Clinical evaluation of a multiplex real-time RT-PCR assay for detection of SARS-CoV-2 in individual and pooled upper respiratory tract samples.

The aim of this study was to identify and validate a sensitive, high-throughput, and cost-effective SARS-CoV-2 real-time RT-PCR assay to be used as a surveillance and diagnostic tool for SARS-CoV-2 in a university surveillance program. We conducted a side-by-side clinical evaluation of a newly developed SARS-CoV-2 multiplex assay (EZ-SARS-CoV-2 Real-Time RT-PCR) with the commercial TaqPath COVID-19 Combo Kit, which has an Emergency Use Authorization from the FDA. The EZ-SARS-CoV-2 RT-PCR incorporates two assays targeting the SARS-CoV-2 N gene, an internal control targeting the human RNase P gene, and a PCR inhibition control in a single reaction. Nasopharyngeal (NP) and anterior nares (AN) swabs were tested as individuals and pools with both assays and in the ABI 7500 Fast and the QuantStudio 5 detection platforms. The analytical sensitivity of the EZ-SARS-CoV-2 RT-PCR assay was 250 copies/ml or approximately 1.75 genome copy equivalents per reaction. The clinical performance of the EZ-SARS-CoV-2 assay was evaluated using NP and AN samples tested in other laboratories. The diagnostic sensitivity of the assay ranged between 94 and 96% across the detection platforms, and the diagnostic specificity was 94.06%. The positive predictive value was 94%, and the negative predictive value ranged from 94 to 96%. Pooling five NP or AN specimens yielded 93% diagnostic sensitivity. The overall agreement between these SARS-CoV-2 RT-PCR assays was high, supported by a Cohen's kappa value of 0.93. The EZ-SARS-CoV-2 RT-PCR assay performance attributes of high sensitivity and specificity with AN sample matrix and pooled upper respiratory samples support its use in a high-throughput surveillance testing program.

Genetics of Immune Disease in the Horse.

Host defenses against infection by viruses, bacteria, fungi, and parasites are critical to survival. It has been estimated that upwards of 7% of the coding genes of mammals function in immunity and inflammation. This high level of genomic investment in defense has resulted in an immune system characterized by extraordinary complexity and many levels of redundancy. Because so many genes are involved with immunity, there are many opportunities for mutations to arise that have negative effects. However, redundancy in the mammalian defense system and the adaptive nature of key immune mechanisms buffer the untoward outcomes of many such deleterious mutations.

Polymorphism at expressed DQ and DR loci in five common equine MHC haplotypes.

The polymorphism of major histocompatibility complex (MHC) class II DQ and DR genes in five common equine leukocyte antigen (ELA) haplotypes was determined through sequencing of mRNA transcripts isolated from lymphocytes of eight ELA homozygous horses. Ten expressed MHC class II genes were detected in horses of the ELA-A3 haplotype carried by the donor horses of the equine bacterial artificial chromosome (BAC) library and the reference genome sequence: four DR genes and six DQ genes. The other four ELA haplotypes contained at least eight expressed polymorphic MHC class II loci. Next generation sequencing (NGS) of genomic DNA of these four MHC haplotypes revealed stop codons in the DQA3 gene in the ELA-A2, ELA-A5, and ELA-A9 haplotypes. Few NGS reads were obtained for the other MHC class II genes that were not amplified in these horses. The amino acid sequences across haplotypes contained locus-specific residues, and the locus clusters produced by phylogenetic analysis were well supported. The MHC class II alleles within the five tested haplotypes were largely non-overlapping between haplotypes. The complement of equine MHC class II DQ and DR genes appears to be well conserved between haplotypes, in contrast to the recently described variation in class I gene loci between equine MHC haplotypes. The identification of allelic series of equine MHC class II loci will aid comparative studies of mammalian MHC conservation and evolution and may also help to interpret associations between the equine MHC class II region and diseases of the horse.

Peptide-binding motifs of two common equine class I MHC molecules in Thoroughbred horses.

