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.

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.

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.

Molecular and biological characterization of a bunyavirus infecting the brown planthopper (Nilaparvata lugens).

A negative-strand symbiotic RNA virus, tentatively named Nilaparvata lugens Bunyavirus (NLBV), was identified in the brown planthopper (BPH, Nilaparvata lugens). Phylogenetic analysis indicated that NLBV is a member of the genus Mobuvirus (family Phenuiviridae, order Bunyavirales). Analysis of virus-derived small interfering RNA suggested that antiviral immunity of BPH was successfully activated by NLBV infection. Tissue-specific investigation showed that NLBV was mainly accumulated in the fat-body of BPH adults. Moreover, NLBV was detected in eggs of viruliferous female BPHs, suggesting the possibility of vertical transmission of NLBV in BPH. Additionally, no significant differences were observed for the biological properties between NLBV-infected and NLBV-free BPHs. Finally, analysis of geographic distribution indicated that NLBV may be prevalent in Southeast Asia. This study provided a comprehensive characterization on the molecular and biological properties of a symbiotic virus in BPH, which will contribute to our understanding of the increasingly discovered RNA viruses in insects.

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.

Field Isolation and Laboratory Vector-Host Studies of Brazoran Virus (Peribunyaviridae: Orthobunyavirus) from Florida.

Brazoran virus was first isolated from Culex mosquitoes in Texas in 2012, yet little is known about this virus. We report the isolation of this virus from Culex erraticus from southern Florida during 2016. The Florida strain had a nucleotide identity of 96.3% (S segment), 99.1% (M segment), and 95.8% (L segment) to the Texas isolate. Culex quinquefasciatus and Aedes aegypti colonies were subsequently fed virus blood meals to determine their vector competence for Brazoran virus. Culex quinquefasciatus was susceptible to midgut infection, but few mosquitoes developed disseminated infections. Aedes aegypti supported disseminated infection, but virus transmission could not be demonstrated. Suckling mice became infected by intradermal inoculation without visible disease signs. The virus was detected in multiple mouse tissues but rarely infected the brain. This study documents the first isolation of Brazoran virus outside of Texas. Although this virus infected Ae. aegypti and Cx. quinquefasciatus in laboratory trials, their vector competence could not be demonstrated, suggesting they are unlikely vectors of Brazoran virus.

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.

Genomic Characterization of a Bataï Orthobunyavirus, Previously Classified as Ilesha Virus, from Field-Caught Mosquitoes in Senegal, Bandia 1969.

Bataï virus (BATV), belonging to the Orthobunyavirus genus, is an emerging mosquito-borne virus with documented cases in Asia, Europe, and Africa. It causes various symptoms in humans and ruminants. Another related virus is Ilesha virus (ILEV), which causes a range of diseases in humans and is mainly found in African countries. This study aimed to genetically identify and characterize a BATV strain previously misclassified as ILEV in Senegal. The strain was reactivated and subjected to whole genome sequencing using an Illumina-based approach. Genetic analyses and phylogeny were performed to assess the evolutionary relationships. Genomic analyses revealed a close similarity between the Senegal strain and the BATV strains UgMP-6830 from Uganda. The genetic distances indicated high homology. Phylogenetic analysis confirmed the Senegal strain's clustering with BATV. This study corrects the misclassification, confirming the presence of BATV in West Africa. This research represents the first evidence of BATV circulation in West Africa, underscoring the importance of genomic approaches in virus classification. Retrospective sequencing is crucial for reevaluating strains and identifying potential public health threats among neglected viruses.

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.

Outbreak of Oropouche virus in frontier regions in western Amazon.

Oropouche virus (OROV) is characterized as a re-emerging arbovirus of great concern for public health, being responsible for several outbreaks of acute fever identified in Latin American countries, registering more than half a million reported cases. The incidence of reports of this virus is intrinsically favored by environmental conditions, in which such characteristics are related to the increase and distribution of the vector population to areas of human traffic. Moreover, there is a problem regarding the lack of diagnosis in Brazil that aggregates the success of the etiologic agent. Thus, by means of molecular techniques, we identified 27 positive cases of the OROV circulating in border locations in western Amazon, with 44.44% (12/27) of the cohort characterized as infected individuals with reported symptoms, mainly ranging from fever, myalgia, and back pain. Among the positive samples, it was possible to obtain a total of 48.14% (13/27) samples to analyze the S and M segments of Oropouche, which showed similarities among the Brazilian sequences. Thus, it was possible to verify the circulation of the OROV in Rondonia and border areas, in which the tracking of neglected arboviruses is necessary for the genomic surveillance of emerging and re-emerging viruses.IMPORTANCEThe western Amazon region is known for outbreaks of acute febrile illnesses, to which the lack of specific diagnostics for different pathogens hinders the management of patients in healthcare units. The Oropouche virus has already been recorded in the region in the 1990s. However, this is the first study, after this record, to perform the detection of individuals with acute febrile illness using a screening test to exclude Zika, dengue, and chikungunya, confirmed by sequencing the circulation of the virus in the state of Rondonia and border areas. We emphasize the importance of including diagnostics for viruses such as Oropouche, which suffers underreporting for years and is related to seasonal periods in Western Amazon locations, a factor that has a direct influence on public health in the region. In addition, we emphasize the importance of genomic surveillance in the elucidation of outbreaks that affect the resident population of these locations.

