Warm winters are associated to more intense West Nile virus circulation in southern Spain.

West Nile virus (WNV) is the most widely distributed mosquito-borne flavivirus in the world. This flavivirus can infect humans causing in some cases a fatal neurological disease and birds are the main reservoir hosts. WNV is endemic in Spain, and human cases have been reported since 2004. Although different studies analyse how climatic conditions can affect the dynamics of WNV infection, very few use long-term datasets. Between 2003 and 2020 a total of 2,724 serum samples from 1,707 common coots (Fulica atra) were analysed for the presence of WNV-specific antibodies. Mean (SD) annual seroprevalence was 24.67% (0.28) but showed high year-to-year variations ranging from 5.06% (0.17) to 68.89% (0.29). Significant positive correlations (p < 0.01) were observed between seroprevalence and maximum winter temperature and mean spring temperature. The unprecedented WNV outbreak in humans in the south of Spain in 2020 was preceded by a prolonged period of escalating WNV local circulation. Given current global and local climatic trends, WNV circulation is expected to increase in the next decades. This underscores the necessity of implementing One Health approaches to reduce the risk of future WNV outbreaks in humans. Our results suggest that higher winter and spring temperatures may be used as an early warning signal of more intense WNV circulation among wildlife in Spain, and consequently highlight the need of more intense vector control and surveillance in human inhabited areas.

Implications of migratory and exotic birds and the mosquito community on West Nile virus transmission.

BACKGROUND: Vector-borne diseases like West Nile virus (WNV) pose a global health challenge, with rising incidence and distribution. Culex mosquitoes are crucial WNV vectors. Avian species composition and bird community diversity, along with vector communities, influence WNV transmission patterns. However, limited knowledge exists on their impact in southwestern Spain, an area with active WNV circulation in wild birds, mosquitoes, and humans. METHODS: To address this, we conducted a comprehensive study investigating the contributions of migratory and exotic bird species to WNV transmission and the influence of mosquito community composition. RESULTS: Analysing 1194 serum samples from 44 avian species, we detected WNV antibodies in 32 samples from 11 species, four for the first time in Europe. Migratory birds had higher WNV exposure likelihood than native and exotic species, and higher phylogenetic diversity in bird communities correlated with lower exposure rates. Moreover, in 5859 female mosquitoes belonging to 12 species, we identified WNV competent vectors like Cx. pipiens s.l. and the Univittatus subgroup. Birds with WNV antibodies were positively associated with competent vector abundance, but negatively with overall mosquito species richness. CONCLUSIONS: These findings highlight the complex interactions between bird species, their phylogenetics, and mosquito vectors in WNV transmission. Understanding these dynamics will help to implement effective disease control strategies in southwestern Spain.

Detection of a new avian bornavirus in barn owl (Tyto alba) by pan-viral microarray.

A barn owl (Tyto alba) died with neurological signs compatible with a viral infection. After discarding other possible infections caused by circulating viruses in the area, analysis of the central nervous system using a pan-viral microarray revealed hybridization to canary bornavirus 2 (CnBV-2). Subsequent sequence analysis confirmed the presence of a virus sharing more than 83% identity with CnBV-2. Surprisingly, the new sequence corresponds to a new virus, here named Barn owl Bornavirus 1 (BoBV-1), within the Orthobornavirus serini species. Moreover, it is the first member of this species that has been detected in a non-passerine bird, indicating that Orthobornavirus serini species comprises viruses with a wider range of hosts than previously presumed. The use of this microarray has proven to be an excellent tool for viral detection in clinical samples, with capacity to detect new viral variants. This allows the diagnosis of a great range of viruses, which can cause similar disease symptoms and which identification by PCR methods might be tedious, probably unsuccessful and, in the long run, expensive. This platform is highly useful for a fast and precise viral detection, contributing to the improvement of diagnostic methods.

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.

Long-term serological surveillance for West Nile and Usutu virus in horses in south-West Spain.

