Neuropathogenic Varicellovirus Equid Alpha-1 Strain associated with mare abortion in BrazilABSTRACT
Varicellovirus equidalpha 1, formerly Equid alphaherpesvirus 1 (EqAHV-1), is prevalent globally, causing persistent infections via latency in horse populations. Viral reactivation under stress leads to new excretion and transmission, resulting in respiratory illness, abortion, neonatal foal death, and myeloencephalopathy. The neuropathogenic variant, associated with a specific neuropathogenic marker (G2254/N752), poses a higher replication rate and longer viremia duration. In Brazil, EqAHV-1's neuropathogenic strain has been identified, causing Equid Herpesvirus Myeloencephalopathy (EHM) in the states of São Paulo and Minas Gerais. The present study aimed to detect and genotype EqAHV-1 strains in abortion cases, exploring other potential etiologic agents. DNA samples from 118 samples were analyzed using PCR assays and sequencing. Results revealed EqAHV-1 DNA in 23.3% of abortion cases, with the neuropathogenic marker in 85.7% of positive samples. Leptospira spp., Brucella abortus, tick-borne pathogens and Trypanosoma evansi were considered in the differential diagnoses. EqAHV-1-positive mares had a vaccination history, but commercial vaccines didn't prevent neurological signs. The present study suggests the neuropathogenic EqAHV-1 as a significant cause of reproductive losses in Brazilian horses. Further research is needed to assess the infection's epidemiological profile and the efficacy of biosafety measures to prevent outbreaks.
Keywords:
reproductive loss; abortion; Varicellovirus equidalpha 1; neuropathogenic marker; differential diagnoses
RESUMO
O Varicellovirus equidalpha 1, anteriormente Equid alphaherpesvirus 1 (EqAHV-1), é prevalente globalmente, causando infecções persistentes em populações de cavalos por meio de latência. A reativação viral sob estresse leva à nova excreção e transmissão, resultando em doenças respiratórias, aborto, morte neonatal de potros e mieloencefalopatia. A variante neuropatogênica, associada a um marcador neuropatogênico específico (G2254/N752), apresenta maior taxa de replicação e maior duração da viremia. No Brasil, foi identificada a cepa neuropatogênica EqAHV-1, causando mieloencefalopatia por herpesvírus equino (EHM) nos estados de São Paulo e Minas Gerais. O presente estudo teve como objetivo detectar e genotipar cepas EqAHV-1 em casos de aborto e morte neonatal, explorando outros potenciais agentes etiológicos. Amostras de DNA de 118 amostras foram analisadas usando-se ensaios de PCR e sequenciamento. Os resultados revelaram DNA EqAHV-1 em 23,3% dos casos de aborto, com o marcador neuropatogênico em 85,7% das amostras positivas. Leptospira spp., Brucella abortus, patógenos transmitidos por carrapatos e Trypanosoma evansi foram considerados para diagnósticos diferenciais. As éguas positivas para EqAHV-1 tinham histórico de vacinação, mas as vacinas comerciais não preveniram os sinais neurológicos. O presente estudo sugere EqAHV-1 neuropatogênica como causa significativa de perdas reprodutivas em cavalos brasileiros. Mais pesquisas são necessárias para avaliar o perfil epidemiológico da infecção e a eficácia das medidas de biossegurança para prevenir surtos.
Palavras-chave:
perda reprodutiva; aborto; Varicellovirus equidalpha1; marcador neuropatogênico; diagnóstico diferencial
INTRODUCTION
Varicellovirus equidalpha1, previously known as Equid alphaherpesvirus 1 (EqAHV-1) or Equine herpesvirus 1, is highly prevalent in global horse populations, establishing persistent infections through latency. Under stressful conditions, viral reactivation occurs, resulting in new viral excretion and transmission to other horses (Allen, 2008). It induces respiratory illness in young horses, leads to abortion, typically in the last trimester of pregnancy, causes neonatal death in foals, and triggers myeloencephalopathy (Pusterla and Hussey, 2014). These manifestations significantly contribute to a substantial economic loss in the equine industry. The increased incidence of EqAHV-1 causing neurological disease is significantly associated with a single nucleotide polymorphism (SNP) in the viral DNA polymerase gene (ORF30). The exchange of adenine (A) to guanine (G) at nucleotide position 2254 results in an N (asparagine) to D (aspartic acid) change at amino acid position 752 (Nugent et al., 2006). The neuropathogenic genotype is associated with a 165-fold higher risk of neurological disease compared to the non-neuropathogenic genotype, and this increase is also observed in vaccinated animals. The role of this mutation was confirmed by reverse genetics experiments in both neuropathogenic (Ab4) and non-neuropathogenic (NY03) strains (Goodman et al., 2007; Van de Walle et al., 2009).
