Crimean-Congo haemorrhagic fever virus in questing non-Hyalomma spp. ticks in Northwest Spain, 2021.

Crimean-Congo haemorrhagic fever (CCHF) unexpectedly emerged in humans in Northwest Spain in 2021, and two additional cases were reported in the region in 2022. The 2021 case was associated with a tick bite on the outskirts of the city where the patient lived. PCR analysis of 95 questing ticks collected in the outskirts of that city in 2021, none of the genus Hyalomma, revealed a prevalence of confirmed CCHF virus (CCHFV) infection of 10.5%. Our results in this emerging scenario suggest the need to consider that CCHFV may be effectively spreading to Northwest Spain and to urgently understand any possible role of non-Hyalomma spp. ticks in the eco-epidemiological dynamics of CCHFV.

Re-Emergence of a West Nile Virus (WNV) Variant in South Spain with Rapid Spread Capacity.

West Nile Virus (WNV) is a mosquito vector-borne zoonosis with an increasing incidence in Europe that has become a public health concern. In Spain, although local circulation has been known for decades, until 2020, when a large outbreak occurred, West Nile Virus cases were scarce and mostly occurred in southern Spain. Since then, there have been new cases every year and the pathogen has spread to new regions. Thus, monitoring of circulating variants and lineages plays a fundamental role in understanding WNV evolution, spread and dynamics. In this study, we sequenced WNV consensus genomes from mosquito pools captured in 2022 as part of a newly implemented surveillance program in southern Spain and compared it to other European, African and Spanish sequences. Characterization of WNV genomes in mosquitoes captured in 2022 reveals the co-circulation of two WNV lineage 1 variants, the one that caused the outbreak in 2020 and another variant that is closely related to variants reported in Spain in 2012, France in 2015, Italy in 2021-2022 and Senegal in 2012-2018. The geographic distribution of these variants indicates that WNV L1 dynamics in southern Europe include an alternating dominance of variants in some territories.

Untargeted metagenomic sequencing identifies Toscana virus in patients with idiopathic meningitis, southern Spain, 2015 to 2019.

BackgroundVarious pathogens, including bacteria, fungi, parasites, and viruses can lead to meningitis. Among viruses causing meningitis, Toscana virus (TOSV), a phlebovirus, is transmitted through sandfly bites. TOSV infection may be suspected if patients with enterovirus- and herpesvirus-negative aseptic (non-bacterial) meningitis recall recent insect bites. Other epidemiological factors (season, rural area) may be considered. The broad range of possible meningitis aetiologies poses considerable diagnosis challenges. Untargeted metagenomic next-generation sequencing (mNGS) can potentially identify pathogens, which are not considered or detected in routine diagnostic panels.AimIn this retrospective, single-centre observational study, we investigated mNGS usefulness to understand the cause of meningitis when conventional approaches fail.MethodsCerebrospinal fluid (CSF) samples from patients hospitalised in southern Spain in 2015-2019 with aseptic meningitis and no aetiology found by conventional testing, were subjected to mNGS. Patients' demographic characteristics had been recorded and physicians had asked them about recent insect bites. Obtained viral genome sequences were phylogenetically analysed.ResultsAmong 23 idiopathic cases, TOSV was identified in eight (all male; median age: 39 years, range: 15-78 years). Five cases lived in an urban setting, three occurred in autumn and only one recalled insect bites. Phylogenetic analysis of TOSV segment sequences supported one intra-genotype reassortment event.ConclusionsOur study highlights the usefulness of mNGS for identifying viral pathogens directly in CSF. In southern Spain, TOSV should be considered regardless of recalling of insect bites or other epidemiological criteria. Detection of a disease-associated reassortant TOSV emphasises the importance of monitoring the spread and evolution of phleboviruses in Mediterranean countries.

Experimental infections in red-legged partridges reveal differences in host competence between West Nile and Usutu virus strains from Southern Spain.

