Development and Validation of Three Triplex Real-Time RT-PCR Assays for Typing African Horse Sickness Virus: Utility for Disease Control and Other Laboratory Applications.

The African horse sickness virus (AHSV) belongs to the Genus Orbivirus, family Sedoreoviridae, and nine serotypes of the virus have been described to date. The AHSV genome is composed of ten linear segments of double-stranded (ds) RNA, numbered in decreasing size order (Seg-1 to Seg-10). Genome segment 2 (Seg-2) encodes outer-capsid protein VP2, the most variable AHSV protein and the primary target for neutralizing antibodies. Consequently, Seg-2 determines the identity of the virus serotype. An African horse sickness (AHS) outbreak in an AHS-free status country requires identifying the serotype as soon as possible to implement a serotype-specific vaccination program. Considering that nowadays 'polyvalent live attenuated' is the only commercially available vaccination strategy to control the disease, field and vaccine strains of different serotypes could co-circulate. Additionally, in AHS-endemic countries, more than one serotype is often circulating at the same time. Therefore, a strategy to rapidly determine the virus serotype in an AHS-positive sample is strongly recommended in both epidemiological situations. The main objective of this study is to describe the development and validation of three triplex real-time RT-PCR (rRT-PCR) methods for rapid AHSV serotype detection. Samples from recent AHS outbreaks in Kenia (2015-2017), Thailand (2020), and Nigeria (2023), and from the AHS outbreak in Spain (1987-1990), were included in the study for the validation of these methods.

Perceived pain and disability but not fear of movement are associated with altered cervical kinematics in people with acute neck pain following a whiplash injury.

OBJECTIVES: To determine if measures of cervical kinematics are altered in people with acute whiplash associated disorders (WAD) and secondarily, to examine whether kinematic variables are associated with self-reported outcomes. METHODS: We recruited people with acute WAD within 15 days after a motor vehicle collision and asymptomatic control participants. All participants performed active neck movements at a self-determined velocity. Maximal range of motion (ROM), peak and mean velocity of movement, smoothness of movement, and cervical joint position error were assessed. Moreover, self-reported measures of perceived pain and disability, pain catastrophising, and fear of movement were obtained. RESULTS: Sixty people participated: 18 with acute WAD (mean age [SD] 38.7 [12.0]) and 42 as asymptomatic controls (mean age [SD] 38.4 [10.2]). Participants with acute WAD showed significantly decreased ROM in all movement directions (p < 0.0001). All participants with acute WAD showed a reduction in the mean and peak velocity of movement in all directions (p < 0.0001) and the number of velocity peaks was significantly higher (i.e., reduced smoothness of movement) in those with acute WAD in all directions (p < 0.0001). Repositioning acuity following cervical rotation was not significantly different between groups. Neck pain-related disability showed the largest number of significant associations with kinematic features, while fear of movement was not associated with measures of cervical kinematics. CONCLUSIONS: Participants with acute WAD presented with altered cervical kinematics compared to asymptomatic participants. Several measures of cervical kinematics were associated with the level of pain and disability in people with acute WAD but not their fear of movement.

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.

Re-emergence of bluetongue virus serotype 4 in Iberian ibex (Capra pyrenaica) and sympatric livestock in Spain, 2018-2019.

Between early October and mid-December 2018, mortalities were detected in Iberian ibex (Capra pyrenaica) populations in southern Spain. In the same region and period, bluetongue virus (BTV) circulation was also reported in sentinel and clinically affected domestic ruminant herds. Molecular analyses confirmed BTV serotype 4 (BTV-4) infection in eight Iberian ibexes from six hunting areas, and in 46 domestic ruminants from seven herds in close proximity to affected hunting estates. Histopathological analyses revealed vascular changes in several organs, pneumonia, lymphoid depletion, inflammatory mononuclear cell infiltrate and fibrosis as the most frequently observed lesions in the affected Iberian ibexes. Epidemiological and laboratory results indicate that BTV-4 was the main aetiological agent involved in outbreaks detected in Iberian ibex populations during the study period. Sequence analyses indicated that the BTV-4 strain detected in Iberian ibex had high homology (99.4%-100%) with strains isolated in livestock during the same period, and with previous isolates (≥98.9%) from Spain and Mediterranean Basin countries. Further studies are warranted to determine the impact of BTV-4 on the health status of Iberian ibex populations after the outbreaks. The inclusion of this species in the surveillance programme may be useful for early detection of BTV, especially in epidemiological scenarios at the wildlife-livestock interface.

Assessment of reproducibility of a VP7 Blocking ELISA diagnostic test for African horse sickness.