Quantitative peptide-binding motifs of MHC class I alleles provide a valuable tool to efficiently identify putative T cell epitopes. Detailed information on equine MHC class I alleles is still very limited, and to date, only a single equine MHC class I allele, Eqca-1*00101 (ELA-A3 haplotype), has been characterized. The present study extends the number of characterized ELA class I specificities in two additional haplotypes found commonly in the Thoroughbred breed. Accordingly, we here report quantitative binding motifs for the ELA-A2 allele Eqca-16*00101 and the ELA-A9 allele Eqca-1*00201. Utilizing analyses of endogenously bound and eluted ligands and the screening of positional scanning combinatorial libraries, detailed and quantitative peptide-binding motifs were derived for both alleles. Eqca-16*00101 preferentially binds peptides with aliphatic/hydrophobic residues in position 2 and at the C-terminus, and Eqca-1*00201 has a preference for peptides with arginine in position 2 and hydrophobic/aliphatic residues at the C-terminus. Interestingly, the Eqca-16*00101 motif resembles that of the human HLA A02-supertype, while the Eqca-1*00201 motif resembles that of the HLA B27-supertype and two macaque class I alleles. It is expected that the identified motifs will facilitate the selection of candidate epitopes for the study of immune responses in horses.

Bone marrow transcriptome and epigenome profiles of equine common variable immunodeficiency patients unveil block of B lymphocyte differentiation.

Common variable immunodeficiency (CVID) is a late-onset humoral deficiency characterized by B lymphocyte dysfunction or loss, decreased immunoglobulin production, and recurrent bacterial infections. CVID is the most frequent human primary immunodeficiency but still presents challenges in the understanding of its etiology and treatment. CVID in equine patients manifests with a natural impairment of B lymphocyte differentiation, and is a unique model to identify genetic and epigenetic mechanisms of disease. Bone marrow transcriptome analyses revealed decreased expression of genes indicative of the pro-B cell differentiation stage, importantly PAX5 (p≤0.023). We hypothesized that aberrant epigenetic regulation caused PAX5 gene silencing, resulting in the late-onset and non-familial manifestation of CVID. A significant increase in PAX5 enhancer region methylation was identified in equine CVID patients by genome-wide reduced-representation bisulfite sequencing and bisulfite PCR sequencing (p=0.000). Thus, we demonstrate that integrating transcriptomics and epigenetics in CVID enlightens potential mechanisms of dysfunctional B lymphopoiesis or function.

The Cornell COVID-19 Testing Laboratory: A Model to High-Capacity Testing Hubs for Infectious Disease Emergency Response and Preparedness.

The unprecedented COVID-19 pandemic posed major challenges to local, regional, and global economies and health systems, and fast clinical diagnostic workflows were urgently needed to contain the spread of SARS-CoV-2. Here, we describe the platform and workflow established at the Cornell COVID-19 Testing Laboratory (CCTL) for the high-throughput testing of clinical samples from the university and the surrounding community. This workflow enabled efficient and rapid detection and the successful control of SARS-CoV-2 infection on campus and its surrounding communities. Our cost-effective and fully automated workflow enabled the testing of over 8000 pooled samples per day and provided results for over 2 million samples. The automation of time- and effort-intensive sample processing steps such as accessioning and pooling increased laboratory efficiency. Customized software applications were developed to track and store samples, deconvolute positive pools, track and report results, and for workflow integration from sample receipt to result reporting. Additionally, quality control dashboards and turnaround-time tracking applications were built to monitor assay and laboratory performance. As infectious disease outbreaks pose a constant threat to both human and animal health, the highly effective workflow implemented at CCTL could be modeled to establish regional high-capacity testing hubs for infectious disease preparedness and emergency response.

Decreased infectivity of a neutralization-resistant equine infectious anemia virus variant can be overcome by efficient cell-to-cell spread.

Two variants of equine infectious anemia virus (EIAV) that differed in sensitivity to broadly neutralizing antibody were tested in direct competition assays. No differences were observed in the growth curves and relative fitness scores of EIAVs of principal neutralizing domain variants of groups 1 (EIAV(PND-1)) and 5 (EIAV(PND-5)), respectively; however, the neutralization-resistant EIAV(PND-5) variant was less infectious in single-round replication assays. Infectious center assays indicated similar rates of cell-to-cell spread, which was approximately 1,000-fold more efficient than cell-free infectivity. These data indicate that efficient cell-to-cell spread can overcome the decreased infectivity that may accompany immune escape and should be considered in studies assessing the relative levels of fitness among lentivirus variants, including HIV-1.