Introduction: West Nile virus (WNV) and Usutu virus (USUV) are emerging zoonotic arboviruses sharing the same life cycle with mosquitoes as vectors and wild birds as reservoir hosts. The main objective of this study was to characterize the pathogenicity and course of infection of two viral strains (WNV/08 and USUV/09) co-circulating in Southern Spain in a natural host, the red-legged partridge (Alectoris rufa), and to compare the results with those obtained with the reference strain WNV/NY99. Methods: WNV inoculated birds were monitored for clinical and analytical parameters (viral load, viremia, and antibodies) for 15 days post-inoculation. Results and discussion: Partridges inoculated with WNV/NY99 and WNV/08 strains showed clinical signs such as weight loss, ruffled feathers, and lethargy, which were not observed in USUV/09-inoculated individuals. Although statistically significant differences in mortality were not observed, partridges inoculated with WNV strains developed significantly higher viremia and viral loads in blood than those inoculated with USUV. In addition, the viral genome was detected in organs and feathers of WNV-inoculated partridges, while it was almost undetectable in USUV-inoculated ones. These experimental results indicate that red-legged partridges are susceptible to the assayed Spanish WNV with pathogenicity similar to that observed for the prototype WNV/NY99 strain. By contrast, the USUV/09 strain was not pathogenic for this bird species and elicited extremely low viremia levels, demonstrating that red-legged partridges are not a competent host for the transmission of this USUV strain.

West Nile virus emergence in humans in Extremadura, Spain 2020.

In Spain, the largest human West Nile virus (WNV) outbreak among humans was reported in 2020, constituting the second most important outbreak in Europe that season. Extremadura (southwestern Spain) was one of the affected areas, reporting six human cases. The first autochthonous human case in Spain was reported in Extremadura in 2004, and no other human cases were reported until 2020. In this work, we describe the first WNV human outbreak registered in Extremadura, focusing on the most important clinical aspects, diagnostic results, and control actions which followed. In 2020, from September to October, human WNV infections were diagnosed using a combination of molecular and serological methods (an in-house specific qRT-PCR and a commercial ELISA for anti-WNV IgM and IgG antibodies) and by analysing serum, urine, and/or cerebrospinal fluid samples. Serological positive serum samples were further tested using commercial kits against related flaviviruses Usutu and Tick-borne encephalitis in order to analyse serological reactivity and to confirm the results by neutralisation assays. In total, six cases of WNV infection (five with neuroinvasive disease and one with fever) were identified. Clinical presentation and laboratory findings are described. No viral RNA was detected in any of the analysed samples, but serological cross-reactivity was detected against the other tested flaviviruses. Molecular and serological methods for WNV detection in various samples as well as differential diagnosis are recommended. The largest number of human cases of WNV infection ever registered in Extremadura, Spain, occurred in 2020 in areas where circulation of WNV and other flaviviruses has been previously reported in humans and animals. Therefore, it is necessary to enhance surveillance not only for the early detection and implementation of response measures for WNV but also for other emerging flaviviruses that could be endemic in this area.

A Novel RT-LAMP for the Detection of Different Genotypes of Crimean-Congo Haemorrhagic Fever Virus in Patients from Spain.

Crimean-Congo haemorrhagic fever (CCHF) is a potentially lethal tick-borne viral disease with a wide distribution. In Spain, 12 human cases of CCHF have been confirmed, with four deaths. The diagnosis of CCHF is hampered by the nonspecific symptoms, the high genetic diversity of CCHFV, and the biosafety requirements to manage the virus. RT-qPCR and serological tests are used for diagnosis with limitations. Reverse-transcription loop-mediated isothermal amplification (RT-LAMP) could be an effective alternative in the diagnosis of the disease. However, none of the few RT-LAMP assays developed to date has detected different CCHFV genotypes. Here, we designed a RT-LAMP using a degenerate primer set to compensate for the variability of the CCHFV target sequence. RT-LAMP was performed in colorimetric and real-time tests on RT-qPCR-confirmed CCHF patient samples notified in Spain in 2020 and 2021. Urine from an inpatient was analysed by RT-LAMP for the first time and compared with RT-qPCR. The amplicons obtained by RT-qPCR were sequenced and African III and European V genotypes were identified. RT-LAMP amplified both genotypes and was more sensitive than RT-qPCR in urine samples. We have developed a novel, rapid, specific, and sensitive RT-LAMP test that allows the detection of different CCHFV genotypes in clinical samples. This pan-CCHFV RT-LAMP detected viral RNA for the first time in urine samples. It can be easily performed as a single-tube isothermal colorimetric method on a portable platform in real time and without the need for expensive equipment, thus bringing molecular diagnostics closer to rural or resource-poor areas, where CCHF usually occurs.