The laboratory diagnosis of African horse sickness (AHS) is important for: (a) demonstrating freedom from infection in a population, animals or products for trade (b) assessing the efficiency of eradication policies; (c) laboratory confirmation of clinical diagnosis; (d) estimating the prevalence of AHS infection; and (e) assessing postvaccination immune status of individual animals or populations. Although serological techniques play a secondary role in the confirmation of clinical cases, their use is very important for all the other purposes due to their high throughput, ease of use and good cost-benefit ratio. The main objective of this study was to support the validation of AHS VP7 Blocking ELISA up to the Stage 3 of the World Animal Health Organization (OIE) assay validation pathway. To achieve this, a collaborative ring trial, which included all OIE Reference Laboratories and other AHS-specialist diagnostic centres, was conducted in order to assess the diagnostic performance characteristics of the VP7 Blocking ELISA. In this trial, a panel of sera of different epidemiological origin and infection status was used. Through this comprehensive evaluation we can conclude that the VP7 Blocking ELISA satisfies the OIE requirements of reproducibility. The VP7 Blocking ELISA, in its commercial version is ready to enter Stage 4 of the validation pathway (Programme Implementation). Specifically, this will require testing the diagnostic performance of the assay using contemporary serum samples collected during control campaigns in endemic countries.

Detection of West Nile virus lineage 2 in North-Eastern Spain (Catalonia).

In September 2017, West Nile virus (WNV) lineage 2 was detected in Catalonia (Northern Spain) in northern goshawks by passive surveillance. The phylogenetic analyses showed that it was related to the Central/Southern European strains, evidencing WNV lineage 2 spread to Western Europe. WNV local transmission was later detected in bearded vultures housed at the Wildlife Recovery center where the goshawk was transferred to. Further studies, before the following period of high mosquito activity, indicated that WNV had circulated intensively in poultry and horses but only surrounding of the area where the virus was detected. In other areas of Catalonia, circulation of flaviviruses different to WNV was identified. Public Health investigations failed to detect WNV infection in humans.

Experimental West Nile virus infection in Gyr-Saker hybrid falcons.

West Nile disease (WND) has become a major public and veterinary health concern since the appearance of West Nile virus (WNV) in New York in 1999. The following panzootic spread in the U.S. and the recent WNV outbreaks in Europe and the Mediterranean Basin have increased interest in WND. Despite considerable investigation of WNV infection in birds, the effects of WNV on avian populations are still largely unknown. In Europe, raptors have been found to be particularly susceptible to WNV infection, but studies in birds of prey are lacking. To our knowledge, the present study is the first to report an experimental infection with WNV in Gyr-Saker hybrid falcons. We show that 10-week-old captive-reared Gyr-Saker (Falco rusticolus × Falco cherrug) hybrid falcons are susceptible to WNV infection. Neither morbidity nor mortality was observed after subcutaneous WNV inoculation with mixed extracts of non-infected mosquito salivary glands. Both the macroscopic and microscopic lesions observed were similar to those previously reported in naturally and experimentally infected North American raptors. The results obtained in the present study demonstrate that although Gyr-Saker hybrid falcons do not seem to be a good reservoir for WNV transmission via mosquito, they can become infected with WNV, develop viremia and antibodies, and are able to shed the virus.

Bagaza virus in partridges and pheasants, Spain, 2010.

In September 2010, an unusually high number of wild birds (partridges and pheasants) died in Cádiz in southwestern Spain. Reverse transcription PCR and virus isolation detected flavivirus infections. Complete nucleotide sequence analysis identified Bagaza virus, a flavivirus with a known distribution that includes sub-Saharan Africa and India, as the causative agent.

Serosurvey of West Nile virus and other flaviviruses of the Japanese encephalitis antigenic complex in birds from Andalusia, southern Spain.

Flaviviruses of the Japanese encephalitis virus (JEV) antigenic complex, including West Nile virus (WNV), are recognized as emerging and reemerging pathogens. Circulation of flaviviruses has been recently detected in different mosquito and vertebrate species in several European countries. A serosurvey study was carried out to evaluate the circulation of WNV and other flaviviruses of the JEV antigenic complex in different wild bird species in Spain between 2006 and 2009. Seropositiviy against JEV using a competitive enzyme-linked immunosorbent assay was found in common coot, Montagu's Harrier, black kite, black vulture, Bonelli's eagle, Spanish imperial eagle, Egyptian vulture, and Eurasian spoonbill. Seropositivity to JEV antigenic complex viruses was significantly higher in samples collected during autumn compared with animals sampled during summer. Significantly higher seroprevalence was also observed in 2007 compared with 2009, whereas there were no significant differences in seropositivity among taxonomic levels, migratory versus resident behavior, body size (large vs. medium), or habitats (free-ranging vs. captivity). Neutralizing antibodies against WNV were detected in common coot and Spanish imperial eagle using a virus-neutralization test. Oral shedding of WNV was not detected in any of the Spanish imperial eagles, Egyptian vultures, Eurasian Spoonbills, Lammergeiers, and the Black vultures analyzed by means of real-time reverse transcription-polymerase chain reaction. The results indicate that WNV and others flaviviruses of the JEV antigenic group circulated in migratory and resident wild bird species in Spain between 2007 and 2008. Further studies are necessary to determine the precise role that each of these wild bird species, some of them cataloged as "near threatened," "vulnerable," or "endangered," play in the epidemiology of those viruses.