First detection of Jingmen tick virus in Corsica, France and development of a real time detection system for multiple tick-associated jingmenviruses.

Jingmen tick virus (JMTV) is a recently discovered segmented RNA virus, closely related to flaviviruses. It was identified for the first time in 2014, in China and subsequently in Brazil. Following this discovery, JMTV-related sequences have been identified in arthropods, vertebrates (including humans), plants, fungus and environmental samples from Asia, America, Africa, Europe and Oceania. Several studies suggest an association between these segmented flavi-like viruses, termed jingmenviruses, and febrile illness in humans. The development of rapid diagnostic assays for these viruses is therefore crucial to be prepared for a potential epidemic, for the early detection of these viruses via vector surveillance or hospital diagnosis. In this study, we designed a RT-qPCR assay to detect tick-associated jingmenviruses, validated it and tested its range and limit of detection with six tick-associated jingmenviruses using in vitro transcripts. Then we screened ticks collected in Corsica (France) from different livestock species, in order to determine the distribution of these viruses on the island. In total, 6,269 ticks from eight species were collected from 763 cattle, 538 horses, 106 sheep and 218 wild boars and grouped in 1,715 pools. We report the first detection of JMTV in Corsica, in Rhipicephalus bursa, Hyalomma marginatum and R. sanguineus ticks collected from cattle and sheep. The highest prevalence was found in the Rhipicephalus genus. The complete genome of a Corsican JMTV was obtained from a pool of Rhipicephalus bursa ticks and shares between 94.7% and 95.1% nucleotide identity with a JMTV sequence corresponding to a human patient in Kosovo and groups phylogenetically with European JMTV strains. These results show that a Mediterranean island such as Corsica could act as a sentinel zone for future epidemics.

Crimean-Congo Hemorrhagic Fever Virus in Ticks Collected from Cattle, Corsica, France, 2023.

We report the detection of Crimean-Congo hemorrhagic fever virus (CCHFV) in Corsica, France. We identified CCHFV African genotype I in ticks collected from cattle at 2 different sites in southeastern and central-western Corsica, indicating an established CCHFV circulation. Healthcare professionals and at-risk groups should be alerted to CCHFV circulation in Corsica.

Comprehensive evaluation of nucleic acid amplification methods widely used for generic detection of sandfly-borne phleboviruses.

Sandfly-borne phleboviruses (SBPs), which cause sandfly fever, aseptic meningitis, encephalitis, and meningoencephalitis, are emerging pathogens of major public health concern. Virus nucleic acid testing is essential for SBP diagnosis, especially in the early stages of infection, and for the discovery of novel SBPs. The efficacy of utilizing generic primers that target conserved nucleotide sequences for the detection of both known and novel SBPs has not been extensively evaluated. We aimed to compare and evaluate the performance of five generic primer sets, widely used to detect S- and L-segments of arthropod-borne phleboviruses and designed as singleplex (n = 3) and nested (n = 2) formats, including both well-known and recently characterized 15 Old World virus strains. Furthermore, we performed in silico analysis to assess the detection capabilities of these generic primer sets. The initial evaluation of previously published generic primer sets for SBP detection yielded two singleplex primer sets with the potential to be adapted for use in real-time or high-throughput detection settings. Studies are ongoing to develop and further optimize a preliminary assay and test various hosts and vectors to assess their capacity to detect known and novel viruses. IMPORTANCE: Virus nucleic acid testing is the primary diagnostic method, particularly in the early stages of illness. Virus-specific or syndromic tests are widely used for this purpose. The use of generic primers has had a considerable impact on the discovery, identification, and detection of Old World sandfly-borne phleboviruses (OWSBP). The study is significant because it is the first to carry out a comparative evaluation of all published OWSBP generic primer sets.

Molecular epidemiology identifies the expansion of the DENV2 epidemic lineage from the French Caribbean Islands to French Guiana and mainland France, 2023 to 2024.