Viral RNA Load and Infectivity of SARS-CoV-2 in Paired Respiratory and Oral Specimens from Symptomatic, Asymptomatic, or Postsymptomatic Individuals.

In the present study, we assessed the diagnostic sensitivity and determined the viral RNA load and infectivity of SARS-CoV-2 in paired respiratory (nasopharyngeal and anterior nares) and oral samples (saliva and sublingual swab). Samples were collected from 77 individuals of which 75 were diagnosed with COVID-19 and classified as symptomatic (n = 29), asymptomatic (n = 31), or postsymptomatic (n = 15). Specimens were collected at one time point from each individual, between day 1 and 23 after the initial COVID-19 diagnosis, and included self-collected saliva (S), or sublingual (SL) swab, and bilateral anterior nares (AN) swab, followed by health care provider collected nasopharyngeal (NP) swab. Sixty-three specimen sets were tested using five assay/platforms. The diagnostic sensitivity of each assay/platform and specimen type was determined. Of the 63 specimen sets, SARS-CoV-2 was detected in 62 NP specimens, 52 AN specimens, 59 saliva specimens, and 31 SL specimens by at least one platform. Infectious SARS-CoV-2 was isolated from 21 NP, 13 AN, 12 saliva, and one SL specimen out of 50 specimen sets. SARS-CoV-2 isolation was most successful up to 5 days after initial COVID-19 diagnosis using NP specimens from symptomatic patients (16 of 24 positives, 66.67%), followed by specimens from asymptomatic patients (5 of 17 positives, 29.41%), while it was not very successful with specimens from postsymptomatic patients. Benefits of self-collected saliva and AN specimens balance the loss of sensitivity relative to NP specimens. Therefore, saliva and AN specimens are acceptable alternatives for symptomatic SARS-CoV-2 diagnostic testing or surveillance with increased sampling frequency of asymptomatic individuals. IMPORTANCE The dynamics of infection with SARS-CoV-2 have a significant impact on virus infectivity and in the diagnostic sensitivity of molecular and classic virus detection tests. In the present study we determined the diagnostic sensitivity of paired respiratory (nasopharyngeal and anterior nares swabs) and oral secretions (saliva and sublingual swab) and assessed infectious virus shedding patterns by symptomatic, asymptomatic, or postsymptomatic individuals. Understanding the diagnostic performance of these specimens and the patterns of infectious virus shedding in these bodily secretions provides critical information to control COVID-19, and may help to refine guidelines on isolation and quarantine of positive individuals and their close contacts identified through epidemiological investigations.

Analysis of MHC class I genes across horse MHC haplotypes.

The genomic sequences of 15 horse major histocompatibility complex (MHC) class I genes and a collection of MHC class I homozygous horses of five different haplotypes were used to investigate the genomic structure and polymorphism of the equine MHC. A combination of conserved and locus-specific primers was used to amplify horse MHC class I genes with classical and nonclassical characteristics. Multiple clones from each haplotype identified three to five classical sequences per homozygous animal and two to three nonclassical sequences. Phylogenetic analysis was applied to these sequences, and groups were identified which appear to be allelic series, but some sequences were left ungrouped. Sequences determined from MHC class I heterozygous horses and previously described MHC class I sequences were then added, representing a total of ten horse MHC haplotypes. These results were consistent with those obtained from the MHC homozygous horses alone, and 30 classical sequences were assigned to four previously confirmed loci and three new provisional loci. The nonclassical genes had few alleles and the classical genes had higher levels of allelic polymorphism. Alleles for two classical loci with the expected pattern of polymorphism were found in the majority of haplotypes tested, but alleles at two other commonly detected loci had more variation outside of the hypervariable region than within. Our data indicate that the equine major histocompatibility complex is characterized by variation in the complement of class I genes expressed in different haplotypes in addition to the expected allelic polymorphism within loci.

Development of a DNA microarray for detection of expressed equine classical MHC class I sequences in a defined population.

