Spatiotemporal analysis of equine infectious anemia and prediction of risk areas in Europe.

Equine Infectious Anemia (EIA) is a vector-borne persistent viral infection in equine animals. The EIA is characterized by recurrent fever, thrombocytopenia, depression, anemia, rapid weight loss, and lower body edema. Control of EIA is achieved through the elimination or isolation of infected animals, resulting in significant economic losses. In recent years, many countries in Europe have experienced outbreaks of EIA, which could potentially develop into a new wave of epidemic and pose a significant threat to the healthy development of the equine industry. This study utilized spatiotemporal analysis techniques and ecological niche modeling to investigate the spatiotemporal distribution characteristics of historical EIA outbreaks and predict risk areas for EIA occurrence in Europe. Spatiotemporal analysis results indicate that from 2005 to 2023, the EIA outbreaks in Europe exhibit five significant spatiotemporal clusters, with hotspots concentrated in southeastern France and northwestern Italy. Ecological niche modeling reveals that western, central, and southern Europe are high-risk areas for EIA outbreaks. Annual mean temperature, annual precipitation, and horse density are important variables that influence the occurrence of EIA. The results of this study can provide decision-makers with valuable insights, helping with EIA monitoring and resource allocation.

Undetection of vector-borne viruses in equids of Galapagos Islands.

Domestic species, including equids, were introduced in the Galapagos Islands in the XIX century. Equine vector-borne diseases are circulating in South America but their occurrence in the Galapagos Island was unknown. The objective of this study was to detect the occurrence of West Nile virus (WNV), Usutu virus (USUV) and equine infectious anemia virus (EIAV) in the four Galapagos Islands raising equids if they were present at a prevalence >1%. Serum samples were collected from 411 equids belonging to 124 owners from April to July 2019. All the results were negative to the ELISA tests used suggesting that WNV, USUV and EIAV are not circulating in the equine population of the Galapagos Islands.

Surveillance and screening of Stomoxyinae flies from Mallorca Island (Spain) reveal the absence of selected pathogens but confirm the presence of the endosymbiotic bacterium Wolbachia pipientis.

Adult brachycera biting flies can significantly impact livestock through both direct effects (reduction of food intake, disturbance, painful bites, and blood loss) and indirect effects (pathogen transmission), leading to substantial economic losses and production damage. This study aimed to assess the presence of blood-sucking flies in six mixed-animal farm environments on the island of Mallorca (Balearic Islands, Spain) by employing multiple trapping methods. Additionally, distribution maps of brachycera biting fly species recorded in Spain were created, based on data extracted thorough review of scientific literature and citizen digital databases. Investigation of several pathogens, including equine infectious anemia virus (EIAV), Anaplasmataceae bacteria, and piroplasm protozoa, was carried out using different PCR targets (18S rRNA, 16S rRNA, groESL, and tat genes). Citizen science databases and literature review corroborated the consistent distribution trend for two Stomoxyinae species, underscoring the importance of citizen collaboration as a complement to traditional entomological surveillance. Our study confirmed the presence of two biting Stomoxyinae species: the prevalent stable fly Stomoxys calcitrans across all sampled farms, and the horn fly Haematobia irritans, which turned out to be less abundant. DNA barcoding techniques validated the identification of the two species. Neither EIAV nor bacterial/protozoan pathogens were detected using the selected PCR targets in either fly species. However, Wolbachia pipientis (clustered in the supergroup A together with the only sequence of W. pipientis from the USA) was identified through PCR targeting 16S rRNA, groESL and wsp genes in all pools of H. irritans (n = 13) collected from two of the examined farms. This study represents the first attempt to investigate pathogens in Stomoxyinae biting flies in Spain. The discovery of the endosymbiotic Wolbachia organism in H. irritans represents the first record in Spain and the second from Europe. This finding holds significant implications for future research on the applications of this bacterium in biocontrol programs.

Development and evaluation of a test strip for the rapid detection of antibody against equine infectious anemia virus.

