Novel serotype of bluetongue virus in South America and first report of epizootic haemorrhagic disease virus in Ecuador

Bluetongue virus (BTV ) and Epizootic haemorrhagic disease virus (EHDV ) are closely related Orbiviruses that affect domestic and wild ruminants. In Ecuador previous serological studies reported the presence of BTV ; however, no data are available about the presence of EHDV . In this study, 295 cattle without symptoms of infection were sampled from two farms located in Andean and Amazonian regions and from a slaughterhouse in the coastal region. ELISA analyses showed high prevalence of BTV (98.9%) and EHDV (81.3%) antibodies, and RT ‐qPCR s revealed the presence of EHDV (24.1%) and BTV (10.2%) genomes in cattle blood samples. Viral isolation allowed to identify EHDV serotype 1 (EHDV 1) and BTV serotypes 9 (BTV 9), 13 and 18. These findings suggest that BTV and EHDV are enzootic diseases in Ecuador.     

Development of Real‐Time RT‐PCR Assays for Detection and Typing of Epizootic Haemorrhagic Disease Virus

Epizootic haemorrhagic disease virus (EHDV) is an emerging arboviral pathogen of wild and domestic ruminants worldwide. It is closely related to bluetongue virus (BTV) and is transmitted by adult females of competent Culicoides vector species. The EHDV genome consists of ten linear double‐stranded (ds)RNA segments, encoding five non‐structural and seven structural proteins. Genome‐segment reassortment contributes to a high level of genetic variation in individual virus strains, particularly in the areas where multiple and distinct virus lineages co‐circulate. In spite of the relatively close relationship between BTV and EHDV herd‐immunity to BTV does not appear to protect against the introduction and infection of animals by EHDV. Although EHDV can cause up to 80% morbidity in affected animals, vaccination with the homologous EHDV serotype is protective. Outer‐capsid protein VP2, encoded by Seg‐2, is the most variable of the EHDV proteins and determines both the specificity of reactions with neutralizing antibodies and consequently the identity of the eight EHDV serotypes. In contrast, VP6 (the viral helicase), encoded by Seg‐9, is highly conserved, representing a virus species/serogroup‐specific antigen. We report the development and evaluation of quantitative (q)RT‐PCR assays targeting EHDV Seg‐9 that can detect all EHDV strains (regardless of geographic origin/topotype/serotype), as well as type‐specific assays targeting Seg‐2 of the eight EHDV serotypes. The assays were evaluated using orbivirus isolates from the ‘Orbivirus reference collection’ (ORC) at The Pirbright Institute and were shown to be EHDV pan‐reactive or type‐specific. They can be used for rapid, sensitive and reliable detection and identification (typing) of EHDV RNA from infected blood, tissue samples, homogenized Culicoides, or tissue culture supernatant. None of the assays detected RNA from closely related but heterologous orbiviruses, or from uninfected host animals or cell cultures. The techniques presented could be used for both surveillance and vaccine matching (serotype identification) as part of control strategies for incursions in wild and domestic animal species.     

Investigating Incursions of Bluetongue Virus Using a Model of Long‐Distance Culicoides Biting Midge Dispersal

Bluetongue virus (BTV) is an economically important pathogen of ruminants that is the aetiological agent of the haemorrhagic disease bluetongue. Bluetongue virus is biologically transmitted by Culicoides biting midges (Diptera: Ceratopogonidae), and long‐range dispersal of infected vector species contributes substantially to the rapid spread of the virus. The range of semi‐passive flights of infected Culicoides on prevailing winds has been inferred to reach several hundred kilometres in a single night over water bodies. In this study, an atmospheric dispersion model was parameterized to simulate Culicoides flight activity based on dedicated entomological data sets collected in the UK. Five outbreaks of BTV in Europe were used to evaluate the model for use as an early warning tool and for retrospective analyses of BTV incursions. In each case, the generated predictions were consistent with epidemiological observations confirming its reliability for use in disease outbreak management. Furthermore, the model aided policy makers to predict, contain and eradicate BTV outbreaks in the UK during 2007 and 2008.     