Genomic Analysis of West Nile Virus Lineage 1 Detected in Mosquitoes during the 2020-2021 Outbreaks in Andalusia, Spain.

Emerging infectious diseases are one of the most important global health challenges because of their impact on human and animal health. The vector-borne West Nile virus (WNV) is transmitted between birds by mosquitos, but it can also infect humans and horses causing disease. The local circulation of WNV in Spain has been known for decades, and since 2010, there have been regular outbreaks in horses, although only six cases were reported in humans until 2019. In 2020, Spain experienced a major outbreak with 77 human cases, which was followed by 6 additional cases in 2021, most of them in the Andalusian region (southern Spain). This study aimed to characterize the genomes of the WNV circulating in wild-trapped mosquitoes during 2020 and 2021 in Andalusia. We sequenced the WNV consensus genome from two mosquito pools and carried out the phylogenetic analyses. We also compared the obtained genomes with those sequenced from human samples obtained during the outbreak and the genomes obtained previously in Spain from birds (2007 and 2017), mosquitoes (2008) and horses (2010) to better understand the eco-epidemiology of WNV in Spain. As expected, the WNV genomes recovered from mosquito pools in 2020 were closely related to those recovered from humans of the same outbreak. In addition, the strain of WNV circulating in 2021 was highly related to the WNV strain that caused the 2020 outbreak, suggesting that WNV is overwintering in the area. Consequently, future outbreaks of the same strain may occur in in the future.

Crimean-Congo Hemorrhagic Fever, Spain, 2013-2021.

Crimean-Congo hemorrhagic fever (CCHF) is a viral infectious disease for which distribution of the main vector, Hyalomma spp. ticks, is expanding. We analyzed all 10 cases of CCHF diagnosed in Spain during 2013-2021; case-patient median age was 56.5 years, and 7 were men. We identified CCHF virus genotypes III and V. Six case-patients acquired the infection in urban areas. Sixty percent of patients were infected in summer and 40% in spring. Two patients met criteria for hemophagocytic syndrome. Seven patients survived. The epidemiologic pattern of CCHF in Spain is based on occasional cases with an elevated mortality rate. Genotype III and, to a less extent also genotype V, CCHF circulates in humans in a common geographic area in Spain. Those data suggest that the expansion pathways are complex and may change over time. Physicians should remain alert to the possibility of new CCHF cases.

A One Health view of the West Nile virus outbreak in Andalusia (Spain) in 2020.

Reports of West Nile virus (WNV) associated disease in humans were scarce in Spain until summer 2020, when 77 cases were reported, eight fatal. Most cases occurred next to the Guadalquivir River in the Sevillian villages of Puebla del Río and Coria del Río. Detection of WNV disease in humans was preceded by a large increase in the abundance of Culex perexiguus in the neighbourhood of the villages where most human cases occurred. The first WNV infected mosquitoes were captured approximately one month before the detection of the first human cases. Overall, 33 positive pools of Cx. perexiguus and one pool of Culex pipiens were found. Serology of wild birds confirmed WNV circulation inside the affected villages, that transmission to humans also occurred in urban settings and suggests that virus circulation was geographically more widespread than disease cases in humans or horses may indicate. A high prevalence of antibodies was detected in blackbirds (Turdus merula) suggesting that this species played an important role in the amplification of WNV in urban areas. Culex perexiguus was the main vector of WNV among birds in natural and agricultural areas, while its role in urban areas needs to be investigated in more detail. Culex pipiens may have played some role as bridge vector of WNV between birds and humans once the enzootic transmission cycle driven by Cx. perexiguus occurred inside the villages. Surveillance of virus in mosquitoes has the potential to detect WNV well in advance of the first human cases.

First Isolation of Punique Virus from Sand Flies Collected in Northern Algeria.