Equine infectious anemia (EIA) is a contagious disease of horses caused by the equine infectious anemia virus (EIAV). The clinical signs at the acute phase include intermittent high fever, thrombocytopenia, hemorrhage, edema, and anemia. The clinical signs at chronic and relapsing subclinical levels include emaciation and progressive weakness. Surviving horses become lifelong carriers because of the integration of the viral genome into that of the host, and these horses can produce and transmit the virus to other animals. This increases the difficulty of imposing practical control measures to prevent epidemics of this disease. Serological tests measuring the antibodies in equine sera are considered to be a reliable tool for the long-term monitoring of EIA. However, the standard serological tests for EIV either have low sensitivity (e.g., agar gel immunodiffusion test, AGID) or are time consuming to perform (e.g., ELISA and western blotting). The development of a rapid and simple method for detecting the disease is therefore critical to control the spread of EIA. In this study, we designed and developed a colloidal gold immunochromatographic (GICG) test strip to detect antibodies against EIAV based on the double-antigen sandwich. Both the p26 and gp45 proteins were used as the capture antigens, which may help to improve the positive detection rate of the strip. We found that the sensitivity of the test strip was 8 to 16 times higher than those of two commercially available ELISA tests and 128 to 256 times higher than AGID, but 8 to 16 times lower than that of western blotting. The strip has good specificity and stability. When serum samples from experimental horses immunized with the attenuated EIAV vaccine (n = 31) were tested, the results of the test strip showed 100% coincidence with those from NECVB-cELISA and 70.97% with AGID. When testing clinical serum samples (n = 1014), the test strip surprisingly provided greater sensitivity and a higher number of "true positive" results than other techniques. Therefore, we believe that the GICG test strip has demonstrated great potential in the field trials as a simple and effective tool for the detection of antibodies against EIAV. KEY POINTS: • A colloidal gold immunochromatographic (GICG) fast test strip was developed with good specificity, sensitivity, stability, and repeatability • The test strip can be used in point-of-care testing for the primary screening of EIAV antibodies • Both the p26 and gp45 proteins were used as the capture antigens, giving a high positive detection rate in the testing of experimentally infected animal and field samples.

Development and evaluation of a real-time quantitative PCR for the detection of equine infectious anemia virus.

IMPORTANCE: Equine infectious anemia (EIA) has a worldwide distribution and causes significant losses to the equine industry worldwide. A reliable detection method is necessary to control the transmission of EIA virus (EIAV). Currently, most of the available real-time PCR assays, including the qPCR of recommended by WOAH, are developed according to the sequences of European or American EIAV strains; however, the primers and probe sequences have low homology with Asian EIAV strains. To the best of our knowledge, no qPCR method capable of the well detection of Asian EIAV strains, especially Chinese EIAV strains, has been published to date. The development of a sensitive, specific, and rapid qPCR assay for the detection of the EIAV strains is therefore of great importance.

Host cell restriction factors of equine infectious anemia virus.

Equine infectious anemia virus (EIAV) is a member of the lentivirus genus in the Retroviridae family and is considered an animal model for HIV/AIDS research. An attenuated EIAV vaccine, which was successfully developed in the 1970s by classical serial passage techniques, is the first and only lentivirus vaccine that has been widely used to date. Restriction factors are cellular proteins that provide an early line of defense against viral replication and spread by interfering with various critical steps in the viral replication cycle. However, viruses have evolved specific mechanisms to overcome these host barriers through adaptation. The battle between the viruses and restriction factors is actually a natural part of the viral replication process, which has been well studied in human immunodeficiency virus type 1 (HIV-1). EIAV has the simplest genome composition of all lentiviruses, making it an intriguing subject for understanding how the virus employs its limited viral proteins to overcome restriction factors. In this review, we summarize the current literature on the interactions between equine restriction factors and EIAV. The features of equine restriction factors and the mechanisms by which the EIAV counteract the restriction suggest that lentiviruses employ diverse strategies to counteract innate immune restrictions. In addition, we present our insights on whether restriction factors induce alterations in the phenotype of the attenuated EIAV vaccine.

Serological Examinations of Significant Viral Infections in Domestic Donkeys at the Special Nature Reserve "Zasavica", Serbia.