Evaluation of an IGM‐specific ELISA for early detection of bluetongue virus infections in domestic ruminants sera

Competitive‐ELISA (c‐ELISA) is the most widely used serological test for the detection of Bluetongue virus (BTV) viral protein 7 (VP7) antibodies (Ab). However, these BTV c‐ELISAs cannot to distinguish between IgG and IgM. IgM Ab are generated shortly after the primary immune response against an infectious agent, indicating a recent infection or exposure to antigens, such as after vaccination. Because the BTV genome or anti‐VP7 Ab can be detected in ruminant blood months after infection, BTV diagnostic tools cannot discriminate between recent and old infections. In this study, we evaluated an IgM‐capture ELISA prototype to detect ruminant anti‐BTV VP7 IgM on 1,650 serum samples from cattle, sheep, or goats. Animals were BTV‐naive, infected, or/and vaccinated with BTV‐1, ‐2, ‐4, ‐8, ‐9, ‐16, or ‐27, and we also included 30 sera from cattle infected with the Epizootic haemorrhagic disease virus (EHDV) serotype 6. Results demonstrated that this ELISA kit is specific and can detect the presence of IgM with satisfactory diagnostic specificity and sensitivity from 1 to 5 weeks after BTV infection in domestic ruminants (for goats and cattle; for sheep, at least up to 24 days). The peak of anti‐VP7 IgM was reached when the level of infectious viruses and BTV RNA in blood were the highest. The possibility of detecting BTV‐RNA in IgM‐positive sera allows the amplification and sequencing of the partial RNA segment 2 (encoding the serotype specific to VP2) to determine the causative BTV serotype/strain. Therefore, BTV IgM ELISA can detect the introduction of BTV (or EHDV) in an area with BTV‐seropositive domestic animals regardless of their serological BTV status. This approach may also be of particular interest for retrospective epidemiological studies on frozen serum samples.     

Diagnosis and Control Measures of the 2010 Outbreak of Foot‐and‐Mouth Disease A Type in the Republic of Korea

In January 2010, foot‐and‐mouth disease (FMD) occurred for the first time in 8 years in Korea. The outbreaks were because of A serotype, different from the O type, which had occurred previously in 2000 and 2002. The FMD outbreaks were identified in seven farms, consisting of six cattle farms where viruses were detected and one deer farm where only FMDV antibody was detected. The seven farms were within 9.3 km of each other. All susceptible animals within 10 km radius of the outbreak farms were placed under movement restrictions for 3–11 weeks. No vaccination took place to facilitate the clinical observation of infected animals and virus detection. After clinical observations and serological tests within the control zones showed no evidence of FMD infection, the movement restrictions were lifted, followed by FMD‐free declaration (23 March) at 80 days after the first outbreak on 2 January. This communication describes the outbreak of FMD A serotype, and control measures applied to eradicate the disease in Korea.     

Epidemiology of RHDV2 (Lagovirus europaeus/GI.2) in free‐living wild European rabbits in Portugal

As the detection of the first outbreak of a novel aetiological agent of rabbit haemorrhagic disease commonly called RHDV 2 or RHDV b (Lagovirus europaeus /GI .2, henceforth GI .2) in France in 2010, the virus rapidly spread throughout continental Europe and nearby islands such as Great Britain, Sardinia, Sicily, the Azores and the Canary Islands among others. The outbreaks of this new lagovirus cause important economic losses in rabbitries, and ecological disruptions by affecting the conservation of rabbit‐sensitive top predators. We analysed 550 rabbit carcasses collected in the field between May 2013 and March 2016, to investigate the epidemiology of GI .2 in free‐living populations and to perform a comparative analysis with the epidemiology of classical rabbit haemorrhagic disease virus forms (RHDV , henceforth GI .1) in Portugal. Rabbits were sexed, aged and liver and blood samples were collected for subsequent RHDV screening and serology. A total of 172 samples were PCR‐positive to GI .2, whereas GI .1 strains were not detected in any of the samples. The outbreaks of GI .2 revealed a marked seasonality, with peaks during the breeding season (November‐May). We also found that approximately, one‐third of free‐ranging European rabbits in Portugal have seroconverted to GI .2. We demonstrate that the GI .2 lagovirus is currently widespread in wild populations in Portugal and is affecting a high proportion of adults and juveniles. Therefore, ongoing monitoring and surveillance are required to assess the effects of GI .2 on wild rabbit populations, its evolution, and to guide management actions aimed at mitigating the impacts of rabbit declines in the ecosystem and in rural economies.     

