Abrogating Type 1 Interferon Signaling in Mice.

Severe fever with thrombocytopenia syndrome (SFTS), a tick-borne zoonotic disease, is caused by infection with SFTS virus (SFTSV). A previous study reported that human-to-human direct transmission of SFTSV can occur. However, potential animal-to-animal transmission of SFTSV without ticks has not been fully clarified. Thus, the objective of this study was to investigate potential mice-to-mice transmission of SFTSV by co-housing three groups of mice [i.e., wild-type mice (WT), mice injected with an anti-type I interferon-α receptor-blocking antibody (IFNAR Ab), and mice with knockout of type I interferon-α receptor (IFNAR KO)] as spreaders or recipients with different immune competence. As a result, co-housed IFNAR Ab and IFNAR KO mice showed body weight loss with SFTS viral antigens detected in their sera, extracorporeal secretions, and various organs. Based on histopathology, white pulp atrophy in the spleen was observed in all co-housed mice except WT mice. These results obviously show that IFNAR Ab and IFNAR KO mice, as spreaders, exhibited higher transmissibility to co-housed mice than WT mice. Moreover, IFNAR KO mice, as recipients, were more susceptible to SFTSV infection than WT mice. These findings suggest that type I interferon signaling is a pivotal factor in mice intraspecies transmissibility of SFTSV in the absence of vectors such as ticks.

IFITM3 inhibits severe fever with thrombocytopenia syndrome virus entry and interacts with viral Gc protein.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging tick-borne hemorrhagic fever disease with high fatality rate of 10%-20%. Vaccines or specific therapeutic measures remain lacking. Human interferon inducible transmembrane protein 3 (hIFITM3) is a broad-spectrum antiviral factor targeting viral entry. However, the antiviral activity of hIFITM3 against SFTS virus (SFTSV) and the functional mechanism of IFITM3 remains unclear. Here we demonstrate that endogenous IFITM3 provides protection against SFTSV infection and participates in the anti-SFTSV effect of type Ⅰ and Ⅲ interferons (IFNs). IFITM3 overexpression exhibits anti-SFTSV function by blocking Gn/Gc-mediated viral entry and fusion. Further studies showed that IFITM3 binds SFTSV Gc directly and its intramembrane domain (IMD) is responsible for this interaction and restriction of SFTSV entry. Mutation of two neighboring cysteines on IMD weakens IFITM3-Gc interaction and attenuates the antiviral activity of IFITM3, suggesting that IFITM3-Gc interaction may partly mediate the inhibition of SFTSV entry. Overall, our data demonstrate for the first time that hIFITM3 plays a critical role in the IFNs-mediated anti-SFTSV response, and uncover a novel mechanism of IFITM3 restriction of SFTSV infection, highlighting the potential of clinical intervention on SFTS disease.

Revisiting the Importance of Orthobunyaviruses for Animal Health: A Scoping Review of Livestock Disease, Diagnostic Tests, and Surveillance Strategies for the Simbu Serogroup.

Orthobunyaviruses (order Bunyavirales, family Peribunyaviridae) in the Simbu serogroup have been responsible for widespread epidemics of congenital disease in ruminants. Australia has a national program to monitor arboviruses of veterinary importance. While monitoring for Akabane virus, a novel orthobunyavirus was detected. To inform the priority that should be given to this detection, a scoping review was undertaken to (1) characterise the associated disease presentations and establish which of the Simbu group viruses are of veterinary importance; (2) examine the diagnostic assays that have undergone development and validation for this group of viruses; and (3) describe the methods used to monitor the distribution of these viruses. Two search strategies identified 224 peer-reviewed publications for 33 viruses in the serogroup. Viruses in this group may cause severe animal health impacts, but only those phylogenetically arranged in clade B are associated with animal disease. Six viruses (Akabane, Schmallenberg, Aino, Shuni, Peaton, and Shamonda) were associated with congenital malformations, neurological signs, and reproductive disease. Diagnostic test interpretation is complicated by cross-reactivity, the timing of foetal immunocompetence, and sample type. Serological testing in surveys remains a mainstay of the methods used to monitor the distribution of SGVs. Given significant differences in survey designs, only broad mean seroprevalence estimates could be provided. Further research is required to determine the disease risk posed by novel orthobunyaviruses and how they could challenge current diagnostic and surveillance capabilities.

Nairovirus polymerase mutations associated with the establishment of persistent infection in human cells.