The paper presents the findings of specific antibodies in the blood sera of donkeys against the following viruses: equine infectious anemia virus (EIAV), African horse sickness virus (AHSV), equine herpesvirus type 1 (EHV-1), equine influenza virus subtype H3N8 (EIV) and equine arteritis virus (EAV). The analyses were conducted during the year 2022. From a total of 199 donkeys bred in "Zasavica", blood was sampled from 53 animals (2 male donkeys and 51 female donkeys), aged 3 to 10 years. Specific antibodies against EIAV were not detected in any of the tested animals using the agar-gel immunodiffusion (AGID) assay. No specific antibodies against AHSV, tested by enzyme-linked immunosorbent assay (ELISA), or antibodies against EAV, tested by the virus neutralization test (VNT) and ELISA were detected in any of these animals. A positive serological result for EHV-1 was determined by the VNT in all animals, with antibody titer values ranging from 1:2 to 1:128, while a very low antibody titer value for EIV (subtype H3N8) of 1:16 was determined in 18 donkeys using the hemagglutination inhibition test (HI test).

Comparison of 4 agar gel immunodiffusion kits for serologic detection of equine infectious anemia virus antibodies.

Using 85 sera collected from horses that had been experimentally infected with equine infectious anemia virus (EIAV) and 200 field sera collected from racehorses in Japan, we compared 4 agar gel immunodiffusion (AGID) kits for serologic detection of EIAV antibodies from Idexx, VMRD, IDvet, and the National Engineering Research Center of Veterinary Biologics, China (NECVB). The positive control lines were sufficiently clear in all kits for evaluation to be made, with slight differences in sharpness: NECVB was the sharpest, followed by VMRD, IDvet, and Idexx. The test results for all 285 samples agreed among the 4 kits, with 62 positives and 223 negatives. The sensitivities and specificities of VMRD, IDvet, and NECVB compared with the Idexx kit were 100%, and the kappa coefficient values between the kits were 1.0 for all combinations. We concluded that the testing capacity of these 4 kits was virtually identical.

Development and evaluation of a blocking ELISA for serological diagnosis of equine infectious anemia.

Equine infectious anemia (EIA) is an important viral disease characterized by persistent infection in equids worldwide. Most EIA cases are life-long virus carriers with low antibody reactions and without the appearance of clinical symptoms. A serological test with high sensitivity and specificity is required to detect inapparent infection. In this study, a B-cell common epitope-based blocking ELISA (bELISA) was developed using a monoclonal antibody together with the EIAV p26 protein labelled with HRP. The test has been evaluated against the standard and with field serum samples globally. This bELISA test can be completed within 75 min, and the sensitivity is higher than those of either the AGID or one commercial cELISA kit. This bELISA assay was 8-16 times more analytically sensitive than AGID, and 2 to 4 times more analytically sensitive than one cELISA kit by testing three sera from the USA, Argentina, and China, respectively. The 353 serum samples from Argentina were tested, in comparison with AGID, the diagnostic sensitivity and specificity of our bELISA assay were 100% (154/154) and 97.0% (193/199), respectively, and the accuracy of the bELISA test was 98.3%. The bELISA test developed in this study is a rapid, sensitive, specific method for the detection of EIAV infection, and could be a promising candidate for use in the monitoring of the EIA epidemic worldwide. KEY POINTS: • A universal epitope-based blocking enzyme-linked immunosorbent assay (bELISA) was developed for detection of antibodies to EIAV. • The bELISA assay can be used to test EIAV serum samples from different regions of the world including North America, South America, Europe, and Asia. • The bELISA assay was evaluated in three different international labs and showed a better performance than other commercial kits.

Key Factors and Parameter Ranges for Immune Control of Equine Infectious Anemia Virus Infection.

Equine Infectious Anemia Virus (EIAV) is an important infection in equids, and its similarity to HIV creates hope for a potential vaccine. We analyze a within-host model of EIAV infection with antibody and cytotoxic T lymphocyte (CTL) responses. In this model, the stability of the biologically relevant endemic equilibrium, characterized by the coexistence of long-term antibody and CTL levels, relies upon a balance between CTL and antibody growth rates, which is needed to ensure persistent CTL levels. We determine the model parameter ranges at which CTL and antibody proliferation rates are simultaneously most influential in leading the system towards coexistence and can be used to derive a mathematical relationship between CTL and antibody production rates to explore the bifurcation curve that leads to coexistence. We employ Latin hypercube sampling and least squares to find the parameter ranges that equally divide the endemic and boundary equilibria. We then examine this relationship numerically via a local sensitivity analysis of the parameters. Our analysis is consistent with previous results showing that an intervention (such as a vaccine) intended to control a persistent viral infection with both immune responses should moderate the antibody response to allow for stimulation of the CTL response. Finally, we show that the CTL production rate can entirely determine the long-term outcome, regardless of the effect of other parameters, and we provide the conditions for this result in terms of the identified ranges for all model parameters.