In 2018, a novel C2254 (H752) genotype was identified during an outbreak in France. A nucleotide substitution to cytosine (C) at position 2254 resulted in the replacement of histidine (H) at amino acid position 752. The outbreak was marked by a rapid onset of severe respiratory symptoms, including cough, nasal discharge, anorexia, and pyrexia, along with hyperemia and hind limb edema, lasting at least two weeks. Some horses also exhibited mild neurological deficits, such as ataxia, weakness, and proprioceptive issues. A similar outbreak with this C2254 (H752) genotype was reported in the United States in 2021 (Sutton et al., 2020; Pusterla et al., 2021).
Infections with these markers (neuropathogenic G2254/N752 and C2254) are associated with longer duration and higher magnitude of leukocyte-associated viremia than observed in horses infected with non-neuropathogenic strains of EqAHV-1 (Smith et al., 2010). Studies indicate that high antibody titers do not reduce the duration or severity of viremia caused by neuropathogenic variants, making the control of cell-associated viremia critical. Current commercial vaccines, which are inactivated and limited by horses' sensitivity to adjuvants that stimulate an efficient cellular immune response, have not been shown to prevent equine herpesvirus myeloencephalopathy (Lunn et al., 2009; Pusterla and Hussey, 2014). No vaccine studies targeting the C2254 variant have been conducted to date.
EqAHV-1 abortion is the result of infection of the endometrium, leading to placental abruption and abortion in the 3rd end of pregnancy. Therefore, abortion may occur even before the virus reaches the fetus. In contrast, when EqAHV-1 infection causes only mild lesions and does not lead to immediate abortion, the virus may reach the fetus and replicate to high titers in fetal tissues. The exact pathogenesis remains to be investigated further (Leon et al., 2008; Gardiner et al., 2012). The frequency of abortion induced by EqAHV-1 has decreased from 1957 to 2008, possibly due to worldwide vaccination practices (Smith et al., 2010). In contrast, cases of Equid Herpesvirus Myeloencephalopathy (EHM) have significantly increased in number and severity since 2000 (Lunn et al., 2009). Although the neuropathogenic strain (G2254/N752) is more commonly associated with EHM cases, the non-neuropathogenic strain is more frequently linked to abortion cases (Lunn et al., 2009). However, retrospective studies on fetal tissues from sporadic abortions have detected the presence of the neuropathogenic strain (G2254/N752) since the 1950s, with an increasing incidence throughout the following decades (3.3% in the 1960s; 14.4% in the 1990s; 19.4% in 2006; 25.8% in 2015) (Smith et al., 2010; Barbić et al., 2012).
This increased incidence of the neuropathogenic strain in abortion cases has also been confirmed in several countries, including Germany, Poland, India, Croatia, Japan, the USA, among others (Smith et al., 2010; Damiani et al., 2014).
In Brazil, the presence of neuropathogenic variants causing EHM has already been identified in the states of São Paulo and Minas Gerais (Costa et al., 2009). Studies conducted in Minas Gerais have revealed that EqAHV-1 is the second-largest viral agent responsible for fatal neurological disease in horses, with a prevalence ranging from 11.8% to 43.68% in cases reported to the Agriculture Defense Agency (Instituto Mineiro de Agropecuária-IMA) (Costa et al., 2009). Additionally, veterinarians have observed an abortion rate of up to 50% in mares vaccinated against EqAHV-1. Therefore, the identification of neuropathogenic markers of EqAHV-1 in abortion outbreaks is crucial for a better understanding of the infection’s epidemiology and to facilitate the adoption of biosafety practices (Smith et al., 2010).
MATERIAL AND METHODS
A total of 118 samples from 35 cases of reproductive loss, collected from August 2017 to October 2018, were submitted for routine diagnostic tests to the Animal Virology Research Laboratory of the Veterinary School at UFMG. Of the 35 reproductive problems, 30 were abortions, 3 cases of neonatal mortality, and 2 cases of stillbirths. The samples originated from 15 properties located in the states of Minas Gerais (MG - 10/15), Rio de Janeiro (RJ - 3/15), São Paulo (SP - 1/15), and Mato Grosso do Sul (MS - 1/15) and were named P1 to P15 (Table 1).