In 2023, dengue virus serotype 2 (DENV2) affected most French overseas territories. In the French Caribbean Islands, viral circulation continues with > 30,000 suspected infections by March 2024. Genome sequence analysis reveals that the epidemic lineage in the French Caribbean islands has also become established in French Guiana but not Réunion. It has moreover seeded autochthonous circulation events in mainland France. To guide prevention of further inter-territorial spread and DENV introduction in non-endemic settings, continued molecular surveillance and mosquito control are essential.

Neutralizing based seroprevalence study of Toscana virus in livestock from Algeria.

Toscana virus is a sandfly-borne human pathogen belonging to Phlebovirus genus into Phenuiviridae family. It is emerging in north Africa posing a complex threat to public health. TOSV is heavily affecting sandfly-exposed people in northern Algeria. A larger distribution has recently been stated in Algeria by using dog sera. Dog exposure to TOSV was repeatedly identified in north Algeria, with 4.56% lately detected to possess respective neutralizing antibodies. However, evidence for TOSV has only been observed in dogs among various species of domestic animals. Therefore, we attempted to assess sera from 221 livestock comprising cattle, sheep, goats, rabbits and horses, to identify the presence of TOSV neutralizing antibodies. The study was conducted during 2017, in 11 areas from the governorates of Blida, Medea, Algiers, Tipaza, Ain Defla, Tissemsilt in the north center, and Setif, Mila, Tizi Ouzou, Jijel in the northeast of Algeria. Positive results were obtained in 14.6% (12/82) cattle, 17.18% (11/64) sheep, 15% (3/20) horses and 3.33% (1/30) goats, whereas rabbits remained negative. Positive samples originated mainly from the north centre, with new areas being first-ever detected. The seroprevalence was noticed to be very strongly related to sample origin (p < 0.01). Females (OR=4.09) were observed to be more likely infected. Our findings represent a further proof of TOSV circulation in Algeria. Moreover, they revealed a potential role of livestock (p = 0.00731) in its natural cycle. This fact emphasize how important is to elucidate the exact contribution of livestock to the epidemiology of sandfly-borne phleboviruses, and their impact on public health.

Comprehensive Cross-Sectional Evaluation of Human Sandfly-Borne Phlebovirus Exposure in an Endemic Region.

Sandfly-borne phleboviruses are endemic in countries around the Mediterranean Basin and pose a significant health threat for populations, with symptoms spanning from febrile diseases to central nervous system involvement. We carried out a comprehensive cross-sectional screening via microneutralization (MN) assays for a quantitative assessment of neutralizing antibodies (NAs) to seven phleboviruses representing three distinct serocomplexes, using samples previously screened via immunofluorescence assays (IFAs) in Turkey, an endemic region with various phleboviruses in circulation. We detected NAs to three phleboviruses: Toscana virus (TOSV), sandfly fever Naples virus (SFNV), and sandfly fever Sicilian virus (SFSV), while assays utilizing Adana virus, Punique virus, Massilia virus, and Zerdali virus remained negative. The most frequently observed virus exposure was due to TOSV, with a total prevalence of 22.6%, followed by SFNV (15.3%) and SFSV (12.1%). For each virus, IFA reactivity was significantly associated with NA detection, and further correlated with NA titers. TOSV and SFSV seroreactivities were co-detected, suggesting exposure to multiple pathogenic viruses presumably due to shared sandfly vectors. In 9.6% of the samples, multiple virus exposure was documented. In conclusion, our findings demonstrate widespread exposure to distinct pathogenic phleboviruses, for which diagnostic testing and serological screening efforts should be directed.

Epidemiology, Isolation, and Genetic Characterization of Toscana Virus in Algerian Patients Displaying Neurological Infection, 2016-2018.