Spillover Events of Infection of Brown Hares (Lepus europaeus) with Rabbit Haemorrhagic Disease Type 2 Virus (RHDV2) Caused Sporadic Cases of an European Brown Hare Syndrome‐Like Disease in Italy and Spain

Rabbit haemorrhagic disease virus (RHDV) is a lagovirus that can cause fatal hepatitis (rabbit haemorrhagic disease, RHD) with mortality of 80–90% in farmed and wild rabbits. Since 1986, RHDV has caused outbreaks in rabbits (Oryctolagus cuniculus) in Europe, but never in European brown hares (Lepus europaeus, EBH). In 2010, a new RHDV‐related virus, called RHDV2, emerged in Europe, causing extended epidemics because it largely overcame the immunity to RHDV present in most rabbit populations. RHDV2 also was identified in Cape hare (Lepus capensis subsp. mediterraneus) and in Italian hare (Lepus corsicanus). Here, we describe two distinct incidents of RHDV2 infection in EBH that occurred in Italy (2012) and Spain (2014). The two RHDV2 strains caused macroscopic and microscopic lesions similar to European brown hare syndrome (EBHS) in hares, and they were genetically related to other RHDV2 strains in Europe. EBHs are common in Europe, often sharing habitat with rabbits. They likely have been exposed to high levels of RHDV2 during outbreaks in rabbits in recent years, yet only two incidents of RHDV2 in EBHs have been found in Italy and Spain, suggesting that EBHs are not a primary host. Instead, they may act as spillover hosts in situations when infection pressure is high and barriers between rabbits and hares are limited, resulting in occasional infections causing EBHS‐like lesions. The serological survey of stocked hare sera taken from Italian and Spanish hare populations provided an understanding of naturally occurring RHDV2 infection in the field confirming its sporadic occurrence in EBH. Our findings increase the knowledge on distribution, host range and epidemiology of RHDV2.     

Serological Study of Exposure to Selected Arthropod‐Borne Pathogens in European Bison (Bison bonasus) in Poland

Bison bonasus is an indigenous species of Central and Eastern Europe with the largest wild population inhabiting Białowieża Primeval Forest; however, free‐living and captive European bison are reared in many countries around the world. Despite that the European bison was rescued from the extinction after the First World War, it remains as endangered species. Changing environment as well as human activity may have contributed to the observed increase of the risk of the emergence and re‐emergence of pathogens. The aim of the survey was to establish the distribution of four pathogens transmitted by arthropods including three arboviruses [Bluetongue disease virus (BTV), Epizootic haemorrhagic disease virus (EHDV) and Schmallenberg virus (SBV)] and a bacteria (Francisella tularensis) in the main populations of European bison in Poland. A total of 251 European bison originating from eight main populations were included in the study and sampled between February 2011 and December 2014. Serum samples originated from chemically immobilized, eliminated or dead by natural causes animals. Additionally, 65 cervids from Białowieża Forest were tested to compare the seroprevalences of other ruminants inhabiting the same environment. The antibodies to SBV and BTV were found in 76.1% and 24.7% of European bison, respectively. In autumn 2012, simultaneous emergence of SBV and BTV in European bison was observed; however, while SBV has spread in all populations scattered around the country, BTV infections were observed only in the north‐eastern part of Poland, where BTV cases have been previously reported in domestic ruminants. European bison age was found to be the only significant risk factor for SBV and BTV seroprevalences; however, this association was connected to the animal size, rather than to the length of exposure. None of the animals tested positive for antibodies against EHDV or F. tularensis. SBV exposure rate of cervids was much lower (35.4%) than in European bison, while BTV seroprevalence was comparable in both groups.     