Review and future perspectives on the integration characteristics for equine lentivirus in the host genome.

Despite over 40 years of research on the human immunodeficiency virus type 1 (HIV-1) vaccine, we still lack a considerable progress. Equine infectious anemia virus (EIAV) is a lentivirus in the Retroviridae family, akin to HIV-1 in genome structure and antigenicity. EIA is an important infectious disease in equids, characterized by anemia, persistent infection, and repeated fevers. The EIAV attenuated vaccine in China is the only lentiviral vaccine used on a large scale. Elucidating the mechanism of waning and induction of protective immunity from this attenuated vaccine strain will provide a critical theoretical basis and reference point for vaccine research, particularly in the development of lentivirus vaccines, with far-reaching scientific value and social significance. In this paper, we summarize the information related to EIAV integration site selection, particularly for the Chinese EIAV attenuated vaccine strains on the equine genome. This may improve our mechanistic understanding of EIAV virulence reduction at the host genome level. The obtained data may help elucidate the biological characteristics of EIAV, particularly the Chinese attenuated EIAV vaccine strain, and provide valuable information regarding retroviral infections, particularly lentiviral infection and associated therapeutic vectors.

Identification of a Novel Post-transcriptional Transactivator from the Equine Infectious Anemia Virus.

All lentiviruses encode a post-transcriptional transactivator, Rev, which mediates the export of viral mRNA from the nucleus to the cytoplasm and which is required for viral gene expression and viral replication. In the current study, we demonstrate that equine infectious anemia virus (EIAV), an equine lentivirus, encodes a second post-transcriptional transactivator that we designate Grev. Grev is encoded by a novel transcript with a single splicing event that was identified using reverse transcription-PCR (RT-PCR) and RNA-seq in EIAV-infected horse tissues and cells. Grev is about 18 kDa in size, comprises the first 18 amino acids (aa) of Gag protein together with the last 82 aa of Rev, and was detected in EIAV-infected cells. Similar to Rev, Grev is localized to the nucleus, and both are able to mediate the expression of Mat (a recently identified viral protein of unknown function from EIAV), but Rev can mediate the expression of EIAV Gag/Pol, while Grev cannot. We also demonstrate that Grev, similar to Rev, specifically binds to rev-responsive element 2 (RRE-2, located in the first exon of mat mRNAs) to promote nuclear export of mat mRNA via the chromosome region maintenance 1 (CRM1) pathway. However, unlike Rev, whose function depends on its multimerization, we could not detect multimerization of Grev using coimmunoprecipitation (co-IP) or bimolecular fluorescence complementation (BiFC) assays. Together, these data suggest that EIAV encodes two post-transcriptional transactivators, Rev and Grev, with similar, but not identical, functions. IMPORTANCE Nuclear export of viral transcripts is a crucial step for viral gene expression and viral replication in lentiviruses, and this export is regulated by a post-transcriptional transactivator, Rev, that is shared by all lentiviruses. Here, we report that the equine infectious anemia virus (EIAV) encodes a novel viral protein, Grev, and demonstrated that Grev, like Rev, mediates the expression of the viral protein Mat by binding to the first exon of mat mRNAs via the chromosome region maintenance 1 (CRM1) pathway. Grev is encoded by a single-spliced transcript containing two exons, whereas Rev is encoded by a multiple-spliced transcript containing four exons. Moreover, Rev is able to mediate EIAV Gag/Pol expression by binding to rev-responsive element (RRE) located within the Env-coding region, while Grev cannot. Therefore, the present study demonstrates that EIAV encodes two post-transcriptional regulators, Grev and Rev, suggesting that post-transcriptional regulation patterns in lentivirus are diverse and complex.

Seroprevalence and risk factors associated with equine infectious anemia in the state of Goiás, Brazil.