West Nile virus (WNV) is a re-emerging zoonotic pathogen with increasing incidence in Europe, producing a recent outbreak in 2020 in Spain with 77 human cases and eight fatalities. However, the factors explaining the observed changes in the incidence of WNV in Europe are not completely understood. Longitudinal monitoring of WNV in wild animals across Europe is a useful approach to understand the eco-epidemiology of WNV in the wild and the risk of spillover into humans. However, such studies are very scarce up to now. Here, we analysed the occurrence of WNV and Usutu virus (USUV) antibodies in 2102 samples collected between 2005 and 2020 from a population of feral horses in Doñana National Park. The prevalence of WNV antibodies varied between years, with a mean seroprevalence of 8.1% (range 0%-25%) and seasonally. Climate conditions including mean minimum annual temperatures and mean rainy days per year were positively correlated with WNV seroprevalence, while the annual rainfall was negatively. We also detected the highest incidence of seroconversions in 2020 coinciding with the human outbreak in southern Spain. Usutu virus-specific antibodies were detected in the horse population since 2011. The WNV outbreak in humans was preceded by a long period of increasing circulation of WNV among horses with a very high exposure in the year of the outbreak. These results highlight the utility of One Health approaches to better understand the transmission dynamics of zoonotics pathogens.

The Detection of SARS-CoV-2 Antibodies in an Exposed Human Population Is Biased by the Immunoassay Used: Implications in Serosurveillance.

The presence of SARS-CoV-2 antibodies was examined over 7 months in a population of essential service workers exposed during the first epidemic wave in Madrid (Spain). Results obtained with different serological assays were compared. Firstly, serum samples obtained in April 2020 were analyzed using eleven SARS-CoV-2 antibody detection methods, including seven ELISAs, two CLIAs and two LFAs. While all of the ELISA tests and the Roche eCLIA method showed good performance, it was poorer for the Abbott CLIA and LFA tests. Sera from 115 workers with serologically positive results in April were collected 2 and 7 months after the first sampling and were analyzed using five of the tests previously assessed. The results showed that while some ELISA tests consistently detected the presence of anti-SARS-CoV-2 antibodies even 7 months after first detection, other methods, such as the Abbott CLIA test, showed an important reduction in sensitivity for these mature antibodies. The sensitivity increased after establishing new cut-off values, calculated taking into account both recent and old infections, suggesting that an adjustment of assay parameters may improve the detection of individuals exposed to the infection.

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.

Evaluation of NS4A, NS4B, NS5 and 3'UTR Genetic Determinants of WNV Lineage 1 Virulence in Birds and Mammals.

West Nile virus (WNV) is amplified in an enzootic cycle involving birds as amplifying hosts. Because they do not develop high levels of viremia, humans and horses are considered to be dead-end hosts. Mosquitoes, especially from the Culex genus, are vectors responsible for transmission between hosts. Consequently, understanding WNV epidemiology and infection requires comparative and integrated analyses in bird, mammalian, and insect hosts. So far, markers of WNV virulence have mainly been determined in mammalian model organisms (essentially mice), while data in avian models are still missing. WNV Israel 1998 (IS98) is a highly virulent strain that is closely genetically related to the strain introduced into North America in 1999, NY99 (genomic sequence homology > 99%). The latter probably entered the continent at New York City, generating the most impactful WNV outbreak ever documented in wild birds, horses, and humans. In contrast, the WNV Italy 2008 strain (IT08) induced only limited mortality in birds and mammals in Europe during the summer of 2008. To test whether genetic polymorphism between IS98 and IT08 could account for differences in disease spread and burden, we generated chimeric viruses between IS98 and IT08, focusing on the 3' end of the genome (NS4A, NS4B, NS5, and 3'UTR regions) where most of the non-synonymous mutations were detected. In vitro and in vivo comparative analyses of parental and chimeric viruses demonstrated a role for NS4A/NS4B/5'NS5 in the decreased virulence of IT08 in SPF chickens, possibly due to the NS4B-E249D mutation. Additionally, significant differences between the highly virulent strain IS98 and the other three viruses were observed in mice, implying the existence of additional molecular determinants of virulence in mammals, such as the amino acid changes NS5-V258A, NS5-N280K, NS5-A372V, and NS5-R422K. As previously shown, our work also suggests that genetic determinants of WNV virulence can be host-dependent.

