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.

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.

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.

Isolation and whole-genome sequence analysis of Balagodu virus in Japan.

Whole-genome sequencing of a virus isolated from Culicoides biting midges in southern Japan in 2020 revealed that it is a strain of Balagodu virus (BLGV; genus Orthobunyavirus; family Peribunyaviridae; order Bunyavirales). A solitary instance of BLGV isolation occurred in India in 1963. All assembled segments comprise complete protein-coding sequences that are similar to those of other orthobunyaviruses. The consensus 3'- and 5'-terminal sequences of orthobunyaviruses' genomic RNAs are also conserved in the Japanese BLGV strain. Here, we update the geographic distribution of BLGV and provide its complete sequence, contributing to the clarification of orthobunyavirus phylogeny.

A Scoping Review on the Epidemiology of Orthobunyaviruses in Canada, in the Context of Human, Wildlife, and Domestic Animal Host Species.

Background: Mosquito-borne orthobunyaviruses in Canada are a growing public health concern. Orthobunyaviral diseases are commonly underdiagnosed and in Canada, likely underreported as surveillance is passive. No vaccines or specific treatments exist for these disease agents. Further, climate change is facilitating habitat expansion for relevant reservoirs and vectors, and it is likely that the majority of the Canadian population is susceptible to these viruses. Methods: A scoping review was conducted to describe the current state of knowledge on orthobunyavirus epidemiology in Canada. The Preferred Reporting Items for Systematic Reviews and Meta-Analyses extension for Scoping Reviews guideline was used. Literature searches were conducted in six databases and in gray literature. The epidemiology of orthobunyaviruses was characterized for studies focusing on host species, including spatiotemporal patterns, risk factors, and climate change impact. Results: A total of 172 relevant studies were identified from 1734 citations from which 95 addressed host species, including humans, wildlife, and domestic animals including livestock. The orthobunyaviruses-Cache Valley virus (CVV), Jamestown Canyon virus (JCV), Snowshoe Hare virus (SHV), and La Crosse virus (LACV)-were identified, and prevalence was widespread across vertebrate species. CVV, JCV, and SHV were detected across Canada and the United States. LACV was reported only in the United States, predominantly the Mid-Atlantic and Appalachian regions. Disease varied by orthobunyavirus and was associated with age, environment, preexisting compromised immune systems, or livestock breeding schedule. Conclusion: Knowledge gaps included seroprevalence data in Canada, risk factor analyses, particularly for livestock, and disease projections in the context of climate change. Additional surveillance and mitigation strategies, especially accounting for climate change, are needed to guide future public health efforts to prevent orthobunyavirus exposure and disease.

Seroprevalence and Risk Factors Associated with Akabane Virus Infection in Sheep and Goats in Fars Province, Iran.

Akabane disease is an arthropod-borne viral disease that affects ruminants. This teratogenic pathogen causes severe economic losses in ruminants worldwide and in Iran; however, it has not received enough attention in Fars province, Iran. Therefore, this study aimed to determine the influence of age, gender, climate, farming system, and history of abortions on the seroprevalence of the Akabane disease in sheep and goats in Fars province. In the present study, Fars province was divided into three climates, and three cities were randomly selected from each climatic region. In each city, two epidemiologic units were selected, and all sheep and goats in each unit were sampled. Overall, 540 serum samples (391 sheep and 149 goats) were collected and examined with the commercial ELISA kit. The results showed that 83 out of 540 (15.4%) samples were seropositive and had antibodies against the Akabane virus (AKAV). The effect of gender and age on the rate of the AKAV was not significant. Animals in warm climates were 4.218 times more likely to have antibodies against the AKAV than animals in cold climates. Females were 1.32 times more likely to exhibit seropositivity. The odds of AKAV infection were higher in animals with an abortion history than in healthy animals. The findings of the present study indicated that the prevalence of the AKAV was high in small ruminants in Fars province. Therefore, it is necessary to conduct more studies to control the risk factors involved in the spread of this virus.

Emergence potential of mosquito-borne arboviruses from the Florida Everglades.