Samples from mares included 34 whole blood samples and 14 nasal swabs. Fetal, stillborn, or neonatal foal samples included 14 lungs, 5 livers, 9 spleens, 8 kidneys, 7 hearts, 7 thymuses, 4 organ pools (kidney + spleen + liver), 6 thoracic fluids, 2 stomach fluids and 8 placenta/umbilical cord remains. Clinical specimens from the mare and fragments of fetal or foal organs from the same reproductive problem were not consistently collected.
These samples were processed (Gardiner et al., 2012) and DNA extraction used the MINISPIN Virus DNA/RNA kit (KASVI), following the manufacturer's recommendations. Nested-PCR assays for EqAHV-1 and hemi-nested PCR for EqAHV-4 were conducted to amplify the DNA polymerase (ORF30) and glycoprotein B (gB) genes, respectively (Varrassoet al., 2001) (Table 2). Positive controls for EqAHV-4, as well as non-neuropathogenic and neuropathogenic (EqAHV-1) strains, were kindly provided by Dr. Mauricio Resende (Veterinary School/UFMG).
For the detection of the neuropathogenic marker (G2254), positive amplicons from the EqAHV-1 PCR assay underwent purification using the DNA Clean & Concentrator kit (Zymo Research), following the manufacturer's instructions. Sequencing was carried out using the BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems) in the ABI3730 DNA Analyzer sequencer (Applied Biosystems). Electropherograms were analyzed using SeqMan Software 7.1.0 (DNAStar Inc.). The EqAHV-1 sequences obtained in the present study were aligned with the sequences of the neuropathogenic reference strain Ab4 (GenBank accession number AY665713), the non-neuropathogenic reference strain V592 (GenBank accession number AY464052) and the new genotype C2254 (H752) reference strain FR56628 (GenBank accession number MT968035), using the Multalin algorithm (http://multalin.toulouse.inra.fr/multalin/).
Spleen and liver samples from aborted fetuses, stillbirths, or neonatal mortality cases in foals were subjected to PCR assays to detect infectious agents considered significant contributors to reproductive problems in Brazil (Table 2). Leptospira spp. should consistently be included in the differential diagnoses of abortions in Brazil, given its high prevalence in the country. Although less prevalent, Brucella abortus is also present and should be considered. Tick-borne diseases and Trypanosoma evansi were also included in the differential diagnoses, considering their considerable detection in Brazil (Silveira, 2012).
RESULTS AND DISCUSSION
EqAHV-1 DNA was detected in 7 out of 30 abortion cases (23.3%). EqAHV-4 was detected in only 1 case, from a thymus sample. None of the infectious agents considered in the differential diagnosis were detected using PCR. The causes of stillborn foals or neonates remains undiagnosed. Following the sequencing of positive EqAHV-1 samples, the neuropathogenic marker (G2254/N752 point mutation) was identified in 6 out of the 7 samples analyzed (Fig. 1). One sample did not reach sufficient quality for sequencing and was discarded. The size of the nucleotide sequences analyzed was 254 bp, and the nucleotide sequences exhibited 100% similarity among themselves and with the neuropathogenic reference sample Ab4 (GenBank accession number AY665713) (Fig. 1). The region of the genome amplified by the PCR EqAHV-1 assay is a highly conserved region of the gene that encodes DNA polymerase (ORF 30), justifying this high degree of similarity (Nugent et al., 2006). The sequences were named: BR17_01_2, BR17_02_2, BR18_18_2, BR18_22_2, BR18_25_2, and BR18_35_2, following previously described nomenclature (Nugent et al., 2006).
Partial alignment of nucleotide and deduced amino acid sequences from EqAHV-1, of the gene amplifying viral DNA polymerase (ORF30). Brazilian samples from abortion cases of the present study: BR17_01_2, BR17_02_2, BR18_18_2, BR18_22_2, BR18_25_2, and BR18_35_2, neuropathogenic reference strain Ab4, GenBank accession number DQ180669 (neuropathogenic marker, single nucleotide polymorphism (SNP) G2254/D752), non-neuropathogenic reference strain V592, GenBank accession number DQ172359 (non-neuropathogenic marker, SNP A2254/N752) and C2254 reference strain V592, GenBank accession number MT968035 (SNP C2254/H752).