Assessing the potential spread and maintenance of foot‐and‐mouth disease virus infection in wild ungulates: general principles and application to a specific scenario in Thrace

Foot‐and‐mouth disease (FMD), due to infection with serotype O virus, occurred in wild boar and within eleven outbreaks in domestic livestock in the south‐east of Bulgaria, Thrace region, in 2011. Hence, the issue of the potential for the spread and maintenance of FMD virus (FMDV) infection in a population of wild ungulates became important. This assessment focused on the spread and maintenance of FMDV infection within a hypothetical wild boar and deer population in an environment, which is characterized by a climate transitional between Mediterranean and continental and variable wildlife population densities. The assessment was based on three aspects: (i) a systematic review of the literature focusing on experimental infection studies to identify the parameters describing the duration of FMDV infection in deer and wild boar, as well as observational studies assessing the occurrence of FMDV infection in wild deer and wild boar populations, (ii) prevalence survey data of wild boar and deer in Bulgaria and Turkey and (iii) an epidemiological model, simulating the host‐to‐host spread of FMDV infections. It is concluded, based on all three aspects, that the wildlife population in Thrace, and so wildlife populations in similar ecological settings, are probably not able to maintain FMD in the long term in the absence of FMDV infection in the domestic host population. However, limited spread of FMDV infection in time and space in the wildlife populations can occur. If there is a continued cross‐over of FMDV between domestic and wildlife populations or a higher population density, virus circulation may be prolonged.     

Outbreaks of Clade 2.3.4.4 H5N8 highly pathogenic avian influenza in 2018 in the northern regions of South Africa were unrelated to those of 2017

Asian‐origin H5N8 highly pathogenic avian influenza (HPAI) viruses of the H5 Goose/Guangdong/96 lineage, clade 2.3.4.4 group B, reached South Africa by June 2017. By the end of that year, 5.4 million layers and broiler chickens died or were culled, with total losses in the poultry industry estimated at US$ 140 million, and thousands of exotic birds in zoological collections, endangered endemic species and backyard poultry and pet birds also perished. The 2017 H5N8 HPAI outbreaks were characterized by two distinct spatial clusters, each associated with specific reassortant viral genotypes. Genotypes 1, 2, 3 and 5 were restricted to the northern regions, spanning the provinces of Limpopo, Gauteng, North West, Mpumalanga, KwaZulu‐Natal and Free State. The second, much larger cluster of outbreaks was in the south, in the Western and Eastern Cape provinces, wherein 2017 and 2018 outbreaks were caused solely by genotype 4. The last confirmed case of H5N8 HPAI in the northern region in 2017 was in early October, and the viruses seemed to disappear over the summer. However, starting in mid‐February 2018, H5N8 HPAI outbreaks resurged in the north. Viruses from two of the eight outbreaks were sequenced, one from an outbreak in quails (Coturnix japonica) in the North West Province, and another from commercial pullets in the Gauteng province. Phylogenetic analysis identified the viruses as a distinct sixth genotype that was most likely a new introduction to South Africa in early 2018.     

Arrival of rabbit haemorrhagic disease virus 2 to northern Europe: Emergence and outbreaks in wild and domestic rabbits (Oryctolagus cuniculus) in Sweden

Incursion of rabbit haemorrhagic disease virus (RHDV ) into Sweden was documented in 1990 and it is now considered endemic in wild rabbit (Oryctolagus cuniculus ) populations. Rabbit haemorrhagic disease virus 2 (RHDV 2), a new, related lagovirus was first detected in France in 2010, and has spread rapidly throughout Europe and beyond. However, knowledge of RHDV 2 in northern Europe is sporadic and incomplete, and in Sweden, routinely available diagnostic methods to detect rabbit haemorrhagic disease (RHD ) do not distinguish between types of virus causing disease. Using RHDV 2‐specific RT ‐qPCR , sequencing of the VP 60 gene and immunological virus typing of archived and prospective case material from the National Veterinary Institute's (SVA ) wildlife disease surveillance programme and diagnostic pathology service, we describe the emergence of RHDV 2 in Sweden in both wild and domestic rabbits. The earliest documented outbreak occurred on 22 May 2013, and from May 2013 to May 2016, 10 separate incidents of RHDV 2 were documented from six different municipalities in the southern half of Sweden. Phylogenetic analysis of the VP 60 gene shows clear clustering of Swedish isolates into three separate clusters within two different clades according to geographic location and time, suggesting viral evolution, multiple introduction events or both. Almost all cases of RHD examined by SVA from May 2013 to May 2016 were caused by RHDV 2, suggesting that RHDV 2 may be replacing RHDV as the predominant cause of RHD in Sweden.     