Multiplex Assay for Simultaneous Detection of Antibodies against Crimean-Congo Hemorrhagic Fever Virus Nucleocapsid Protein and Glycoproteins in Ruminants.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a widespread tick-borne zoonotic virus that causes Crimean-Congo hemorrhagic fever (CCHF). CCHF is asymptomatic in infected animals but can develop into severe illness in humans, with high case-fatality rates. Due to complex environmental and socio-economic factors, the distribution of CCHFV vectors is changing, leading to disease occurrence in previously unaffected countries. Neither an effective treatment nor a vaccine has been developed against CCHFV; thus, surveillance programs are essential to limit and control the spread of the virus. Furthermore, the WHO highlighted the need of assays that can cover a range of CCHFV antigenic targets, DIVA (differentiating infected from vaccinated animals) assays, or assays for future vaccine evaluation. Here, we developed a multiplex assay, based on a suspension microarray, able to detect specific antibodies in ruminants to three recombinantly produced CCHFV proteins: the nucleocapsid (N) protein and two glycoproteins, GN ectodomain (GNe), and GP38. This triplex assay was used to assess the antibody response in naturally infected animals. Out of the 29 positive field sera to the N protein, 40% showed antibodies against GNe or GP38, with 11 out of these 12 samples being positive to both glycoproteins. To determine the diagnostic specificity of the test, a total of 147 sera from Spanish farms free of CCHFV were included in the study. This multiplex assay could be useful to detect antibodies to different proteins of CCHFV as vaccine target candidates and to study the immune response to CCHFV in infected animals and for surveillance programs to prevent the further spread of the virus. IMPORTANCE Crimean-Congo hemorrhagic fever virus (CCHFV) causes Crimean-Congo hemorrhagic fever, which is one of the most important tick-borne viral diseases of humans and has recently been found in previously unaffected countries such as Spain. The disease is asymptomatic in infected animals but can develop into severe illness in humans. As neither an effective treatment nor a vaccine has been developed against CCHFV, surveillance programs are essential to limit and control the spread of the virus. In this study, a multiplex assay detecting antibodies against different CCHFV antigens in a single sample and independent of the ruminant species has been developed. This assay could be very useful in surveillance studies, to control the spread of CCHFV and prevent future outbreaks, and to better understand the immune response induced by CCHFV.

Risk Factors for Exposure of Wild Birds to West Nile Virus in A Gradient of Wildlife-Livestock Interaction.

West Nile virus (WNV) transmission rate is shaped by the interaction between virus reservoirs and vectors, which may be maximized in farm environments. Based on this hypothesis, we screened for WNV in wild birds in three scenarios with decreasing gradient of interaction with horses: (i) the farm (A1); (ii) the neighborhood (A2); and (iii) a wild area (A3). We captured wild birds and analyzed their sera for WNV antibodies by blocking ELISA and micro-virus neutralization test. Flavivirus infections were tested with generic and specific PCR protocols. We parameterized linear mixed models with predictors (bird abundance and diversity, vector abundance, vector host abundance, and weather quantities) to identify Flavivirus spp. and WNV exposure risk factors. We detected a low rate of Flavivirus infections by PCR (0.8%) and 6.9% of the birds were seropositive by ELISA. Exposure to Flavivirus spp. was higher in A1 (9%) than in A2 and A3 (5.6% and 5.8%, respectively). Bird diversity was the most relevant predictor of exposure risk and passerines dominated the on-farm bird community. Our results suggest that measures deterring the use of the farm by passerines should be implemented because the environmental favorability of continental Mediterranean environments for WNV is increasing and more outbreaks are expected.

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.

Autophagy-linked plasma and lysosomal membrane protein PLAC8 is a key host factor for SARS-CoV-2 entry into human cells.

Better understanding on interactions between SARS-CoV-2 and host cells should help to identify host factors that may be targetable to combat infection and COVID-19 pathology. To this end, we have conducted a genome-wide CRISPR/Cas9-based loss-of-function screen in human lung cancer cells infected with SARS-CoV-2-pseudotyped lentiviruses. Our results recapitulate many findings from previous screens that used full SARS-CoV-2 viruses, but also unveil two novel critical host factors: the lysosomal efflux transporter SPNS1 and the plasma and lysosomal membrane protein PLAC8. Functional experiments with full SARS-CoV-2 viruses confirm that loss-of-function of these genes impairs viral entry. We find that PLAC8 is a key limiting host factor, whose overexpression boosts viral infection in eight different human lung cancer cell lines. Using single-cell RNA-Seq data analyses, we demonstrate that PLAC8 is highly expressed in ciliated and secretory cells of the respiratory tract, as well as in gut enterocytes, cell types that are highly susceptible to SARS-CoV-2 infection. Proteomics and cell biology studies suggest that PLAC8 and SPNS1 regulate the autophagolysosomal compartment and affect the intracellular fate of endocytosed virions.