In the context of 7 EqAHV-1-positive abortion cases, 34 samples underwent analysis, comprising 26 fetal tissues and 9 maternal samples. Of these, EqAHV-1 DNA was detected in 17 samples, with 10/17 - 58.8% originating from fetal tissues and 7/17 - 41.2% originating from maternal samples. (Table 3). The fetal tissues with the highest viral detection were the lung and pool of organs containing liver, kidney and spleen (3/9 - 22.2%), followed by the liver and spleen (2/9 - 22.2%). No viral DNA was detected in fragments of the kidney (only), placenta, umbilical cord, and heart. In mare samples, viral DNA was detected in 6/9 (66.7%) blood samples and 3/9 nasal swabs (33.3%) (Table 3).
Some experimental studies with neuropathogenic variants have demonstrated that in challenges in pregnant mares in the final third of gestation (270-290 days of gestation) high viral loads can be observed in the lung, liver, spleen, and thymus from fetuses and placental tissues (Gardiner et al., 2012).
However, since abortion due to EHV-1 infection can result from endometrial infection leading to premature placental detachment, abortion may occur before the virus even reaches the fetus. There are several reports of infected mares that aborted virologically negative fetuses (Smith et al., 2004). These findings have raised concerns about whether similar events could occur in some spontaneous abortions, leading to false-negative diagnoses if only fetal tissues are examined for virus detection. In such cases, placental histology is recommended to check for lesions associated with multifocal chorionic necrosis, microthrombosis, or vasculitis of some chorionic blood vessels, along with virus screening via PCR or immunohistochemistry. For this, it is necessary to collect samples from different areas of the placenta (Smith et al., 2004). Therefore, the absence of virus detection in the placenta, in the present study, may be attributed to sampling errors, as only a random placental fragment was collected for diagnosis.
The detection of viral DNA in samples from mares confirms the replication of EqAHV-1 at the time of abortion. In two of these cases (2/9 - 22.2%), only clinical samples from the mare (nasal swab and whole blood) were submitted for diagnosis. Although EqAHV-1 may be the causative agent of abortion in these cases, a definitive diagnosis should ideally be based on its detection in the placenta, amniotic fluid and/or fragments of fetal organs (Pusterla and Hussey, 2014). However, in cases where the fetus is not viable due to difficulties in locating it or destruction by vultures or carnivorous animals, the collection of whole blood and nasal swabs from mares is crucial for directing the diagnosis (Pusterla and Hussey, 2014).
Positive cases for EqAHV-1 were reported in five properties, four of which were in municipalities in the state of Minas Gerais (Esmeraldas, Itapecerica, Matozinhos and Pará de Minas), in the southeast region of Brazil, and one in the state of Mato Grosso do Sul (municipality of Campo Grande), in the southern region. The property with a positive case for EqAHV-4 was situated in the state of Rio de Janeiro (municipality of Macaé), in the southeast region (Fig. 2).
The ages of the mares that experienced abortion ranged from 8 to 19 years. Reproductive techniques varied between artificial insemination (AI) (60%) and embryo transfer (ET) (40%). The breeds involved in positive cases for EqAHV-1 were Quarter Horses (QM), followed by Campolina, Paint Horse, and those with no defined breed (Table 1). Studies suggest that factors such as breed, age, and sex are associated with differences in clinical signs, along with infection by neuropathogenic variants of EqAHV-1 (Lunn et al., 2009). EHM in females and older animals tend to be more severe. Some studies report the predisposition of Quarter horses to abortion and neurological clinical signs caused by EqAHV-1 infection. Therefore, there is limited consensus among studies (Pritchard, 2011). Among the reproductive techniques used in positive cases, there was variation between artificial insemination (AI) (60%) and embryo transfer (ET) (40%). Stress-inducing management procedures and extensive movement and mixing of animals, often associated with embryo transfer and artificial insemination practices, can increase the risk of spreading EqAHV-1 infection, with devastating clinical consequences (Barrandeguy et al., 2002).
Map showing locations of the five properties where EqAHV-1 abortions were confirmed, with the property where EqAHV-4 was detected. Municipalities with EqAHV-1 positive cases detected by PCR include Pará de Minas, Itapecerica, Matozinhos, and Esmeraldas in the state of Minas Gerais (MG), as well as Campo Grande in the state of Mato Grosso do Sul (MS) (States are highlighted in purple). The property testing positive for EqAHV-4 is in Macaé, Rio de Janeiro (RJ) (State is highlighted in pink).