In the last decade, several phleboviruses transmitted by sand flies were detected in the Mediterranean countries, with the health impact of some of them being unknown. From September to October 2020, a total of 3351 sand flies were captured in Kherrata (Bejaia, northern Algeria) and identified by sex, grouped in 62 pools, which were tested for the presence of phlebovirus RNA using endpoint RT-PCR. Two pools (male and female, respectively) were positive. The genome sequencing and phylogenetic analysis showed that the two phleboviruses detected were closely related to the Punique virus (PUNV) isolated in Tunisia and detected in Algeria. Both PUNV strains were isolated on VERO cells from positive pools. Morphological identification of 300 sand flies randomly selected, showed a clear dominance of Phlebotomus perniciosus (98.67%). The dominance of this species in the study area was confirmed by PCR targeting the mitochondrial DNA. Our result represents the first isolation of PUNV and the second report in Algeria from two distinct regions which confirm its large circulation in the country and more broadly in North Africa. Further studies are needed to measure the impact on public health through seroprevalence studies in humans as well as animals and to investigate its potential involvement in neurological viral diseases.

Monkeypox outbreak in Madrid (Spain): Clinical and virological aspects.

BACKGROUND: Monkeypox is the most prevalent Orthopoxvirus zoonosis infection since the eradication of smallpox. The current multi-country outbreak involves five WHO regions affecting mainly Europe. Accurate clinical and virological aspects of the disease outside endemic areas are needed. METHODS: We performed an observational study of cases diagnosed in Madrid (Spain) (May/June 2022). Confirmation from vesicular lesions swabs, Orthopoxvirus real-time PCR, sequencing, phylogenetic analysis, and direct detection by Electron microscopy was performed. In addition, a structured epidemiological questionnaire was completed systematically to gather sociodemographic, clinical, and behavioral data from all confirmed cases. FINDINGS: We extracted data from 48 patients, all cisgender men. The median age was 35 years (IQR 29 - 44), and 87.5% were MSM. The most prevalent symptoms were the presence of vesicular-umbilicated and pseudo-pustular skin lesions (93.8%), asthenia (66.6%), and fever (52.1%). In addition, the location of the lesions in the genital or perianal area was related to the role in sexual intercourse (p<0.001). Sequencing analysis indicated the virus circulating in Spain belongs to the western African clade. Like the other European cases in the outbreak, the Spanish isolates are a direct descendant of viruses previously detected in Nigeria, the UK, Singapore, and Israel in 2017-2018. CONCLUSIONS: Monkeypox is an emerging infectious disease in Europe where community transmission is reported, mainly in MSM. The first symptom was skin lesions instead of classical fever and rash. The disease follows a self-limited course, and there have been no cases with a serious presentation or severe complications.

Molecular Characterization of Imported and Autochthonous Dengue in Northeastern Spain.

Dengue is the most significant arbovirus worldwide and a public health threat to non-endemic areas in which Aedes vectors are present. Autochthonous dengue transmission has been reported in several European countries in the last decade. Infected travelers from endemic regions arriving to areas colonized by Aedes albopictus in Europe need to be monitored in surveillance and control programs. We aimed to perform molecular characterization of RT-PCR-positive dengue cases detected in Catalonia, northeastern Spain, from 2013 to 2018. The basic demographic information and the geographical regions of importation were also analyzed. One-hundred four dengue cases were studied (103 imported infections and the first autochthonous case in our region). The dengue virus strains detected were serotyped and genotyped using molecular methods, and phylogenetic analyses were conducted. All four dengue serotypes were detected in travelers, including up to 10 different genotypes, reflecting the global circulation of dengue in endemic areas. The primary travel-related case of the 2018 autochthonous transmission was not identified, but the molecular analysis revealed dengue serotype 1, genotype I of Asian origin. Our results highlight the diversity of imported dengue virus strains and the role of molecular epidemiology in supporting arbovirus surveillance programs.

Unprecedented increase of West Nile virus neuroinvasive disease, Spain, summer 2020.