Purpose: The current study reports the results of the diagnosis of neuro-invasive Toscana virus (TOSV) infection in Algeria between 2016 and 2018 and describes the first isolation of TOSV strain from human samples in North Africa. Materiel and methods: Cerebrospinal fluid (CSF) and sera samples were obtained from 720 hospitalized patients displaying neurological infection symptoms of unknown etiology, of which 604 were screened for TOSV. The diagnosis was performed by serological and/or RT-PCR tests. In addition, TOSV was isolated in vivo and in vitro from CSF and genetically characterized. Results: 23 cases of TOSV neurological infections were detected. Cases were located in 11 Wilayas (administrative provinces), mainly in northern Algeria. In addition, we report the isolation of TOSV strain belonging to lineage A from human samples with its complete coding sequence. Conclusion: Even though the number of infections is probably underestimated, TOSV is endemic in Algeria, with several cases of neuro-invasive diseases in humans recorded each year. Therefore, the diagnosis of TOSV should be included in the differential diagnosis of neurological diseases, especially aseptic meningitis, during the period of activity of the phlebotomine vector. Further studies are required to measure precisely the nationwide prevalence of TOSV in Algeria.

Diversity and Genetic Reassortment of Keystone Virus in Mosquito Populations in Florida.

Keystone orthobunyavirus (KEYV), a member of the genus Orthobunyavirus, was first isolated in 1964 from mosquitoes in Keystone, Florida. Although data on human infections are limited, the virus has been linked to a fever/rash syndrome and, possibly, encephalitis, with early studies suggesting that 20% of persons in the Tampa, Florida, region had antibodies to KEYV. To assess the distribution and diversity of KEYV in other regions of Florida, we collected > 6,000 mosquitoes from 43 sampling sites in St. Johns County between June 2019 and April 2020. Mosquitoes were separated into pools by species and collection date and site. All pools with Aedes spp. (293 pools, 2,171 mosquitoes) were screened with a real-time reverse transcriptase polymerase chain reaction (rRT-PCR) assay that identifies KEYV and other closely related virus species of what was previously designated as the California encephalitis serogroup. In 2020, screening for KEYV was expanded to include 211 pools of Culex mosquitoes from sites where KEYV-positive Aedes spp. had been identified. rRT-PCR-positive samples were inoculated into cell cultures, and five KEYV isolates from Aedes atlanticus pools were isolated and sequenced. Analyses of the KEYV large genome segment sequences revealed two distinct KEYV clades, whereas analyses of the medium and small genome segments uncovered past reassortment events. Our data documented the ongoing seasonal circulation of multiple KEYV clades within Ae. atlanticus mosquito populations along the east coast of Florida, highlighting the need for further studies of the impact of this virus on human health.

Deep Proteomics Network and Machine Learning Analysis of Human Cerebrospinal Fluid in Japanese Encephalitis Virus Infection.

Japanese encephalitis virus is a leading cause of neurological infection in the Asia-Pacific region with no means of detection in more remote areas. We aimed to test the hypothesis of a Japanese encephalitis (JE) protein signature in human cerebrospinal fluid (CSF) that could be harnessed in a rapid diagnostic test (RDT), contribute to understanding the host response and predict outcome during infection. Liquid chromatography and tandem mass spectrometry (LC-MS/MS), using extensive offline fractionation and tandem mass tag labeling (TMT), enabled comparison of the deep CSF proteome in JE vs other confirmed neurological infections (non-JE). Verification was performed using data-independent acquisition (DIA) LC-MS/MS. 5,070 proteins were identified, including 4,805 human proteins and 265 pathogen proteins. Feature selection and predictive modeling using TMT analysis of 147 patient samples enabled the development of a nine-protein JE diagnostic signature. This was tested using DIA analysis of an independent group of 16 patient samples, demonstrating 82% accuracy. Ultimately, validation in a larger group of patients and different locations could help refine the list to 2-3 proteins for an RDT. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD034789 and 10.6019/PXD034789.

Persistence of Toscana virus in sugar and blood meals of phlebotomine sand flies: epidemiological and experimental consequences.