Clinical, Virological and Immunological Responses after Experimental Infection with African Horse Sickness Virus Serotype 9 in Immunologically Naïve and Vaccinated Horses.

This study described the clinical, virological, and serological responses of immunologically naïve and vaccinated horses to African horse sickness virus (AHSV) serotype 9. Naïve horses developed a clinical picture resembling the cardiac form of African horse sickness. This was characterized by inappetence, reduced activity, and hyperthermia leading to lethargy and immobility-recumbency by days 9-10 post-infection, an end-point criteria for euthanasia. After challenge, unvaccinated horses were viremic from days 3 or 4 post-infection till euthanasia, as detected by serogroup-specific (GS) real time RT-PCR (rRT-PCR) and virus isolation. Virus isolation, antigen ELISA, and GS-rRT-PCR also demonstrated high sensitivity in the post-mortem detection of the pathogen. After infection, serogroup-specific VP7 antibodies were undetectable by blocking ELISA (b-ELISA) in 2 out of 3 unvaccinated horses during the course of the disease (9-10 dpi). Vaccinated horses did not show significant side effects post-vaccination and were largely asymptomatic after the AHSV-9 challenge. VP7-specific antibodies could not be detected by the b-ELISA until day 21 and day 30 post-inoculation, respectively. Virus neutralizing antibody titres were low or even undetectable for specific serotypes in the vaccinated horses. Virus isolation and GS-rRT-PCR detected the presence of AHSV vaccine strains genomes and infectious vaccine virus after vaccination and challenge. This study established an experimental infection model of AHSV-9 in horses and characterized the main clinical, virological, and immunological parameters in both immunologically naïve and vaccinated horses using standardized bio-assays.

West Nile Virus Lineage 2 Spreads Westwards in Europe and Overwinters in North-Eastern Spain (2017-2020).

West Nile virus lineage 2 (WNV-L2) emerged in Europe in 2004; since then, it has spread across the continent, causing outbreaks in humans and animals. During 2017 and 2020, WNV-L2 was detected and isolated from four northern goshawks in two provinces of Catalonia (north-eastern Spain). In order to characterise the first Spanish WNV-L2 isolates and elucidate the potential overwintering of the virus in this Mediterranean region, complete genome sequencing, phylogenetic analyses, and a study of phenotypic characterisation were performed. Our results showed that these Spanish isolates belonged to the central-southern WNV-L2 clade. In more detail, they were related to the Lombardy cluster that emerged in Italy in 2013 and has been able to spread westwards, causing outbreaks in France (2018) and Spain (2017 and 2020). Phenotypic characterisation performed in vitro showed that these isolates presented characteristics corresponding to strains of moderate to high virulence. All these findings evidence that these WNV-L2 strains have been able to circulate and overwinter in the region, and are pathogenic, at least in northern goshawks, which seem to be very susceptible to WNV infection and may be good indicators of WNV-L2 circulation. Due to the increasing number of human and animal cases in Europe in the last years, this zoonotic flavivirus should be kept under extensive surveillance, following a One-Health approach.

First evidence of Crimean-Congo haemorrhagic fever virus circulation in Bosnia and Herzegovina.

BACKGROUND: Crimean-Congo haemorrhagic fever (CCHF) is a widespread tick-borne zoonosis with reported detection of virus and/or virus-specific antibodies from over 57 countries across Africa, Asia, Europe and the Middle East and is endemic in the Balkans. Detection of Crimean-Congo Haemorrhagic Fever Virus (CCHFV) antibodies in domestic ruminants has been important in providing initial evidence of virus circulation and in localising CCHFV high-risk spots for human infection. OBJECTIVES: The present study investigated the possible exposure of sheep to CCHFV in Bosnia and Herzegovina (B&H). METHODS: To investigate the presence of anti-CCHFV antibodies in sheep, all sera (n = 176) were tested using multi-species double antigen enzyme-linked immunosorbent assay (ELISA). Reactive sera were further complementary tested by adapted commercial indirect immunofluorescence assay (IFA) using FITC-conjugated protein G instead of anti-human immunoglobulins. RESULTS: CCHFV specific antibodies were detected in 17 (9.66%) animals using ELISA test. All negative sera were determined as negative by both tests, while 13 out of 17 ELISA-positive reactors were also determined as unambiguously positive by IFA test. The age group with the highest proportion of seropositive rectors were the oldest animals. CONCLUSIONS: This is the first report of anti-CCHFV antibodies in sheep from B&H providing the evidence of CCHFV circulation in the country's sheep population. So far, these findings indicate the circulation of the virus in the westernmost region of the Balkans and point to the potential CCHFV spread further out of this endemic area.