The Greater Everglades Region of South Florida is one of the largest natural wetlands and the only subtropical ecosystem found in the continental United States. Mosquitoes are seasonally abundant in the Everglades where several potentially pathogenic mosquito-borne arboviruses are maintained in natural transmission cycles involving vector-competent mosquitoes and reservoir-competent vertebrate hosts. The fragile nature of this ecosystem is vulnerable to many sources of environmental change, including a wetlands restoration project, climate change, invasive species and residential development. In this study, we obtained baseline data on the distribution and abundance of both mosquitos and arboviruses occurring in the southern Everglades region during the summer months of 2013, when water levels were high, and in 2014, when water levels were low. A total of 367,060 mosquitoes were collected with CO2-baited CDC light traps at 105 collection sites stratified among the major landscape features found in Everglades National Park, Big Cypress National Preserve, Fakahatchee State Park Preserve and Picayune State Forest, an area already undergoing restoration. A total of 2,010 pools of taxonomically identified mosquitoes were cultured for arbovirus isolation and identification. Seven vertebrate arboviruses were isolated: Everglades virus, Tensaw virus, Shark River virus, Gumbo Limbo virus, Mahogany Hammock virus, Keystone virus, and St. Louis encephalitis virus. Except for Tensaw virus, which was absent in 2013, the remaining viruses were found to be most prevalent in hardwood hammocks and in Fakahatchee, less prevalent in mangroves and pinelands, and absent in cypress and sawgrass. In contrast, in the summer of 2014 when water levels were lower, these arboviruses were far less prevalent and only found in hardwood hammocks, but Tensaw virus was present in cypress, sawgrass, pinelands, and a recently burned site. Major environmental changes are anticipated in the Everglades, many of which will result in increased water levels. How these might lead to the emergence of arboviruses potentially pathogenic to both humans and wildlife is discussed.

Characterization and reverse genetic establishment of cattle derived Akabane virus in China.

BACKGROUND: Akabane virus (AKAV) is an important insect-borne virus which is widely distributed throughout the world except the Europe and is considered as a great threat to herbivore health. RESULTS: An AKAV strain defined as TJ2016 was firstly isolated from the bovine sera in China in 2016. Sequence analysis of the S and M segments suggested that the isolated AKAV strain was closely related to the AKAV strains JaGAr39 and JaLAB39, which belonged to AKAV genogroup II. To further study the pathogenic mechanism of AKAV, the full-length cDNA clone of TJ2016 S, M, and L segment was constructed separately into the TVT7R plasmid at the downsteam of T7 promoter and named as TVT7R-S, TVT7R-M, and TVT7R-L, respectively. The above three plasmids were further transfected into the BSR-T7/5 cells simultaneously with a ratio of 1:1:1 to produce the rescued virus AKAV. Compared with the parental wild type AKAV (wtAKAV), the rescued virus (rAKAV) was proved to be with similar cytopathic effects (CPE), plaque sizes and growth kinetics in BHK-21 cells. CONCLUSION: We successfully isolated a AKAV strain TJ2016 from the sera of cattle and established a reverse genetic platform for AKAV genome manipulation. The established reverse genetic system is also a powerful tool for further research on AKAV pathogenesis and even vaccine studies.

Mammals Preferred: Reassortment of Batai and Bunyamwera orthobunyavirus Occurs in Mammalian but Not Insect Cells.

Reassortment is a viral genome-segment recomposition known for many viruses, including the orthobunyaviruses. The co-infection of a host cell with two viruses of the same serogroup, such as the Bunyamwera orthobunyavirus and the Batai orthobunyavirus, can give rise to novel viruses. One example is the Ngari virus, which has caused major outbreaks of human infections in Central Africa. This study aimed to investigate the potential for reassortment of Bunyamwera orthobunyavirus and the Batai orthobunyavirus during co-infection studies and the replication properties of the reassortants in different mammalian and insect cell lines. In the co-infection studies, a Ngari-like virus reassortant and a novel reassortant virus, the Batunya virus, arose in BHK-21 cells (Mesocricetus auratus). In contrast, no reassortment was observed in the examined insect cells from Aedes aegypti (Aag2) and Aedes albopictus (U4.4 and C6/36). The growth kinetic experiments show that both reassortants are replicated to higher titers in some mammalian cell lines than the parental viruses but show impaired growth in insect cell lines.

First evidence of Schmallenberg virus infection in domestic ruminants in Kosovo and Albania.

Schmallenberg virus (SBV), a novel Orthobunyavirus, emerged in European domestic ruminants in 2011 causing abortions and malformations in newborns and none or mild clinical symptoms in adult animals. Here, a total of 364 bovine, ovine and caprine serum samples were collected in Kosovo and Albania between May 2014 and August 2016 and analyzed for the presence of anti­SBV antibodies. Sera were tested using an enzyme­linked immunosorbent assay (ELISA), and 48 ELISA­positive samples were subsequently analyzed by serum neutralization test (SNT). The overall percentage of ELISA positive results was 17.9%; 23.1% (53/229) was the prevalence observed in Kosovo (cattle 45.5%, sheep 19.2% and goat 6.8%), while 8.9% (12/135) was that observed in Albania (cattle 11.1%, sheep 0% and goat 20.0%). SNT confirmed the presence of neutralizing antibodies against SBV in all samples tested. This is the first study reporting SBV circulation in domestic ruminants in Kosovo and Albania, with indication that this virus has been present in Kosovo and Albania at least since 2014 without being detected.