Many virological studies have tested the persistence of enveloped RNA viruses in various environmental and laboratory conditions and shown their short-term persistence. In this article, we analyzed Toscana virus (TOSV) infectivity, a pathogenic sandfly-borne phlebovirus, in two different conditions: in the sugar meal and blood meal of sand flies. Our results showed that TOSV RNA was detectable up to 15 days in sugar solution at 26 °C and up to 6 h in blood at 37 °C. Moreover, TOSV remains infective for 7 days in sugar solution and for minimum 6 h in rabbit blood. TOSV has shown persistent infectivity/viability under different conditions, which may have important epidemiological consequences. These results strengthen new hypotheses about the TOSV natural cycle, such as the possibility of horizontal transmission between sand flies through infected sugar meal.

Presence of the sandfly-borne phlebovirus (Toscana virus) in different bio-geographical regions of Algeria demonstrated by a microneutralisation-based seroprevalence study in owned dogs.

Toscana virus (TOSV) is major meningitis and meningoencephalitis agent in the Mediterranean basin. Dogs are frequently exposed to TOSV; thereby they can contribute to estimating its circulation. In Algeria, little is known about its circulation, and available data are restricted to the Kabylian region. To investigate the current situation in Algeria, a total of 205 dog sera collected from 13 different wilayas over the country were analyzed by using in-house Enzyme Linked Immunosorbent Assay (ELISA) and microneutralization test (MNT). An overall seroprevalence rate of 20% (14.5-25.5%) was observed by ELISA. Whereas, a seroprevalence rate of 4.56% (1.65-7.43%) was recorded by microneutralization test elucidating the exact occurrence of TOSV exposure in dogs, in Algeria. Positive dogs were detected from the areas of Algiers, Bejaia, Blida, Bouira, Medea, Setif, and Tlemcen in the north; Laghouat in the high lands and Tamanrasset in great Sahara. Only one serum, originating from Bejaia in the north east, was positive for both testing methods, while 8/9 positive sera in MNT remained negative in ELISA. MNT negative/ELISA positive result of 40/41 might suggest evidence for dog transmission, and circulation of phleboviruses other than TOSV. Noticeably, TOSV and antigenically related viruses are largely prevalent. Thus, they are not only confined to Kabylia region, but are widespread in Algeria, despite its climate diversity.

Detection and Isolation of Sindbis Virus from Field Collected Mosquitoes in Timimoun, Algeria.

Sindbis virus (SINV) is a zoonotic alphavirus (family Togaviridae, genus Alphavirus) that causes human diseases in Africa, Europe, Asia, and Australia. Occasionally, SINV outbreaks were reported in South Africa and northern Europe. Birds are the main amplifying hosts of SINV, while mosquitoes play the role of the primary vector. Culex mosquitoes were collected in Algeria and subsequently tested for SINV. SINV RNA was detected in 10 pools out of 40, from a total of 922 mosquitoes tested. A strain of SINV was isolated from a pool displaying high viral load. Whole-genome sequencing and phylogenetic analysis showed that the SINV Algeria isolate was most closely related to a Kenyan strain. This was the first record of SINV in Algeria and more broadly in northwestern Africa, which can be a potential risk for human health in the circulating area. Further studies are needed to measure the impact on public health through seroprevalence studies in Algeria.

Immunoglobulin M seroneutralization for improved confirmation of Japanese encephalitis virus infection in a flavivirus-endemic area.

BACKGROUND: The mainstay of diagnostic confirmation of acute Japanese encephalitis (JE) involves detection of anti-JE virus (JEV) immunoglobulin M (IgM) by enzyme-linked immunosorbent assay (ELISA). Limitations in the specificity of this test are increasingly apparent with the introduction of JEV vaccinations and the endemicity of other cross-reactive flaviviruses. Virus neutralization testing (VNT) is considered the gold standard, but it is challenging to implement and interpret. We performed a pilot study to assess IgG depletion prior to VNT for detection of anti-JEV IgM neutralizing antibodies (IgM-VNT) as compared with standard VNT. METHODS: We evaluated IgM-VNT in paired sera from anti-JEV IgM ELISA-positive patients (JE n=35) and negative controls of healthy flavivirus-naïve (n=10) as well as confirmed dengue (n=12) and Zika virus (n=4) patient sera. IgM-VNT was subsequently performed on single sera from additional JE patients (n=76). RESULTS: Anti-JEV IgG was detectable in admission serum of 58% of JE patients. The positive, negative and overall percentage agreement of IgM-VNT as compared with standard VNT was 100%. A total of 12/14 (86%) patient samples were unclassified by VNT and, with sufficient sample available for IgG depletion and IgG ELISA confirming depletion, were classified by IgM-VNT. IgM-VNT enabled JE case classification in 72/76 (95%) patients for whom only a single sample was available. CONCLUSIONS: The novel approach has been readily adapted for high-throughput testing of single patient samples and it holds promise for incorporation into algorithms for use in reference centres.