Equine infectious anemia (EIA) is an infectious disease affecting equine in most countries and represents a notifiable disease with compulsory euthanasia of positive animals. The present study aimed to determine the prevalence of EIAV infected equines in herds of the state of Goiás (Central Brazil) and to evaluate the risk factors associated with the occurrence of the disease. Blood samples were collected from 1170 equids from 332 randomly selected farms divided into three different strata according to their herd characteristics. Also, an epidemiological questionnaire was applied during the visit to the farm. Of the 332 farms evaluated, 12 (3.1%; 95% CI: 1.24 - 6.00) had at least one positive equine for EIA, and of the 1170 evaluated equines, 14 (2%; 95% CI: 0.31-3.00) were positive in agar gel immunodiffusion. Multivariate analysis revealed that the use of a vaccination pistol (p < 0.001) and the presence of water bodies inside the farm (p < 0.01) were risk factors associated with the occurrence of EIA. Thus, the present study demonstrated a low but widespread prevalence of EIAV infected animals in the herds of Goiás state and that iatrogenic and environmental risk factors were associated with the occurrence of the disease.

Equine Infectious Anemia Virus (EIAV): Evidence of Circulation in Donkeys from the Brazilian Northeast Region.

Equine infectious anemia (EIA) is listed by the World Organization for Animal Health (OIE) as one of the equine diseases that must be notified. No effective treatment or vaccine is available. EIA control is based on segregation and euthanasia of positive equids. The disease is caused by the equine infectious anemia virus (EIAV), a member of the genus Lentivirus of the Retroviridae family. Despite the importance of this disease in equids, EIA has been poorly studied in donkeys (Equus asinus). We evaluate the sanitary conditions related to EIAV in donkeys from a shelter of abandoned animals captured on the roads of the Ceará. A total of 124 donkeys were randomly selected, and three horses lived at the same shelter. The animals were clinically evaluated, and a group of the 20 animals was submitted to hematological tests. Three diagnostic tests for EIA were used, agar gel immunodiffusion (AGID), enzyme-linked immunosorbent assay (ELISA) using EIAV recombinant protein gp90 (rgp90) and recombinant protein p26 (rp26) ELISA, and polymerase chain reaction (PCR) for detection of the EIAV tat-gag gene. From the donkeys, only 1 animal was positive using AGID 0.81% (1/124), compared to 21.8% (27/124) in the rgp90 and 10.5% (13/124) in the rp26 ELISA. Proviral DNA was detected by PCR tat-gag in 8.8% (11/124), and phylogenetic analysis confirms that the EIAV sequences of donkeys from the Brazilian Northeast grouped with Pantanal Brazilian sequences. Thus, in light of the results, we conclude that donkeys are carriers of EIAV and could be sources of infection.

Modelling Mutation in Equine Infectious Anemia Virus Infection Suggests a Path to Viral Clearance with Repeated Vaccination.

Equine infectious anemia virus (EIAV) is a lentivirus similar to HIV that infects horses. Clinical and experimental studies demonstrating immune control of EIAV infection hold promise for efforts to produce an HIV vaccine. Antibody infusions have been shown to block both wild-type and mutant virus infection, but the mutant sometimes escapes. Using these data, we develop a mathematical model that describes the interactions between antibodies and both wild-type and mutant virus populations, in the context of continual virus mutation. The aim of this work is to determine whether repeated vaccinations through antibody infusions can reduce both the wild-type and mutant strains of the virus below one viral particle, and if so, to examine the vaccination period and number of infusions that ensure eradication. The antibody infusions are modelled using impulsive differential equations, a technique that offers insight into repeated vaccination by approximating the time-to-peak by an instantaneous change. We use impulsive theory to determine the maximal vaccination intervals that would be required to reduce the wild-type and mutant virus levels below one particle per horse. We show that seven boosts of the antibody vaccine are sufficient to eradicate both the wild-type and the mutant strains. In the case of a mutant virus infection that is given infusions of antibodies targeting wild-type virus (i.e., simulation of a heterologous infection), seven infusions were likewise sufficient to eradicate infection, based upon the data set. However, if the period between infusions was sufficiently increased, both the wild-type and mutant virus would eventually persist in the form of a periodic orbit. These results suggest a route forward to design antibody-based vaccine strategies to control viruses subject to mutant escape.

Identification and genetic characterization of equine infectious anemia virus in Western Balkans.