Cases of West Nile neuroinvasive disease (WNND) in Spain increased in summer 2020. Here we report on this increase and the local, regional and national public health measures taken in response. We analysed data from regional surveillance networks and the National Epidemiological Surveillance Network, both for human and animal West Nile virus (WNV) infection. During the 2020 season, a total of 77 human cases of WNV infection (median age 65 years; 60% males) were detected in the south-west of Spain; 72 (94%) of these cases developed WNND, presenting as meningoencephalitis, seven of which were fatal. In the previous two decades, only six human cases of WNND were detected in Spain. Reduced activities for vector control this season, together with other factors, might have contributed to the massive increase. Public health measures including vector control, campaigns to raise awareness among physicians and the general population, and interventions to ensure the safety of donations of blood products, organs, cells and tissues were effective to reduce transmission. Going forward, maintenance of vector control activities and an update of the vector-borne diseases response plan in Spain is needed.

Retrospective Identification of Early Autochthonous Case of Crimean-Congo Hemorrhagic Fever, Spain, 2013.

Before this report, 7 autochthonous human cases of Crimean-Congo hemorrhagic fever had been reported in Spain, all occurring since 2016. We describe the retrospective identification of an eighth case dating back to 2013. This study highlights that the earliest cases of an emerging disease are often difficult to recognize.

New circulation of genotype V of Crimean-Congo haemorrhagic fever virus in humans from Spain.

BACKGROUND: Crimean-Congo haemorrhagic fever (CCHF) is a widespread tick-borne viral disease caused by the Crimean-Congo haemorrhagic fever virus (CCHFV). CCHFV has been implicated in severe viral haemorrhagic fever outbreaks. During the summer of 2016, the first two cases with genotype III (Africa 3) were reported in Spain. The first objective of our study was to determine the presence of CCHFV among patients with febrile illness during the spring and summer periods in 2017 and 2018. Finally, we perform a phylogenetic analysis to determine the genotype of the virus. METHODOLOGY: We prospectively evaluated patients aged 18 years and older who came to the emergency department at the Salamanca's University Hospital (HUS) with fever. Specific IgM and IgG antibodies against CCHFV by ELISA and one immunofluorescence assay against two different proteins (nucleoprotein and glycoprotein C) was done. Moreover, molecular detection by Real Time PCR was performed in all collected samples. A phylogenetic analysis was carried out to genetically characterize CCHFV detected in this study. PRINCIPAL FINDINGS: A total of 133 patients were selected. The mean age was 67.63 years and 60.9% were male. One-third of the patients presented an acute undifferentiated febrile illness. Three patients had anti-CCHFV IgG antibodies, suggesting a previous infection. One patient had anti-CCHFV IgM antibodies and a confirmatory RT-PCR. Phylogenetic analysis indicated that the virus corresponds to the European genotype V. This patient came to the emergency department at HUS in August 2018 presenting an acute febrile syndrome with thrombopenia and liver impairment. CONCLUSIONS: We describe a new circulation of European genotype V CCHFV in Spain. Moreover, this study suggests that CCHFV is an identifiable cause of febrile illness of unknown origin in Spain. Thus, CCHF could be suspected in patients with fever, liver damage, and/or haemorrhagic disorders, particularly in people with risk activities who present in the spring or summer.

Widespread Detection of Multiple Strains of Crimean-Congo Hemorrhagic Fever Virus in Ticks, Spain.

Human cases of Crimean-Congo hemorrhagic fever (CCHF) were first detected in Spain in 2016. National human and animal health authorities organized a large, multidisciplinary study focusing on ticks as sentinels to determine the nationwide distribution of ticks with CCHF virus. Ticks were collected from animals and vegetation, samples pooled (12,584 ticks; 4,556 pools), and molecular methods used to look for the virus. We detected the virus in 135 pools from most of the regions studied, indicating that it is widespread in Spain. We found sequences of CCHF virus genotypes I, III, and IV in the tick species collected, most commonly in Hyalomma lusitanicum, suggesting this tick has a prominent role in the virus's natural cycle. The red deer (Cervus elaphus) was the host that most frequently yielded positive ticks. Our study highlights the need for larger studies in Spain to ascertain the complete risk to public health.