Crimean-Congo hemorrhagic fever virus (CCHFV), a tick-borne virus of the Orthonairovirus genus, persistently infects tick cells. It has been reported to establish persistent infection in non-human primates, but virological analysis has not yet been performed in human cells. Here, we investigated whether and how nairoviruses persistently infect human cells using Hazara orthonairovirus (HAZV), a surrogate model for CCHFV. We established a human cell line that was persistently infected with HAZV. Surprisingly, virions of persistently infected HAZV (HAZVpi) were not observed in the culture supernatants. There were five mutations (mut1, mut2, mut3, mut4, and mut5) in L protein of HAZVpi. Mutations in L protein of HAZVpi contribute to non-detection of virion in the supernatants. Lmut4 was found to cause low viral growth rate, despite its high polymerase activity. The low growth rate was restored by Lmut2, Lmut3, and Lmut5. The polymerase activity of Lmut1 was extremely low, and recombinant HAZV carrying Lmut1 (rHAZV/Lmut1) was not released into the supernatants. However, genomes of rHAZV/Lmut1 were retained in the infected cells. All mutations (Lmut1-5) found in L protein of HAZVpi were required for experimental reproduction of HAZVpi, and only Lmut1 and Lmut4 were insufficient. We demonstrated that point mutations in viral polymerase contribute to the establishment of persistent HAZV infection. Furthermore, innate immunity was found to be suppressed in HAZVpi-infected cells, which also potentially contributes to viral persistence. This is the first presentation of a possible mechanism behind how nairoviruses establish persistent infection in human cells. IMPORTANCE: We investigated whether and how nairoviruses persistently infect human cells, using Hazara orthonairovirus (HAZV), a surrogate model for Crimean-Congo hemorrhagic fever virus. We established a human cell line that was persistently infected with HAZV. Five mutations were found in L protein of persistently infected HAZV (HAZVpi): mut1, mut2, mut3, mut4, and mut5. Among them, Lmut1 and Lmut4 restricted viral growth by low polymerase activity and low growth rate, respectively, leading to inhibition of viral overgrowth. The restriction of viral growth caused by Lmut1 and Lmut4 was compensated by other mutations, including Lmut2, Lmut3, and Lmut5. Each of the mutations found in L protein of HAZVpi was concluded to cooperatively modulate viral growth, which facilitates the establishment of persistent infection. Suppression of innate immunity also potentially contributes to virus persistence. This is the first presentation of a possible mechanism behind how nairoviruses establish persistent infection in human cells.

Skin muscle is the initial site of viral replication for arboviral bunyavirus infection.

The first step in disease pathogenesis for arboviruses is the establishment of infection following vector transmission. For La Crosse virus (LACV), the leading cause of pediatric arboviral encephalitis in North America, and other orthobunyaviruses, the initial course of infection in the skin is not well understood. Using an intradermal (ID) model of LACV infection in mice, we find that the virus infects and replicates nearly exclusively within skin-associated muscle cells of the panniculus carnosus (PC) and not in epidermal or dermal cells like most other arbovirus families. LACV is widely myotropic, infecting distal muscle cells of the peritoneum and heart, with limited infection of draining lymph nodes. Surprisingly, muscle cells are resistant to virus-induced cell death, with long term low levels of virus release progressing through the Golgi apparatus. Thus, skin muscle may be a key cell type for the initial infection and spread of arboviral orthobunyaviruses.

Inhibition of SFTSV replication in humanized mice by a subcutaneously administered anti-PD1 nanobody.

Severe fever with thrombocytopenia syndrome (SFTS) is a life-threatening disease caused by a novel bunyavirus (SFTSV), mainly transmitted by ticks. With no effective therapies or vaccines available, understanding the disease's mechanisms is crucial. Recent studies found increased expression of programmed cell death-1 (PD-1) on dysfunctional T cells in SFTS patients. However, the role of the PD-1/programmed cell death-ligand 1 (PD-L1) pathway in SFTS progression remains unclear. We investigated PD-1 blockade as a potential therapeutic strategy against SFTSV replication. Our study analyzed clinical samples and performed in vitro experiments, revealing elevated PD-1/PD-L1 expression in various immune cells following SFTSV infection. An anti-PD-1 nanobody, NbP45, effectively inhibited SFTSV infection in peripheral blood mononuclear cells (PBMCs), potentially achieved through the mitigation of apoptosis and the augmentation of T lymphocyte proliferation. Intriguingly, subcutaneous administration of NbP45 showed superior efficacy compared to a licensed anti-PD-1 antibody in an SFTSV-infected humanized mouse model. These findings highlight the involvement of the PD-1/PD-L1 pathway during acute SFTSV infection and suggest its potential as a host target for immunotherapy interventions against SFTSV infection.