BACKGROUND: Equine infectious anemia (EIA) is a viral disease, caused by the Equine Infectious Anemia virus (EIAV) belonging to the Retroviridae family, genus Lentivirus. Horses (or equids) infected with EIAV are lifelong carriers and they remain contagious for other horses even in the absence of clinical signs. So far, EIAV infection has been reported among horses in North and South America, France, Germany, Italy, Hungary and Romania, with no publication regarding the presence of EIAV in horses in Serbia. To determine the circulation of EIAV among, approximately, the 5000 horses of the Vojvodina region, northern part of Serbia, 316 serum undergone serological testing for EIA. Then, identification and full genome sequencing using next generation sequencing was performed from one EIA positive horse. RESULTS: the 316 sera were tested with 3 different commercial agar gel immunodiffusion (AGID) tests and two different commercial enzyme-linked immunosorbent assay (ELISA). With the three AGID kits, 311 (98.4%) among the 316 tested sera were negative and only five (1.6%) sera were positive for EIA. Some discrepancies were seen for the two ELISA kits tested since one exhibited the same results as AGID test and the second gave 295 sera with negative results, five with a positive result and 16 with doubtful outcome. Phylogenetic analysis performed using the full genome sequence showed that EIAV characterized from a horse in Serbia is different from those identify so fare around the world and form a distinct and separate group together with another EIAV strain. CONCLUSIONS: This study demonstrate for the first time that EIAV is circulating at a low level in the horse population from the Northern part of Serbia. Interestingly, phylogenetic data indicates that this EIAV from the western Balkan region of Europe belongs to a new cluster.

Molecular detection of equine infectious anemia virus in clinically normal, seronegative horses in an endemic area of Mexico.

Equine infectious anemia (EIA) is a highly infectious disease in members of the Equidae family, caused by equine infectious anemia virus (EIAV). The disease severity ranges from subclinical to acute or chronic, and causes significant economic losses in the equine industry worldwide. Serologic tests for detection of EIAV infection have some concerns given the prolonged seroconversion time. Therefore, molecular methods are needed to improve surveillance programs for this disease. We attempted detection of EIAV in 6 clinical and 42 non-clinical horses in Nuevo Leon State, Mexico, using the agar gel immunodiffusion (AGID) test for antibody detection, and nested and hemi-nested PCR for detection of proviral DNA. We found that 6 of 6, 5 of 6, and 6 of 6 clinical horses were positive by AGID, nested PCR, and hemi-nested PCR, respectively, whereas 0 of 42, 1 of 42, and 9 of 42 non-clinical horses were positive by these tests, respectively. BLAST analysis of the 203-bp 5'-LTR/tat segment of PCR product revealed 83-93% identity with EIAV isolates in GenBank and reference strains from other countries. By phylogenetic analysis, our Mexican samples were grouped in a different clade than other sequences reported worldwide, indicating that the LRT/tat region represents an important target for the detection of non-clinical horses.

True prevalence and spatial distribution of equine infectious anemia virus (EIAV) in horses from northeast region of Brazil