Bluetongue Dynamics in French Wildlife: Exploring the Driving Forces

Bluetongue (BT) was monitored in wildlife in France during two consecutive years corresponding to contrasting incidence rates in livestock: in 2008 at the peak of domestic outbreaks and in 2009 when very few outbreaks were observed. The disease status of 2 798 ruminants comprising 837 red deer (Cervus elaphus) was explored using ELISA test on serum and real‐time RT‐PCR test on blood or spleen. A large proportion of red deer were seropositive and positive to RT‐PCR in 2008, but also in 2009 (seroprevalence: 47.1% and 24.3%), suggesting that red deer could maintain infection when domestic incidence was negligible. By contrast, low seroprevalence (<3%) and few RT‐PCR positive results were observed in other wild ruminant species, which rather appeared thus as dead‐end hosts. The risk factors of bluetongue circulation during the periods of high (2008) and low (2009) domestic incidence were explored in red deer using logistic mixed models. In this species, prevalence has been mainly influenced by the initial peak of BT in livestock, but also by environmental factor such as elevation and edge density between forest and pastures. Surprisingly, cattle density has a negative influence on prevalence in red deer, possibly due to the protective effect of cattle regarding midges' bites and/or to still unexplained factors dealing with the host/midge interface. To our knowledge, this study is the first attempt at measuring the effect of landscape and wildlife/domestic interface on BT prevalence in wildlife in Europe.     

Highly pathogenic avian influenza subtype H5Nx clade 2.3.4.4 outbreaks in Dutch poultry farms, 2014–2018: Clinical signs and mortality

In recent years, different subtypes of highly pathogenic avian influenza (HPAI) viruses caused outbreaks in several poultry types worldwide. Early detection of HPAI virus infection is crucial to reduce virus spread. Previously, the use of a mortality ratio threshold to expedite notification of suspicion in layer farms was proposed. The purpose of this study was to describe the clinical signs reported in the early stages of HPAI H5N8 and H5N6 outbreaks on chicken and Pekin duck farms between 2014 and 2018 in the Netherlands and compare them with the onset of an increased mortality ratio (MR). Data on daily mortality and clinical signs from nine egg‐producing chicken farms and seven Pekin duck farms infected with HPAI H5N8 (2014 and 2016) and H5N6 (2017–2018) in the Netherlands were analysed. In 12 out of 15 outbreaks for which a MR was available, MR increase preceded or coincided with the first observation of clinical signs by the farmer. In one chicken and two Pekin duck outbreaks, clinical signs were observed prior to MR increase. On all farms, veterinarians observed clinical signs of general disease. Nervous or locomotor signs were reported in all Pekin duck outbreaks, but only in two chicken outbreaks. Other clinical signs were observed less frequently in both chickens and Pekin ducks. Compared to veterinarians, farmers observed and reported clinical signs, especially respiratory and gastrointestinal signs, less frequently. This case series suggests that a MR with a set threshold could be an objective parameter to detect HPAI infection on chicken and Pekin duck farms at an early stage. Observation of clinical signs may provide additional indication for farmers and veterinarians for notifying a clinical suspicion of HPAI infection. Further assessment and validation of a MR threshold in Pekin ducks are important as it could serve as an important tool in HPAI surveillance programs.     

Rift Valley Fever in Kruger National Park: Do Buffalo Play a Role in the Inter‐Epidemic Circulation of Virus?

Rift Valley fever (RVF) is a zoonotic mosquito‐borne virus disease of livestock and wild ruminants that has been identified as a risk for international spread. Typically, the disease occurs in geographically limited outbreaks associated with high rainfall events and can cause massive losses of livestock. It is unclear how RVF virus persists during inter‐epidemic periods but cryptic cycling of the virus in wildlife populations may play a role. We investigated the role that free‐living African buffalo (Syncerus caffer caffer) might play in inter‐epidemic circulation of the virus and looked for geographic, age and sex patterns of Rift Valley fever virus (RVFV) infection in African buffalo. Buffalo serum samples were collected (n = 1615) in Kruger National Park (KNP), South Africa, during a period of 1996–2007 and tested for antibodies to RVF. We found that older animals were more likely to be seropositive for anti‐RVFV antibody than younger animals, but sex was not correlated with the likelihood of being anti‐RVFV antibody positive. We also found geographic variation within KNP; herds in the south were more likely to have acquired anti‐RVFV antibody than herds farther north – which could be driven by host or vector ecology. In all years of the study between 1996 and 2007, we found young buffalo (under 2 years of age) that were seropositive for anti‐RVFV antibody, with prevalence ranging between 0 and 27% each year, indicating probable circulation. In addition, we also conducted a 4‐year longitudinal study on 227 initially RVFV seronegative buffalo to look for evidence of seroconversion outside known RVF outbreaks within our study period (2008–2012). In the longitudinal study, we found five individuals that seroconverted from anti‐RVFV antibody negative to anti‐RVFV antibody positive, outside of any detected outbreak. Overall, our results provide evidence of long‐term undetected circulation of RVFV in the buffalo population.     