Lack of Evidence for Crimean-Congo Hemorrhagic Fever Virus in Ticks Collected from Animals, Corsica, France.

In Corsica, France, 9.1% of livestock serum samples collected during 2014-2016 were found to have antibodies against Crimean-Congo hemorrhagic fever virus (CCHFV), an emerging tickborne zoonotic disease. We tested 8,051 ticks for CCHFV RNA and Nairovirus RNA. The results indicate that Corsica is not a hotspot for CCHFV.

Exposure to Phlebotomus perniciosus sandfly vectors is positively associated with Toscana virus and Leishmania infantum infection in human blood donors in Murcia Region, southeast Spain.

Antibodies against Phlebotomus perniciosus sandfly salivary gland homogenate (SGH) and recombinant protein rSP03B, sandfly-borne Toscana virus (TOSV), Sandfly Fever Sicilian virus (SFSV) and Leishmania, as well as DNA of the latter parasite, were investigated in 670 blood samples from 575 human donors in Murcia Region, southeast Spain, in 2017 and 2018. The estimated SGH and rSP03B seroprevalences were 69% and 88%, respectively, although correlation between test results was relatively low (ρ = 0.39). Similarly, TOSV, SFSV and Leishmania seroprevalences were 26%, 0% and 1%, respectively, and Leishmania PCR prevalence was 2%. Prevalences were significantly greater in 2017, overdispersed and not spatially related to each other although both were positively associated with SGH but not to rSP03B antibody optical densities, questioning the value of the latter as a diagnostic marker for these infections in humans.

Molecular detection of parapoxvirus in Ixodidae ticks collected from cattle in Corsica, France.

BACKGROUND: Several viruses belonging to the family Poxviridae can cause infections in humans and animals. In Corsica, livestock farming (sheep, goats, pigs, and cattle) is mainly mixed, leading to important interactions between livestock, wildlife, and human populations. This could facilitate the circulation of zoonotic diseases, and makes Corsica a good example for studies of tick-borne diseases. OBJECTIVES: To gain understanding on the circulation of poxviruses in Corsica, we investigated their presence in tick species collected from cattle, sheep, horses, and wild boar, and characterized them through molecular techniques. METHODS: Ticks were tested using specific primers targeting conserved regions of sequences corresponding to two genera: parapoxvirus and orthopoxvirus. RESULTS: A total of 3555 ticks were collected from 1549 different animals (687 cattle, 538 horses, 106 sheep, and 218 wild boars). They were tested for the presence of parapoxvirus DNA on one hand and orthopoxvirus DNA on the other hand using Pangeneric real-time TaqMan assays. Orthopoxvirus DNA was detected in none of the 3555 ticks. Parapoxvirus DNA was detected in 6.6% (36/544) of ticks collected from 23 cows from 20 farms. The remaining 3011 ticks collected from horses, wild boars, and sheep were negative. The infection rate in cow ticks was 8.0% (12/148) in 2018 and 6.0% (24/396) in 2019 (p = 0.57). Parapoxvirus DNA was detected in 8.5% (5/59) of Hyalomma scupense pools, 8.2% (15/183) of Hyalomma marginatum pools, and 6.7% (16/240) of Rhipicephalus bursa pools (p = 0.73). We successfully amplified and sequenced 19.4% (7/36) of the positive samples which all corresponded to pseudocowpox virus. CONCLUSIONS: Obviously, further studies are needed to investigate the zoonotic potential of pseudocowpox virus and its importance for animals and public health.