Characterization of antibodies targeting severe fever with thrombocytopenia syndrome virus glycoprotein Gc.

Severe fever with thrombocytopenia syndrome (SFTS) is a hemorrhagic fever caused by SFTS virus (SFTSV), which is primarily found in East Asian countries. Despite its high mortality rate and increasing incidence, no vaccines or therapeutics have yet been approved for use against SFTS. Antibody drugs have shown promise in treating lethal infectious diseases that currently have no established treatments. In the case of SFTS, however, only a limited amount of research has been done on SFTSV-neutralizing antibodies targeting the transmembrane proteins Gn and Gc, which play critical roles in viral infection. This study focuses on the production and characterization of antibodies targeting the SFTSV Gc protein. Monoclonal antibodies against Gc were generated through immunization of mice, and their antiviral activity was evaluated. Three out of four anti-Gc antibody clones from this study demonstrated dose-dependent SFTSV neutralization activity, two of which exhibited a synergistic effect on the neutralization activity of the anti-Gn antibody clone Mab4-5. Further studies are necessary to identify key sites on the SFTSV glycoprotein and to develop novel agents as well as antibodies with diverse mechanisms of action against SFTSV.

Oropouche virus: A neglected global arboviral threat.

The Oropouche virus is an important arthropod-borne virus in the Peribunyaviridae family that can cause febrile illnesses, and it is widely distributed in tropical regions such as Central and South America. Since the virus was first identified, a large number of related cases are reported every year. No deaths have been reported to date, however, the virus can cause systemic infections, including the nervous and blood systems, leading to serious complications. The transmission of Oropouche virus occurs through both urban and sylvatic cycles, with the anthropophilic biting midge Culicoides paraensis serving as the primary vector in urban areas. Direct human-to-human transmission of Oropouche virus has not been observed. Oropouche virus consists of three segments, and the proteins encoded by the different segments enables the virus to replicate efficiently in the host and to resist the host's immune response. Phylogenetic analyses showed that Oropouche virus sequences are geographically distinct and have closer homologies with Iquitos virus and Perdoes virus, which belong to the family Peribunyaviridae. Despite the enormous threat it poses to public health, there are currently no licensed vaccines or specific antiviral treatments for the disease it causes. Recent studies have utilised imJatobal virusmunoinformatics approaches to develop epitope-based peptide vaccines, which have laid the groundwork for the clinical use of vaccines. The present review focuses on the structure, epidemiology, immunity and phylogeny of Oropouche virus, as well as the progress of vaccine development, thereby attracting wider attention and research, particularly with regard to potential vaccine programs.

Downregulation of transcription 1 hinders the replication of Dabie bandavirus by promoting the expression of TLR7, TLR8, and TLR9 signaling pathway.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is a bunyavirus that causes SFTS, with a case fatality rate of up to 30 %. The innate immune system plays a crucial role in the defense against SFTSV; however, the impact of viral propagation of STFSV on the innate immune system remains unclear. Although proteomics analysis revealed that the expression of the downregulator of transcription 1 (DR1) increased after SFTSV infection, the specific change trend and the functional role of DR1 during viral infection remain unelucidated. In this study, we demonstrate that DR1 was highly expressed in response to SFTSV infection in HEK 293T cells using qRT-PCR and Western blot analysis. Furthermore, viral replication significantly increased the expression of various TLRs, especially TLR9. Our data indicated that DR1 positively regulated the expression of TLRs in HEK 293T cells, DR1 overexpression highly increased the expression of numerous TLRs, whereas RNAi-mediated DR1 silencing decreased TLR expression. Additionally, the myeloid differentiation primary response gene 88 (MyD88)-dependent or TIR-domain-containing adaptor inducing interferon-ß (TRIF)-dependent signaling pathways were highly up- and downregulated by the overexpression and silencing of DR1, respectively. Finally, we report that DR1 stimulates the expression of TLR7, TLR8, and TLR9, thereby upregulating the TRIF-dependent and MyD88-dependent signaling pathways during the SFTSV infection, attenuating viral replication, and enhancing the production of type I interferon and various inflammatory factors, including IL-1ß, IL-6, and IL-8. These results imply that DR1 defends against SFTSV replication by inducing the expression of TLR7, TLR8, and TLR9. Collectively, our findings revealed a novel role and mechanism of DR1 in mediating antiviral responses and innate immunity.