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We evaluated a lyophilized CRISPR-Cas12 assay for SARS-CoV-2 detection (Lyo-CRISPR SARS-CoV-2 kit) based on reverse transcription, isothermal amplification, and CRISPR-Cas12 reaction. From a total of 210 RNA samples extracted from nasopharyngeal swabs using spin columns, the Lyo-CRISPR SARS-CoV-2 kit detected 105/105 (100%, 95% confidence interval (CI): 96.55–100) positive samples and 104/105 (99.05%, 95% CI: 94.81–99.97) negative samples that were previously tested using commercial RT-qPCR. The estimated overall Kappa index was 0.991, reflecting an almost perfect concordance level between the two diagnostic tests. An initial validation test was also performed on 30 nasopharyngeal samples collected in lysis buffer, in which the Lyo-CRISPR SARS-CoV-2 kit detected 20/21 (95.24%, 95% CI: 76.18–99.88) positive samples and 9/9 (100%, 95% CI: 66.37–100) negative samples. The estimated Kappa index was 0.923, indicating a strong concordance between the test procedures. The Lyo-CRISPR SARS-CoV-2 kit was suitable for detecting a wide range of RT-qPCR-positive samples (cycle threshold range: 11.45–36.90) and dilutions of heat-inactivated virus (range: 2.5–100 copies/µL), no cross-reaction was observed with the other respiratory pathogens tested. We demonstrated that the performance of the Lyo-CRISPR SARS-CoV-2 kit was similar to that of commercial RT-qPCR, as the former was highly sensitive and specific, timesaving (1.5 h), inexpensive, and did not require sophisticated equipment. The use of this kit would reduce the time taken for diagnosis and facilitate molecular diagnosis in low-resource laboratories.

Equid alphaherpesvirus 3 (EHV-3) is the etiological agent of equine coital exanthema (ECE). Because no vaccines or antiviral therapies are available, prevention consists of clinical examination of mares and stallions before mating or semen collection and resting from breeding activities when lesions are present. However, this methodology does not identify subclinically infected animals. Ganciclovir is the most potent compound known to reduce EHV-3 replication. This study aimed to evaluate the efficacy of topical ganciclovir application to reduce EHV-3 replication in experimentally infected mares. A pilot study, after a double-blind completely randomized design, was carried out. Twenty mares were randomly divided into five groups (three treated with ganciclovir with different regimen of doses, one treated with a placebo, and one nontreated). Mares were experimentally infected with EHV-3 on day 0. Rectal temperature, clinical signs, and lesions were recorded. Daily perineal and vaginal swabs were evaluated by quantitative polymerase chain reaction for virus detection. The antibody response was assessed by a virus neutralization test in serum samples collected weekly. Mares experimentally infected with EHV-3 and treated with ganciclovir twice a day for 13 days showed reduced levels and duration of viral excretion and less severe lesions. The viral excretion period was reduced from 18 to nine days compared with the untreated groups. We concluded that ganciclovir had an antiviral effect on EHV-3 replication when topically administered in mares showing clinical signs of ECE. Further trials should be performed to optimize the dose of the antiviral for a definitive formulation.

To facilitate the temporary importation of horses for competition and racing purposes, with a minimum risk of transmitting equine influenza, the World Organisation for Animal Health (Office International des Epizooties, or OIE), formally engaged in a public&ndash, private partnership with the Federation Equestre Internationale (FEI) and the International Federation for Horseracing Authorities (IFHA) to establish, within the context of existing OIE standards, a science-based rationale to identify the ideal time period for equine influenza vaccination prior to shipment. Field trials using vaccines based on different technologies were carried out on three continents. The antibody response post-booster vaccination at intervals aligned with the different rules/recommendations of the OIE, FEI, and IFHA, was monitored by single radial haemolysis. It was determined that 14 days was the optimum period necessary to allow horses adequate time to respond to booster vaccination and for horses that have previously received four or more doses of vaccine and are older than four years, it is adequate to allow vaccination within 180 days of shipment. In contrast, the results indicate that there is a potential benefit to younger (four years old or younger) horses in requiring booster vaccination within 90 days of shipment, consistent with the current OIE standard.