Orthobunyaviruses in the Caribbean: Melao and Oropouche virus infections in school children in Haiti in 2014.

We report the identification of two orthobunyaviruses, Melao virus (MELV) and Oropouche virus (OROV), in plasma specimens from Haitian children with acute febrile illness who presented during outbreaks caused by alpha- and flaviviruses in 2014. Heretofore not described as a human pathogen, MELV was isolated in cell culture from the plasma of five case patients. OROV RNA was detected in the plasma of an additional child, using an unbiased sequencing approach, with phylogenetic inference suggesting a close relationship with strains from Brazil. Abdominal pain was reported by four case patients with MELV infections, with lymphadenopathy noted in two cases. Our findings document the occurrence of these orthobunyaviruses within the Caribbean region and highlight the critical importance of surveillance with viral genome sequence analyses to identify outbreaks caused by these and other emerging viruses.

Arbovirus serosurvey (Orthobunyavirus, Flavivirus, and Alphavirus) in a draft horse population from Santa Fe, Argentina (2013-2016).

In the present study, we serosurveyed the exposure of 222 draft horses to different arboviruses in the city of Santa Fe, Argentina. Plaque reduction neutralization tests confirmed exposure to Fort Sherman virus (FSV), Saint Louis encephalitis virus (SLEV), West Nile virus (WNV), and Río Negro virus (RNV). Apparently, Western and Eastern equine encephalitis viruses did not circulate in the population tested. The confirmation of five seroconversions for WNV, FSV, and SLEV and the association between prevalence and age are indicative of recent circulation. These results highlight the importance of considering draft horses in arboviral surveillance in urban and rural areas of developing countries.

Vector Competence of Peruvian Mosquitoes for Two Orthobunyaviruses Isolated From Mosquitoes Captured in Peru.

We evaluated the potential for mosquitoes collected in the Amazon Basin, near Iquitos, Peru, to become infected with and transmit Murutucu (MURV) and Itaqui viruses (ITQV) (Order Bunyavirales, Family: Peribunyaviridae, Genus: Orthobunyavirus). Viremia levels in Syrian hamsters peaked 2 d after infection with either virus, and both viruses were highly lethal in hamsters with virtually all hamsters dying prior to 3-d postinfection. For almost all of the mosquito species tested some individuals were susceptible to infection and some developed a disseminated infection after oral exposure to either MURV or ITQV. However, only the Culex species (Culex (Culex) coronator Dyar and Knab [Diptera, Culicidae], Culex (Melanoconian) gnomatos Sallum, Huchings, and Ferreira [Diptera, Culicidae], Culex (Mel.) pedroi Sirivanakarn and Belkin [Diptera, Culicidae], and Culex (Mel.) vomerifer Komp [Diptera, Culicidae]) successfully transmitted virus by bite. However, even among these species, only about 37% of the individuals with a disseminated infection successfully transmitted these viruses, indicating a significant salivary gland barrier. Although little is known about the medical or veterinary importance of many members of the genus Orthobunyavirus, we have demonstrated that Culex spp. (Diptera, Culicidae) could be potential vectors.

Ngari virus (Orthobunyavirus, Peribunyaviridae) in ixodid ticks collected from cattle in Guinea.

Ngari virus is a mosquito-borne virus belonging to the genus Orthobunyavirus (Peribunyaviridae family). This virus is pathogenic to humans and causes severe illness. Ngari virus is present in several African countries, including Madagascar. Here, we report the detection of Ngari virus in ixodid ticks collected from cows in Guinea. A tick survey was conducted in March-November of 2018 in six regions of Guinea. The sample comprised 710 pools, with a total of 2067 ticks belonging to five species collected from 197 cows. At the initial stage, we screened a subsample of tick pools of vector-borne viruses with a multiplex genus-specific primer panel. In the second stage of the study, we narrowed the search and screened all the samples by qPCR for the detection of Ngari virus. All positive samples were sequenced with primers flanking Ngari virus-specific fragments on the S and M segments. We found Ngari virus in 12 pools that were formed from engorged ticks collected from livestock in three villages of the Kindia and Kankan regions. Sequencing of the S and M segments confirmed that the detected viruses belong to Ngari virus, and the viruses were most similar to the strain Adrar, which was isolated in Mauritania. We detected viral RNA in ticks of the following species: Amblyomma variegatum, Rhipicephalus geigyi, and Rh. (Boophilus) spp. There is no evidence that ixodid ticks are competent vectors of the Ngari virus. Most likely, the ticks obtained the virus through blood from an infected host. The study of engorged ticks can be recommended as a simpler approach for the wide screening of the Ngari virus and subsequent testing of cattle and mosquitos in those locations where the PCR-positive ticks were collected.