Updated phylogenetic analysis of the spike gene and identification of a novel recombinant porcine epidemic diarrhoea virus strain in Taiwan

New variants of porcine epidemic diarrhoea virus (PEDV) causing a highly contagious intestinal disease, porcine epidemic diarrhoea virus (PED), have resulted in high mortality in suckling pigs across several countries since 2013. After 2015, the prevalence of the genogroup 2b (G2b) PEDVs decreased in a cyclical pattern with endemic seasonal outbreaks occasionally seen. To better understand the genetic diversity of PEDVs recently circulating in Taiwan, full‐length spike (S) genes of 31 PEDV strains from 28 pig farms collected during 2016–2018 were sequenced. While the majority of S gene sequences (from 27/28 farms) were closely related to the previous G2b PEDV strains, increased genetic diversities leading to several nonsynonymous mutations scattering in the neutralizing epitopes of the S gene were detected in PEDVs recently circulating in Taiwan. Furthermore, novel recombinant variants, the PEDV TW/Yunlin550/2018 strains exhibiting recombinant events between a previously isolated Taiwan PEDV G2b strain and a wild‐type PEDV G1a strain, were identified and further classified into a new genogroup, G1c. These results provide updated information about the genetic diversity of currently circulating PEDVs in the field and could help to develop more suitable strategies for controlling this disease.     

Worldwide rapid spread of the novel rabbit haemorrhagic disease virus (GI.2/RHDV2/b)

We describe the extremely rapid worldwide spread of the Lagovirus europaeus/GI.2/RHDV2/b (henceforth GI.2), the causative infectious agent of the so‐called ‘novel’ rabbit haemorrhagic disease of the European rabbit (Oryctolagus cuniculus). We tracked down all novel confirmed detections of GI.2 between May 2010 and November 2018 by carrying out a two‐step in‐depth review. We suggest that such spread would not have been possible without anthropogenic involvement. Our results also point out the importance of reviewing and adapting the protocols of virus detection and management in order to control, mitigate and contain properly, not only GI.2, but also new viruses that may emerge in the future.     

Analysis of Recent Serotype O Foot‐and‐Mouth Disease Viruses from Livestock in Kenya: Evidence of Four Independently Evolving Lineages

Foot‐and‐mouth disease (FMD) is endemic in Kenya where four serotypes (O, A, SAT 1 and SAT 2) of the virus are currently in circulation. Within 2010 and 2011, the National Laboratory recorded an increase in the number of FMD outbreaks caused by serotype O virus. The characteristics of these viruses were determined to ascertain whether these were independent outbreaks or one single strain spreading throughout the country. The sequences of the complete VP1‐coding region were analysed from viruses sampled within different areas of Kenya during 2010 and 2011. The results indicated that the 2010 to 2011 outbreaks in Kenya were caused by four independent strains. By comparison with earlier type O isolates from Eastern Africa, it was apparent that the outbreaks were caused by viruses from three different lineages of topotype EA‐2 and a fourth virus strain belonging to topotype EA‐4. The topotypes EA‐1 and EA‐3 were not detected from these outbreaks. Implications of these results for FMD control in Eastern Africa are discussed.     