A new cluster of West Nile virus lineage 1 isolated from a northern goshawk in Spain.

West Nile Virus (WNV; family Flaviviridae, genus flavivirus) is a zoonotic arbovirus worldwide spread. Its genetic diversity has allowed the definition of at least seven lineages, being lineages 1 and 2 the most widely distributed. Western Mediterranean region has been affected by WNV since decades. In Spain, WNV is actively circulating, provoking annual outbreaks in birds, horses and lately in humans. Lineage 1 is responsible for outbreaks that occurred in central and southern regions, while lineage 2 has been recently described in wild birds in north-eastern part of the country. During 2017 season, a disease outbreak in captive raptors was reported in southern Spain and WNV was isolated from a dead northern goshawk. Full genome sequencing was followed by phylogenetic analyses and analyses of the amino acidic substitutions. This strain, named Spain/2017/NG-b, highly differs from those which have been circulating both in Spain and in the neighbouring Mediterranean countries, constituting a new distinct group, tentatively classified in a newly defined cluster 7 within the WNV clade 1a, supporting a new, independent introduction of the virus in the Western Mediterranean region from an unknown origin. Besides, circumstantial evidence indicates that this emerging WNV strain could be behind the subsequent outbreak occurred nearby in horses. Overall, the reinforcement of surveillance programs, especially in wild birds, is essential to early detect the circulation of WNV and other related flaviviruses that could cause outbreaks in wild or domestic birds, equine and human populations.

Widespread Circulation of Flaviviruses in Horses and Birds in Northeastern Spain (Catalonia) between 2010 and 2019.

The surveillance for West Nile virus (WNV) in Catalonia (northeastern Spain) has consistently detected flaviviruses not identified as WNV. With the aim of characterizing the flaviviruses circulating in Catalonia, serum samples from birds and horses collected between 2010 and 2019 and positive by panflavivirus competition ELISA (cELISA) were analyzed by microneutralization test (MNT) against different flaviviruses. A third of the samples tested were inconclusive by MNT, highlighting the limitations of current diagnostic techniques. Our results evidenced the widespread circulation of flaviviruses, in particular WNV, but also Usutu virus (USUV), and suggest that chicken and horses could serve as sentinels for both viruses. In several regions, WNV and USUV overlapped, but no significant geographical aggregation was observed. Bagaza virus (BAGV) was not detected in birds, while positivity to tick-borne encephalitis virus (TBEV) was sporadically detected in horses although no endemic foci were observed. So far, no human infections by WNV, USUV, or TBEV have been reported in Catalonia. However, these zoonotic flaviviruses need to be kept under surveillance, ideally within a One Health framework.

The C-Terminal Half of SARS-CoV-2 Nucleocapsid Protein, Industrially Produced in Plants, Is Valid as Antigen in COVID-19 Serological Tests.

BACKGROUND: The fight against the current coronavirus disease 2019 (COVID-19) pandemic has created a huge demand of biotechnological, pharmaceutical, research and sanitary materials at unprecedented scales. One of the most urgent demands affects the diagnostic tests. The growing need for rapid and accurate laboratory diagnostic tests requires the development of biotechnological processes aimed at producing reagents able to cope with this demand in a scalable, cost-effective manner, with rapid turnaround times. This is particularly applicable to the antigens employed in serological tests. Recombinant protein expression using plants as biofactories is particularly suitable for mass production of protein antigens useful in serological diagnosis, with a neat advantage in economic terms. METHODS: We expressed a large portion of the nucleoprotein (N) derived from SARS-CoV-2 in Nicotiana benthamiana plants. After purification, the recombinant N protein obtained was used to develop an indirect enzyme-linked immunosorbent assay (ELISA) for detection of antibodies to SARS-CoV-2 in human sera. To validate the ELISA, a panel of 416 sera from exposed personnel at essential services in Madrid City Council were tested, and the results compared to those obtained by another ELISA, already validated, used as reference. Furthermore, a subset of samples for which RT-PCR results were available were used to confirm sensitivity and specificity of the test. RESULTS: The performance of the N protein expressed in plants as antigen in serologic test for SARS-CoV-2 antibody detection was shown to be highly satisfactory, with calculated diagnostic sensitivity of 96.41% (95% CI: 93.05-98.44) and diagnostic specificity of 96.37 (95% CI: 93.05-98.44) as compared to the reference ELISA, with a kappa (K) value of 0.928 (95% CI:0.892-0.964). Furthermore, the ELISA developed with plant-derived N antigen detected SARS-CoV-2 antibodies in 84 out of 93 sera from individuals showing RT-PCR positive results (86/93 for the reference ELISA). CONCLUSION: This study demonstrates that the N protein part derived from SARS-CoV-2 expressed in plants performs as a perfectly valid antigen for use in COVID-19 diagnosis. Furthermore, our results support the use of this plant platform for expression of recombinant proteins as reagents for COVID-19 diagnosis. This platform stands out as a convenient and advantageous production system, fit-for-purpose to cope with the current demand of this type of biologicals in a cost-effective manner, making diagnostic kits more affordable.