Absence of IgG antibodies among high-risk contacts of two confirmed cases of Crimean-Congo haemorrhagic fever in the autonomous region of Madrid (Spain).

Crimean-Congo haemorrhagic fever (CCHF) is a widely distributed tick-borne disease. In Spain, the disease has emerged as outbreak associated with high-risk exposures. Our goal was to evaluate the prevalence of antibodies against the CCHF virus (CCHFV) in high-risk contacts. A cross-sectional study was conducted. Three hundred eighty-six high-risk contacts were identified comprising family contacts and hospital workers who had attended the cases. Fifty-seven cases with closer exposure were selected. However, forty-nine cases participated in the study. IgG antibodies were detected by immunoenzymatic techniques. All determinations tested negative for anti-CCHFV IgG antibodies. Most of the responders were women (73.5%), and belong to the intensive care department (53.1%). In relation to other possible sources of exposures, 18.4% travelled to countries with CCHF transmission risk. No CCHF positivity was recorded among selected high-risk contacts. This highlights the importance of standard precautions which might have protected healthcare workers and care providers from CCHF infection.

Real-time RT-PCR assay to detect Granada virus and the related Massilia and Arrabida phleboviruses.

BACKGROUND: Granada virus belongs to the genus Phlebovirus within the Naples serocomplex and was detected for the first time in sand flies from Spain in 2003. Seroprevalence studies have revealed that Granada virus may infect humans with most cases being asymptomatic. Moreover, recent studies in vector samples revealed that the related Massilia and Arrabida phleboviruses could be also circulating in Spain. The objective of this study was to develop and assess a new sensitive real-time RT-PCR assay for Granada virus diagnosis able to detect the related phleboviruses Massilia and Arrabida. METHODS: Two specific primers and one unique probe to detect Granada, Massilia and Arrabida viruses, without differentiating between them, were designed targeting the conserved L-segment of their genome. Sensitivity was assessed using 10-fold serial dilutions of quantified in vitro DNA samples. Specificity was evaluated by testing different genomic RNA extracted from other representative phleboviruses. The new assay was used for virus detection in sand flies collected in 2012 from the Balearic Archipelago, a touristic hotspot in the Mediterranean. RESULTS: The real-time RT-PCR assay exhibited a sensitivity per reaction of 19 copies for Granada and Arrabida, and 16 copies for Massilia. No other related phleboviruses were detected. From the 37 pools of sand fly samples studied from four different Balearic Islands, we detected one positive in the island of Cabrera. CONCLUSIONS: To our knowledge, the method described here is the first real-time RT-PCR designed to detect Granada virus and the related Massilia and Arrabida phleboviruses. The study demonstrated that this is a rapid, robust and reliable assay for the accurate diagnosis of human infections as well as for virus surveillance in vectors.

Clinical Outcomes of a Zika Virus Mother-Child Pair Cohort in Spain.

BACKGROUND: Zika virus (ZIKV) infection has been associated with congenital microcephaly and other neurodevelopmental abnormalities. There is little published research on the effect of maternal ZIKV infection in a non-endemic European region. We aimed to describe the outcomes of pregnant travelers diagnosed as ZIKV-infected in Spain, and their exposed children. METHODS: This prospective observational cohort study of nine referral hospitals enrolled pregnant women (PW) who travelled to endemic areas during their pregnancy or the two previous months, or those whose sexual partners visited endemic areas in the previous 6 months. Infants of ZIKV-infected mothers were followed for about two years. RESULTS: ZIKV infection was diagnosed in 163 PW; 112 (70%) were asymptomatic and 24 (14.7%) were confirmed cases. Among 143 infants, 14 (9.8%) had adverse outcomes during follow-up; three had a congenital Zika syndrome (CZS), and 11 other potential Zika-related outcomes. The overall incidence of CZS was 2.1% (95%CI: 0.4-6.0%), but among infants born to ZIKV-confirmed mothers, this increased to 15.8% (95%CI: 3.4-39.6%). CONCLUSIONS: A nearly 10% overall risk of neurologic and hearing adverse outcomes was found in ZIKV-exposed children born to a ZIKV-infected traveler PW. Longer-term follow-up of these children is needed to assess whether there are any later-onset manifestations.