All positive mares had a vaccination history against EqAHV-1, following a protocol in the 5th, 7th, and 9th months of pregnancy. While commercial inactivated vaccines do not shield against neurological signs induced by neuropathogenic variants, there is a significant decrease in viral excretion in vaccinated horses. Mares vaccinated either twice or three times during pregnancy were equally affected, although challenge experiments using the same vaccine have demonstrated a notable reduction in the frequency of abortion (Damiani et al., 2014). Therefore, the emerging status of EqAHV-1 neuropathogenic variants as an etiological agent for reproductive problems underscores the imperative to heighten awareness regarding the challenges in preventing and mitigating EqAHV-1 outbreaks in Brazil.
CONCLUSION
Prevention and control measures are categorized into actions to diminish the likelihood of outbreaks and actions to constrain the disease's spread during an outbreak. Key strategies include segregating pregnant mares, isolating new arrivals for 21 days, grouping pregnant mares by pregnancy stage, minimizing stress, and reducing viral load in the environment (Lunn et al., 2009). Studies show placental tissue from aborted fetuses and healthy-born foals can carry similar viral levels, indicating a source of infection. Prompt disposal of placental tissues is crucial (Gardiner et al., 2012; Stasiak et al., 2015). These measures can apply to other horses on the farm. Control is challenging due to mild cases and latently infected animals. Introducing asymptomatic EqAHV-1-positive horses into an immunologically naive and unvaccinated herd without proper quarantine measures can result in rapid dissemination, initially manifesting as mild respiratory symptoms and progressing to abortions within 7 to 10 days post-introduction (Barbić et al., 2012). Asymptomatic stallions can transmit EHV-1 through semen, as detected by PCR in stallions from herds with neuropathogenic genotype and cases of neurological disease and abortion. However, the role of stallions in transmitting neuropathogenic strains requires caution, as available data is limited (Hebia-Fellah et al., 2009; Fritsche and Borchers, 2011).
Immediate steps are needed to diagnose and control outbreaks, focusing on early diagnosis and managing clinical cases to prevent further viral spread (Lunn et al., 2009). Monitoring viral excretion in infected mares is crucial. Experimental infection studies with neuropathogenic EqAHV-1 in mares in the final third of pregnancy demonstrate that reproductive tract samples (vaginal and cervical specimens) remain positive for 7 days after abortion. Additionally, detection of EqAHV-1 DNA in nasal swabs can occur between days 1-9 post-challenge, and mares become viremic between days 5 and 8 post-challenge, lasting 1 to 3 days (Gardiner et al., 2012).
ACKNOWLEDGMENTS
We express our appreciation to the field veterinarians who were responsible for sample collection, as well as to our colleagues at the Animal Virology Research Laboratory - LPVA and Protozoology Veterinary Laboratory - PROTOVET - and the Equine Clinic Sector of the UFMG Veterinary School for their invaluable support.
REFERENCES
- ALLEN, G.P. Risk factors for development of neurologic disease after experimental exposure to equine herpesvirus-1 in horses. Am. J. Vet. Res., v.69, p.1595-1600, 2008.
- BARBIĆ, L.; LOJKIĆ, I.; STEVANOVIĆ, V. et al. Two outbreaks of neuropathogenic equine herpesvirus type 1 with breed-dependent clinical signs. Vet. Rec., v.170, p.227-227, 2012.
- BARRANDEGUY, M.E.; LASCOMBES, F.; LLORENTE, J. et al. High case-rate Equine herpesvirus-1 abortion outbreak in vaccinated polo mares in Argentina. Equine Vet. Educ., v.14, p.132-135, 2002.
- COSTA E.A.; VASCONCELOS, A.C.; BOMFIM, M.R.Q. et al. Epidemiological and clinical aspects of equine Herpesvirus encephalitis infection in horses that died with neurological signs from Minas Gerais state, Brazil. Braz. J. Vet. Res. Anim. Sci., v.46, p.262-272, 2009.
- DAMIANI A.M.; VRIES, M.; REIMERS, G. et al. A severe equine herpesvirus type 1 (EHV-1) abortion outbreak caused by a neuropathogenic strain at a breeding farm in northern Germany. Vet. Microbiol., v.172, p.555-562, 2014.