Pathogenesis of Two Western Mediterranean West Nile Virus Lineage 1 Isolates in Experimentally Infected Red-Legged Partridges (Alectoris rufa).

West Nile virus (WNV) is the most widespread flavivirus in the world with a wide vertebrate host range. Its geographic expansion and activity continue to increase with important human and equine outbreaks and local bird mortality. In a previous experiment, we demonstrated the susceptibility of 7-week-old red-legged partridges (Alectoris rufa) to Mediterranean WNV isolates Morocco/2003 and Spain/2007, which varied in virulence for this gallinaceous species. Here we study the pathogenesis of the infection with these two strains to explain the different course of infection and mortality. Day six post-inoculation was critical in the course of infection, with the highest viral load in tissues, the most widespread virus antigen, and more severe lesions. The most affected organs were the heart, liver, and spleen. Comparing infections with Morocco/2003 and Spain/2007, differences were observed in the viral load, virus antigen distribution, and lesion nature and severity. A more acute and marked inflammatory reaction (characterized by participation of microglia and CD3+ T cells) as well as neuronal necrosis in the brain were observed in partridges infected with Morocco/2003 as compared to those infected with Spain/2007. This suggests a higher neurovirulence of Morocco/2003, probably related to one or more specific molecular determinants of virulence different from Spain/2007.

A field test of the dilution effect hypothesis in four avian multi-host pathogens.

The Dilution Effect Hypothesis (DEH) argues that greater biodiversity lowers the risk of disease and reduces the rates of pathogen transmission since more diverse communities harbour fewer competent hosts for any given pathogen, thereby reducing host exposure to the pathogen. DEH is expected to operate most intensely in vector-borne pathogens and when species-rich communities are not associated with increased host density. Overall, dilution will occur if greater species diversity leads to a lower contact rate between infected vectors and susceptible hosts, and between infected hosts and susceptible vectors. Field-based tests simultaneously analysing the prevalence of several multi-host pathogens in relation to host and vector diversity are required to validate DEH. We tested the relationship between the prevalence in house sparrows (Passer domesticus) of four vector-borne pathogens-three avian haemosporidians (including the avian malaria parasite Plasmodium and the malaria-like parasites Haemoproteus and Leucocytozoon) and West Nile virus (WNV)-and vertebrate diversity. Birds were sampled at 45 localities in SW Spain for which extensive data on vector (mosquitoes) and vertebrate communities exist. Vertebrate censuses were conducted to quantify avian and mammal density, species richness and evenness. Contrary to the predictions of DEH, WNV seroprevalence and haemosporidian prevalence were not negatively associated with either vertebrate species richness or evenness. Indeed, the opposite pattern was found, with positive relationships between avian species richness and WNV seroprevalence, and Leucocytozoon prevalence being detected. When vector (mosquito) richness and evenness were incorporated into the models, all the previous associations between WNV prevalence and the vertebrate community variables remained unchanged. No significant association was found for Plasmodium prevalence and vertebrate community variables in any of the models tested. Despite the studied system having several characteristics that should favour the dilution effect (i.e., vector-borne pathogens, an area where vector and host densities are unrelated, and where host richness is not associated with an increase in host density), none of the relationships between host species diversity and species richness, and pathogen prevalence supported DEH and, in fact, amplification was found for three of the four pathogens tested. Consequently, the range of pathogens and communities studied needs to be broadened if we are to understand the ecological factors that favour dilution and how often these conditions occur in nature.