Equine influenza virus (EIV) is considered the most important respiratory pathogen of horses as outbreaks of the disease lead to substantial economic losses. The H3N8 EIV has caused respiratory disease in horses across the world, including South American countries. Nucleotide and deduced amino acid sequences for the complete haemagglutinin gene of the H3N8 EIV detected in South America since 1963 were analyzed. Phylogenetic and Bayesian coalescent analyses were carried out to study the origin, the time of the most recent common ancestors (tMRCA), the demographic and the phylogeographic patterns of the H3N8 EIV. The phylogenetic analysis demonstrated that the H3N8 EIV detected in South America grouped in 5 well-supported monophyletic clades, each associated with strains of different origins. The tMRCA estimated for each group suggested that the virus was circulating in North America at least one year before its effective circulation in the South American population. Phylogenetic and coalescent analyses revealed a polyphyletic behavior of the viruses causing the outbreaks in South America between 1963 and 2012, possibly due to the introduction of at least 4 different EIVs through the international movement of horses. In addition, phylodynamic analysis suggested South America as the starting point of the spread of the H3N8 EIV in 1963 and showed migration links from the United States to South America in the subsequent EIV irruptions. Further, an increase in the relative genetic diversity was observed between 2006 and 2007 and a subsequent decline since 2009, probably due to the cocirculation of different lineages and as a result of the incorporation of the Florida clade 2 strain in vaccines, respectively. The observed data highlight the importance of epidemiological surveillance and the implementation of appropriate quarantine procedures to prevent outbreaks of the disease. Inst.de Virología Fil: Olguin Perglione, Cecilia. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Virología; Argentina Fil: Golemba, Marcelo D. Hospital de Pediatría. S.A.M.I.C. “Prof. Dr. Juan P.Garrahan”; Argentina Fil: Torres, Carolina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Cátedra de Virología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Barrandeguy, Maria Edith. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Virología; Argentina. Universidad del Salvador. Escuela de Veterinaria; Argentina

Background: In 2012, equine influenza (EI) virus was confirmed as the cause of outbreaks of respiratory disease in horses throughout South America. In Uruguay and Argentina, hundreds of vaccinated thoroughbred horses in training and racing facilities were clinically affected. Objective: To characterise the EI viruses detected during the outbreak in Uruguay and Argentina. Methods: Virus was detected in nasopharyngeal swabs by a pan-reactive influenza type A real-time RT-PCR. The nucleotide sequence of the HA1 gene was determined and analysed phylogenetically using mega 5 software. Amino acid sequences alignments were constructed and virus was antigenically characterised with specific ferret antisera. Paired serum samples were tested by haemagglutination inhibition and single radial haemolysis. Results: The diagnosis of EIV was confirmed by real-time RT-PCR, virus isolation and serological testing. The phylogenetic analysis of HA1 gene sequences of 18 EI viruses indicated that all of them belong to clade 1 of the Florida sublineage of the American lineage and are closely related to viruses isolated in the United States in 2012. The HA1 of viruses identified in horses in racing facilities in Maroñas, Uruguay, and in Palermo, Argentina, displayed 100% amino acid sequence identity and were identical to that of a virus isolated in Dubai in 2012, from vaccinated endurance horses recently imported from Uruguay. Conclusions: The surveillance data reported illustrate the international spread of EI viruses and support the recommendations of the OIE expert surveillance panel to include viruses of the Florida sublineage in vaccines. Fil: Olguin Perglione, Cecilia. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Virología; Argentina Fil: Rimondi, Agustina. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Virología; Argentina Fil: Gildea, Sarah. The Irish Equine Centre. Virology Unit; Irlanda Fil: Miño, Samuel. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Virología; Argentina Fil: Vissani, Aldana. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Virología; Argentina Fil: Carossino, Mariano. Universidad del Salvador. Escuela de Veterinaria. Cátedra de Enfermedades Infecciosas; Argentina. University of Kentucky. Department of Veterinary Science. Maxwell H. Gluck Equine Research Center; Estados Unidos Fil: Cullinane, Ann. The Irish Equine Centre. Virology Unit; Irlanda Fil: Barrandeguy, Maria Edith. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Virología; Argentina. Universidad del Salvador. Escuela de Veterinaria. Cátedra de Enfermedades Infecciosas; Argentina