- FRITSCHE A.K.; BORCHERS, K. Detection of neuropathogenic strains of Equid Herpesvirus 1 (EHV-1) associated with abortions in Germany. Vet. Microbiol., v.147, p.176-180, 2011.
- GARDINER D.W.; LUNN, D.P.; GOEHRING, L.S. et al. Strain impact on equine herpesvirus type 1 (EHV-1) abortion models: viral loads in fetal and placental tissues and foals. Vaccine, v.30, p.6564-6572, 2012.
- GOODMAN, L.B.; LOREGIAN, A.; PERKINS, G.A. et al. A point mutation in a herpesvirus polymerase determines neuropathogenicity. PLoS Pathog., v.3, p.e160, 2007.
- HEBIA-FELLAH, I.; LE´AUTE´, A.; FIE´NI, F. et al. Evaluation of the presence of equine viral herpesvirus 1 (EHV-1) and equine viral herpesvirus 4 (EHV-4) DNA in stallion semen using polymerase chain reaction (PCR). Theriogenology, v.71, p.1381-1389, 2009.
- LÉON, A.; FORTIER, G.; FORTIER, C. et al. Detection of equine herpesviruses in aborted foetuses by consensus PCR. Vet. Microbiol., v.126, p.20-29, 2008.
- LUNN, D.P.; DAVIS-POYNTER, N.; FLAMINIO, M.J.B.F. et al. Equine herpesvirus-1 consensus statement. J. Vet. Intern. Med., v.23, p.450-461, 2009.
- NUGENT, J.; BIRCH-MACHIN, I.; SMITH, K.C. et al. Analysis of equid herpesvirus 1 strain variation reveals a point mutation of the DNA polymerase strongly associated with neuropathogenic versus nonneuropathogenic disease outbreaks. J. Virol., v.80, p.4047-4060, 2006.
-
PRITCHARD, W.R. Awakening the dormant dragon: neurological form of equine herpesvirus-1. UCDavis Veterinary Medicine, 2011. Available in: https://ag.colorado.gov/sites/ag/files/documents/Awakening%20the%20Dormant%20Dragon-%20Neurological%20Form%20of%20EHV-1_0.pdf Accessed in: 1 May 2024.
» https://ag.colorado.gov/sites/ag/files/documents/Awakening%20the%20Dormant%20Dragon-%20Neurological%20Form%20of%20EHV-1_0.pdf - PUSTERLA, N.; HUSSEY, G.S. Equine herpesvirus 1 myeloencephalopathy. Vet. Clin. North Am. Equine Pract., v.30, p.489-506, 2014.
- SILVEIRA, J.A.G. Ocorrência de hemoparasitos e ectoparasitos em veado-catingueiro (Mazama gouazoubira Fischer, 1814), veado-campeiro (Ozotocerus bezoarticus Linnaeus, 1758) e cervo-do-pantanal (Blastocerus dichotomus Illiger, 1815): utilização de métodos parasitológicos e moleculares. 2012. 133f. Tese (Doutorado em Parasitologia) - Escola de Veterinária, Universidade Federal de Minas Gerais, Belo Horizonte, MG.
- STASIAK, K.; ROLA, J.; PLOSZAY, G. et al. Detection of the neuropathogenic variant of equine herpesvirus 1 associated with abortions in mares in Poland. BMC Vet. Res., v.11, p.102, 2015.
- SMITH, K.C.; WHITWELL, K.E.; BLUNDEN, A.S. et al. Equine herpesvirus-1 abortion: atypical cases with lesions largely or wholly restricted to the placenta. Equine Vet J., v.36, p.79-82, 2004.
- SMITH, K.L.; ALLEN, G.P.; BRANSCUM, A.J. et al. The increased prevalence of neuropathogenic strains of EHV-1 in equine abortions. Vet. Microbiol., v.141, p.5-11, 2010.
- VAN DE WALLE, G.R.; GOUPIL, R.; WISHON, C. et al. A single-nucleotide polymorphism in a herpesvirus DNA polymerase is sufficient to cause lethal neurological disease. J. Infect. Dis., v.200, p.20-25, 2009.
- VARRASSO, A.; DYNON, K.; FICORILLI, N. et al. Identification of equine herpesviruses 1 and 4 by polymerase chain reaction. Aust. Vet. J., v.79, p.563-569, 2001.
Publication Dates
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Publication in this collection
28 Apr 2025 -
Date of issue
May-Jun 2025
History
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Received
07 Apr 2024 -
Accepted
26 Oct 2024