Equine infectious anemia (EIA) is a viral infection, caused by a lentivirus of the Retroviridae family, Orthoretrovirinael subfamily and its occurrence generates significant economic losses due to culling of positive animals as a measure of infection control. The objective of this work was to determine the prevalence of horses positive for equine infectious anemia virus (EIAV) and to identify the occurrence of areas with higher densities of cases in the states of Paraíba (PB), Pernambuco (PE), Rio Grande do Norte (RN) and Ceará (CE), Northeast region of Brazil, during the rainy (May and June) and dry (October and November) periods of 2017 and 2018. Serum samples from 6,566 horses from the states of PB, PE, RN and CE, Brazil, provided by the Laboratório Veterinária Diagnóstico - Ltda., were used. Serological diagnosis of EIA was performed using indirect enzyme-linked immunosorbent assay (ELISA) as a screening test and agar gel immunodiffusion test (AGID) as a confirmatory test. The apparent prevalence was obtained by dividing the number of seroreactive animals by the total number of animals, while the true prevalence was estimated by adjusting the apparent prevalence, considering the sensitivity (100%) and specificity (98.6%) of the diagnostic protocol used. For the construction of Kernel estimates, the Quartic function was used. In the dry season, of the 1,564 animals sampled, 28 were serologically positive, of which 19 belonged to the state of Ceará, 7 to Paraíba and 2 to Rio Grande do Norte. In 2018, it was observed that, during the rainy season, 26 of the 1,635 horses were seroreactive, with 19 cases resulting from Ceará, 4 from Paraíba and 3 from Pernambuco. In the dry season, 32 of the 1,526 animals were seroreactive to EIAV, of which 26 were from Ceará, 3 from Paraíba, 1 from Rio Grande do Norte and 2 from Pernambuco. In the dry period of 2017, the CE had a real prevalence of 1.22% (95% CI = 0.05 - 2.99%). In 2018, during the rainy season, prevalences of 0.03% (95% CI = 0 - 1.18%) were identified in CE and 1.69% (95% CI = 0 - 8.38%) in PE. Regarding the 2018 dry period, a prevalence of 1.32% (95% CI = 0.26 - 2.84%) was found in the state of CE. In both dry and rainy periods of 2017, the presence of spatial clusters of animals positive for EIA was observed, mainly in the border areas among the states of CE, PE, PB and RN. In 2018, there was a variation in the distribution of areas with higher densities of cases between the rainy and dry periods. The state of CE had the highest prevalence of positive animals and the presence of areas with higher densities of EIA cases in both climatic periods, in the years 2017 and 2018. In some municipalities of the CE, important sporting events of agglomeration of animals take place, which can favor the transmission of EIAV by facilitating the contact of infected and susceptible animals. Population density may be a factor associated with the higher prevalence observed in this region, as it has the second largest herd among the states studied. Higher densities indirectly contribute to the occurrence of infectious diseases, as they favor the contact of infected and susceptible animals. The occurrence of higher densities of cases in the border areas of the states of PE, RN, CE, and PB may be related to the greater movement of animals in these regions, favoring the indirect contact of infected horses with susceptible ones. The observed results demonstrate the circulation of the EIAV in four states in the Northeast region of Brazil.(AU)

Comparative analysis of CpG islands in equine infectious anemia virus strains.

Increasing evidence suggests that DNA methylation has key roles in the replication of retroviruses, including lentiviruses, and pathogenesis of diseases. However, the precise characteristics of CpG islands are not known for many retroviruses. In this study, we compared the distribution of CpG islands among strains of equine infectious anemia virus (EIAV), a lentivirus in the family Retroviridae and a model for HIV research. We identified CpG islands in 32 full-length EIAV genomic sequences obtained from the GenBank database using MethPrimer. Only one CpG island, from 100 to 120 bp, was identified in the genomes of EIAV strains DV10, DLV3-A, and DLV5-10 from China, V26 and V70 from Japan, and IRE H3, IRE F2, IRE F3, and IRE F4 from Ireland. Importantly, the CpG island was located within the Rev gene, which is required for the expression of viral cis-acting elements and the production of new virions. These results suggest that the distribution, length, and genetic properties of CpG islands differ among EIAV strains. Future research should focus on the biological significance of this CpG island within rev to improve our understanding of the precise roles of CpG islands in epigenetic regulation in the species.

High Genomic Variability in Equine Infectious Anemia Virus Obtained from Naturally Infected Horses in Pantanal, Brazil: An Endemic Region Case.

Equine infectious anemia virus (EIAV) is a persistent lentivirus that causes equine infectious anemia (EIA). In Brazil, EIAV is endemic in the Pantanal region, and euthanasia is not mandatory in this area. All of the complete genomic sequences from field viruses are from North America, Asia, and Europe, and only proviral genomic sequences are available. Sequences from Brazilian EIAV are currently available only for gag and LTR regions. Thus, the present study aimed for the first time to sequence the entire EIAV genomic RNA in naturally infected horses from an endemic area in Brazil. RNA in plasma from naturally infected horses was used for next-generation sequencing (NGS), and gaps were filled using Sanger sequencing methodology. Complete viral genomes of EIAV from two horses were obtained and annotated (Access Number: MN560970 and MN560971). Putative genes were analyzed and compared with previously described genes, showing conservation in gag and pol genes and high variations in LTR and env sequences. Amino acid changes were identified in the p26 protein, one of the most common targets used for diagnosis, and p26 molecular modelling showed surface amino acid alterations in some epitopes. Brazilian genome sequences presented 88.6% nucleotide identity with one another and 75.8 to 77.3% with main field strains, such as EIAV Liaoning, Wyoming, Ireland, and Italy isolates. Furthermore, phylogenetic analysis suggested that this Brazilian strain comprises a separate monophyletic group. These results may help to better characterize EIAV and to overcome the challenges of diagnosing and controlling EIA in endemic regions.