Zoo animals as sentinels for Schmallenberg virus monitoring in Spain.

Schmallenberg virus (SBV) is a newly emerged vector-borne pathogen that affects many domestic and wild animal species. A serosurvey was carried out to assess SBV exposure in zoo animals in Spain and to determine the dynamics of seropositivity in longitudinally sampled individuals. Between 2002 and 2019, sera from 278 animals belonging to 73 different species were collected from five zoos (A-E). Thirty-one of these animals were longitudinally sampled at three of these zoo parks during the study period. Seropositivity was detected in 28 (10.1 %) of 278 animals analyzed by blocking ELISA. Specific anti-SBV antibodies were confirmed in 20 (7.2 %; 95 %CI: 4.2-10.3) animals of six different species using virus neutralization test (VNT). The multiple logistic regression model showed that "order" (Artiodactyla) and "zoo provenance" (zoo B; southern Spain) were risk factors potentially associated with SBV exposure. Two (8.7 %) of the 31 longitudinally-sampled individuals showed specific antibodies against SBV at all samplings whereas seroconversion was detected in one mouflon (Ovis aries musimon) and one Asian elephant (Elephas maximus) in 2016 and 2019, respectively. To the best of the author's knowledge, this is the first surveillance conducted on SBV in zoos in Spain. The results confirm SBV exposure in zoo animals in this country and indicate circulation of the virus before the first Schmallenberg disease outbreak was reported in Spain. Surveillance in zoological parks could be a complementary approach to monitoring SBV activity. Further studies are warranted to assess the impact of this virus on the health status of susceptible zoo animals.

Emerging orthobunyaviruses associated with CNS disease.

The Orthobunyavirus genus comprises a wide range of arthropod-borne viruses which are prevalent worldwide and commonly associated with central nervous system (CNS) disease in humans and other vertebrates. Several orthobunyaviruses have recently emerged and increasingly more will likely do so in the future. Despite this large number, an overview of these viruses is currently lacking, making it challenging to determine importance from a One Health perspective. Causality is a key feature of determining importance, yet classical tools are unfit to evaluate the causality of orthobunyaviral CNS disease. Therefore, we aimed to provide an overview of orthobunyaviral CNS disease in vertebrates and objectify the causality strength of each virus. In total, we identified 27 orthobunyaviruses described in literature to be associated with CNS disease. Ten were associated with disease in multiple host species of which seven included humans. Seven viruses were associated with both congenital and postnatal CNS disease. CNS disease-associated orthobunyaviruses were spread across all known Orthobunyavirus serogroups by phylogenetic analyses. Taken together, these results indicate that orthobunyaviruses may have a common tendency to infect the CNS of vertebrates. Next, we developed six tailor-made causality indicators and evaluated the causality strength of each of the identified orthobunyaviruses. Nine viruses had a 'strong' causality score and were deemed causal. Eight had a 'moderate' and ten a 'weak' causality score. Notably, there was a lack of case-control studies, which was only available for one virus. We, therefore, stress the importance of proper case-control studies as a fundamental aspect of proving causality. This comprehensible overview can be used to identify orthobunyaviruses which may be considered causal, reveal research gaps for viruses with moderate to low causality scores, and provide a framework to evaluate the causality of orthobunyaviruses that may newly emerge in the future.

Development and validation of SYBR Green- and probe-based reverse-transcription real-time PCR assays for detection of the S and M segments of Schmallenberg virus.

Schmallenberg virus (SBV), discovered in Germany in 2011, causes congenital malformations in ruminants. Reverse-transcription real-time PCR (RT-rtPCR) assays based on various segments of SBV have been developed for molecular detection. We developed alternative RT-rtPCR assays for SBV detection to avoid earlier reported mutations and hypervariable regions of the S and M segments of the viral genome. For SYBR Green-based detection of the S segment, the R2 value and efficiency of the developed assay were 0.99 and 99%, respectively. For probe-based S segment detection, 2 assays were developed; the first had an R2 value of 0.99 and 102% efficiency, and the second had a R2 value of 0.98 and 86% efficiency. The probe-based M segment assay had an R2 value of 1.00 and 103% efficiency. Detection limits of the RT-rtPCR assays with new primer sets were 102 and 101 copies/µL for the S and M segments, respectively. Field samples from cattle and sheep were also used for primary validation of the developed assays. Our assays should be suitable for SBV detection in ruminants and for in vitro studies of various SBV strains.