Glucosylceramide in bunyavirus particles is essential for virus binding to host cells.

Hexosylceramides (HexCer) are implicated in the infection process of various pathogens. However, the molecular and cellular functions of HexCer in infectious cycles are poorly understood. Investigating the enveloped virus Uukuniemi (UUKV), a bunyavirus of the Phenuiviridae family, we performed a lipidomic analysis with mass spectrometry and determined the lipidome of both infected cells and derived virions. We found that UUKV alters the processing of HexCer to glycosphingolipids (GSL) in infected cells. The infection resulted in the overexpression of glucosylceramide (GlcCer) synthase (UGCG) and the specific accumulation of GlcCer and its subsequent incorporation into viral progeny. UUKV and several pathogenic bunyaviruses relied on GlcCer in the viral envelope for binding to various host cell types. Overall, our results indicate that GlcCer is a structural determinant of virions crucial for bunyavirus infectivity. This study also highlights the importance of glycolipids on virions in facilitating interactions with host cell receptors and infectious entry of enveloped viruses.

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.

Illuminating bunyavirus entry into host cells with fluorescence.

Bunyavirales constitute the largest order of enveloped RNA viruses, many members of which cause severe diseases in humans and domestic animals. In recent decades, innovative fluorescence-based methods have paved the way to visualize and track single fluorescent bunyaviral particles in fixed and live cells. This technological breakthrough has enabled imaging of the early stages of infection and the quantification of every step in the bunyavirus cell entry process. Here, we describe the latest procedures for rendering bunyaviral particles fluorescent and discuss the advantages and disadvantages of each approach in light of the most recent advances in fluorescence detection and monitoring of bunyavirus entry. In this mini-review, we also illustrate how fluorescent viral particles are a powerful tool for deciphering the cellular entry process of bunyaviruses, the vast majority of which have not yet been analyzed.

Congenital malformations caused by Akabane virus in porcine fetuses in southern Japan.

Akabane virus (AKAV) is known as a major teratogenic agent of ruminant fetuses. In this study, we investigated the relationship between porcine abnormal deliveries and AKAV by serology, pathology, and virology investigations using specimens from 16 stillborn fetuses delivered in southern Japan between 2013 and 2015. The major clinical manifestations in stillborn fetuses were hydranencephaly, arthrogryposis, spinal curvature, and both skeletal muscle and subcutaneous edema. Histologic examination of the specimens identified atrophy of skeletal muscle fibers accompanied by adipose replacement. Nonsuppurative encephalomyelitis and decreased neuronal density in the ventral horn of the spinal cord were shown in two separate fetuses, respectively. Neutralizing antibody titers to AKAV were detected in most of the tested fetuses (13/16). The AKAV sequences detected in the affected fetuses in 2013 and 2015 were highly identical and closely related to Japanese AKAV isolates which were isolated in 2013 and sorted into genogroup I of AKAV. Immunohistochemistry visualized AKAV antigens in the neuronal cells of the central nervous system of the fetuses. These findings indicate that AKAV was involved in the birth of abnormal piglets at the affected farm. The clinical manifestations and histopathological features in the stillborn fetuses were very similar to those in ruminant neonates affected by AKAV. To avoid misdiagnosis and to evaluate the precise impact of AKAV on pig reproduction, AKAV should be considered in differential diagnoses of reproductive failures in pigs.

Development and evaluation of RT-qPCR assays for two neglected orthobunyaviruses: Oya virus and Ebinur Lake virus.

OBJECTIVES: Oya virus (OYAV) and Ebinur lake virus (EBIV) belong to the genus Orthobunyavirus within the Peribunyaviridae family, and both are recognized as the novel virus with potential threat to the animal or public health. Given their potential to cause outbreaks and their detection in diverse samples across different regions, the need for a reliable and efficient molecular detection method for OYAV and EBIV becomes imperative. METHODS: The S-segment of OYAV and EBIV was used for designing specific primer and probe sets, which were employed in a real-time reverse transcription quantitative PCR (RT-qPCR) assay. The analytical performance of these assays, encompassing specificity, sensitivity, reproducibility, and fitness for purpose, was thoroughly evaluated across various sample matrices. RESULTS: The developed RT-qPCR assays were very specific to their respective targets. Both assays were highly reproducible (%CV<3) and sensitive with the 95% limit of detection (LOD) of 0.80 PFU/mL for OYAV primer probe set and 0.37 PFU/mL for EBIV primer probe set. Furthermore, the assays fitness for purpose was good as it could detect the specific viruses in virus-spiked serum samples, virus-inoculated mosquito samples, field caught mosquitoes and biting midge samples. CONCLUSIONS: Our study has successfully developed specific, sensitive, and reliable RT-qPCR assays for the detection of OYAV and EBIV. These assays hold great promise for their potential application in clinical and field samples in the future.