High rates of antibodies against Toscana and Sicilian phleboviruses in common quail Coturnix coturnix birds.

Introduction: Birds are involved natural cycle of a number of vector-borne viruses in both rural and urban areas. Toscana (TOSV) and Sicilian (SFSV) phleboviruses are sandfly-borne viruses in the genus Phlebovirus that can cause diseases in human. However, there is limited information on the role of the birds in sandfly-borne phleboviruses natural cycle and reservoirs ofthese viruses remain unknown. Methods: In this study, we analyzed Common Quail (Coturnix coturnix) sera from Spain to identify the seroprevalence of these two phleboviruses. We tested respectively, 106 and 110 quail serum against TOSV and SFSV from 2018, 2019, and 2021 from two locations in northern Spain with using virus neutralization test. Results: We identified high neutralizing antibody rates for SFSV (45.45%) and TOSV (42.45%) with yearly fluctuation. Discussion: This is the first identification of SFSV and TOSV neutralizing antibodies in wild birds. High seroprevalence rates of TOSV and SFSV in quail birds raises the question whether birds have a role as amplifying hosts in the natural cycle of phleboviruses.

Human seroprevalence of Toscana virus and Sicilian phlebovirus in the southwest of Portugal.

Toscana virus (TOSV) is emergent in the Mediterranean region and responsible for outbreaks of encephalitis or meningoencephalitis. Sicilian phlebovirus (SFSV) cause epidemics of febrile illness during the summer. The aim of this study was to evaluate the presence of antibodies against TOSV and SFSV in humans in the southwest of Portugal. Neutralizing antibodies to TOSV and SFSV were respectively detected in 5.3% and 4.3% out of 400 human sera tested highlighting the need to increase public health awareness regarding phleboviruses and to include them in the differential diagnosis in patients presenting with fever of short duration and neurological manifestations.

Field surveys in Croatia and North Macedonia reveal two novel phleboviruses circulating in sandflies.

Sandfly-borne phleboviruses are distributed widely throughout the Mediterranean Basin, presenting a threat to public health in areas where they circulate. However, the true diversity and distribution of pathogenic and apathogenic sandfly-borne phleboviruses remains a key issue to be studied. In the Balkans, most published data rely on serology-based studies although virus isolation has occasionally been reported. Here, we report the discovery of two novel sandfly-borne phleboviruses, provisionally named Zaba virus (ZABAV) and Bregalaka virus (BREV), which were isolated in Croatia and North Macedonia, respectively. This constitutes the first isolation of phleboviruses in both countries. Genetic analysis based on complete coding sequences indicated that ZABAV and BREV are distinct from each other and belong to the genus Phlebovirus, family Phenuiviridae. Phylogenetic and amino acid modelling of viral polymerase shows that ZABAV and BREV are new members of the Salehabad phlebovirus species and the Adana phlebovirus species, respectively. Moreover, sequence-based vector identification suggests that ZABAV is mainly transmitted by Phlebotomus neglectus and BREV is mainly transmitted by Phlebotomus perfiliewi. BREV neutralizing antibodies were detected in 3.3% of human sera with rates up to 16.7% in certain districts, demonstrating that BREV frequently infects humans in North Macedonia. In vitro viral growth kinetics experiments demonstrated viral replication of both viruses in mammalian and mosquito cells. In vivo experimental studies in mice suggest that ZABAV and BREV exhibit characteristics making them possible human pathogens.

Incubation Period for Neuroinvasive Toscana Virus Infections.

Toscana virus (TOSV) is an emerging pathogen in the Mediterranean area and is neuroinvasive in its most severe form. Basic knowledge on TOSV biology is limited. We conducted a systematic review on travel-related infections to estimate the TOSV incubation period. We estimated the incubation period at 12.1 days.