Post‐epidemic investigation of Schmallenberg virus in wild ruminants in Slovenia

Schmallenberg virus (SBV) is a vector‐borne virus belonging to the genus Orthobunyavirus within the Bunyaviridae family. SBV emerged in Europe in 2011 and was characterized by epidemics of abortions, stillbirths and congenital malformations in domestic ruminants. The first evidence of SBV infection in Slovenia was from an ELISA‐positive sample from a cow collected in August 2012; clinical manifestations of SBV disease in sheep and cattle were observed in 2013, with SBV RNA detected in samples collected from a total of 28 herds. A potential re‐emergence of SBV in Europe is predicted to occur when population‐level immunity declines. SBV is also capable of infecting several wild ruminant species, although clinical disease has not yet been described in these species. Data on SBV‐positive wild ruminants suggest that these species might be possible sources for the re‐emergence of SBV. The aim of this study was to investigate whether SBV was circulating among wild ruminants in Slovenia and whether these species can act as a virus reservoir. A total of 281 blood and spleen samples from wild ruminants, including roe deer, red deer, chamois and European mouflon, were collected during the 2017–2018 hunting season. Serum samples were tested for antibodies against SBV by ELISA; the overall seroprevalence was 18.1%. Seropositive samples were reported from all over the country in examined animal species from 1 to 15 years of age. Spleen samples from the seropositive animals and serum samples from the seronegative animals were tested for the presence of SBV RNA using real‐time RT‐PCR; all the samples tested negative. Based on the results of the seropositive animals, it was demonstrated that SBV was circulating in wild ruminant populations in Slovenia even after the epidemic, as almost half (23/51) of the seropositive animals were 1 or 2 years old.     

Serosurvey Reveals Exposure to West Nile Virus in Asymptomatic Horse Populations in Central Spain Prior to Recent Disease Foci

West Nile fever/encephalitis (WNF) is an infectious disease affecting horses, birds and humans, with a cycle involving birds as natural reservoirs and mosquitoes as transmission vectors. It is a notifiable disease, re‐emerging in Europe. In Spain, it first appeared in horses in the south (Andalusia) in 2010, where outbreaks occur every year since. However, in 2014, an outbreak was declared in horses in central Spain, approximately 200 km away from the closest foci in Andalusia. Before that, evidence of West Nile virus (WNV) circulation in central Spain had been obtained only from wildlife, but never in horses. The purpose of this work was to perform a serosurvey to retrospectively detect West Nile virus infections in asymptomatic horses in central Spain from 2011 to 2013, that is before the occurrence of the first outbreaks in the area. For that, serum samples from 369 horses, collected between September 2011 and November 2013 in central Spain, were analysed by ELISA (blocking and IgM) and confirmed by virus neutralization, proving its specificity using parallel titration with another flavivirus (Usutu virus). As a result, 10 of 369 horse serum samples analysed gave positive results by competitive ELISA, 5 of which were confirmed as positive to WNV by virus neutralization (seropositivity rate: 1.35%). One of these WNV seropositive samples was IgM‐positive. Chronologically, the first positive samples, including the IgM‐positive, corresponded to sera collected in 2012 in Madrid province. From these results, we concluded that WNV circulated in asymptomatic equine populations of central Spain at least since 2012, before the first disease outbreak reported in this area.     

Pathogenicity of porcine reproductive and respiratory syndrome virus (ORF5 RFLP 1‐7‐4 viruses) in China

Porcine reproductive and respiratory syndrome virus (PRRSV) is an RNA virus that causes reproductive failure in sows and respiratory problems in piglets. PRRSV infection leads to substantial pig mortality and causing huge economic losses so that disease outbreaks caused by the new PRRSV strain from other regions have caused great concern in China. In this study, we analysed the pathogenicity of the novel ORF5 RFLP 1‐7‐4‐like PRRSV strain, named PRRSV‐ZDXYL‐China‐2018‐1 in pigs. The viral challenge test showed that PRRSV‐ZDXYL‐China‐2018‐1 infection can cause persistent fever, moderate dyspnoea, serum viraemia and interstitial pneumonia in piglets. The levels of viral loads in serum and PRRSV‐specific antigen were also detected in lung tissues were used one‐step Taq‐Man RT‐qPCR and Immunohistochemistry, respectively. At 28dpi, the level of specific antibodies was increased among infected piglets. Importantly, the new virus appeared be a moderately virulent isolate with pathogenicity compared to HP‐PRRSV strain LQ (JXA1‐like strain). Histological examination revealed severe monocyte haemorrhage and interstitial pneumonia associated with monocyte infiltration in the lung tissue of pigs infected with PRRSV‐ZDXYL‐China‐2018‐1 and LQ‐JXA1 strains. Immunohistochemistry (IHC) results showed positive brown‐red epithelial cells and macrophages in pig lungs. Therefore, it is critical to establish an effective strategy to control the spread of PRRSV in China.