Development of an accurate and efficient molecular-based equine MHC class I typing method would facilitate the study of T lymphocyte immune responses in horses. Here, a DNA microarray was designed to detect expressed classical MHC class I genes comprising serologically defined equine leukocyte antigen (ELA)-A haplotypes represented in a closed Arabian horse breeding herd. Initially, cloning and sequencing of RT-PCR products were used to identify sequences associated with the ELA-A1, A4, and W11 haplotypes, and one undefined haplotype, in six horses. Subsequently, sequence-specific, conserved (positive control), and random nucleotide (negative control) 23- to 27-mer oligonucleotide microarray probes were designed and spotted onto an epoxy-coated masked slide using a robotic arrayer. Bulk RT-PCR products from each horse were biotinylated by nick translation, hybridized to the array, and detected using tyramide signal amplification. The microarray consistently detected eight of nine classical MHC class I transcripts and allowed ELA haplotypic associations to be made. Cloning and sequencing of RT-PCR products were then performed in a group of ELA disparate horses and ponies, in which six novel sequences were identified. This group was used to determine the specificity of the array. Overall, the microarray was more efficient than cloning and sequencing for detecting expressed classical MHC class I sequences in this defined population of horses, and was significantly more specific than serology. These results confirmed the utility of a microarray-based method for high-resolution MHC class I typing in the horse. With additional probes the array could be useful in a broader population.

Inhibition monitoring in veterinary molecular testing.

Many of the sample matrices typically used for veterinary molecular testing contain inhibitory factors that can potentially reduce analytic sensitivity or produce false-negative results by masking the signal produced by the nucleic acid target. Inclusion of internal controls in PCR-based assays is a valuable strategy not only for monitoring for PCR inhibitors, but also for monitoring nucleic acid extraction efficiency, and for identifying technology errors that may interfere with the ability of an assay to detect the intended target. The Laboratory Technology Committee of the American Association of Veterinary Laboratory Diagnosticians reviewed the different types of internal controls related to monitoring inhibition of PCR-based assays, and provides information here to encourage veterinary diagnostic laboratories to incorporate PCR internal control strategies as a routine quality management component of their molecular testing.

Best practices for performance of real-time PCR assays in veterinary diagnostic laboratories.

The exquisite sensitivity of in vitro amplification assays such as real-time polymerase chain reaction (rtPCR) requires the establishment of thorough and robust laboratory practices. To this end, an American Association of Veterinary Laboratory Diagnosticians (AAVLD) committee of subject matter experts was convened to develop a set of best practices for performance of nucleic acid amplification assays. Consensus advice for the performance of preanalytical, analytical, and postanalytical steps is presented here, along with a review of supporting literature.

Characterization of a novel Mycoplasma cynos real-time PCR assay.

Mycoplasma cynos is recognized as an emerging causative pathogen of canine infectious respiratory disease (CIRD) worldwide. We developed a new open-source real-time PCR (rtPCR) assay for M. cynos that performs well under standard rtPCR conditions. Primers and probes were designed to target the M. cynos tuf gene. Reaction efficiencies for the M. cynos tuf gene assay on 2 platforms were based on amplification of standard curves spanning 8 orders of magnitude: ABI 7500 platform, 94.3-97.9% (r2 ≥ 0.9935); QuantStudio OpenArray platform, 119.1-122.5% (r2 = 0.9784). The assay performed very well over a range of template input, from 109 copies to the lower limit of quantification at 4 copies of the M. cynos genome on the ABI 7500 platform. Diagnostic performance was estimated by comparison with an in-house legacy assay on clinical specimens as well as testing isolates that were characterized previously by intergenic spacer region (ISR) sequencing. Exclusivity was established by testing 12 other Mycoplasma species. To substantiate the high specificity of the M. cynos tuf gene assay, sequence confirmation was performed on ISR PCR amplicons obtained from clinical specimens. One ISR amplicon sequence revealed M. mucosicanis rather than M. cynos. The complete protocol of the newly developed M. cynos tuf assay is provided to facilitate assay harmonization.

Suggested guidelines for validation of real-time PCR assays in veterinary diagnostic laboratories.

This consensus document presents the suggested guidelines developed by the Laboratory Technology Committee (LTC) of the American Association of Veterinary Laboratory Diagnosticians (AAVLD) for development, validation, and modification (methods comparability) of real-time PCR (rtPCR) assays. These suggested guidelines are presented with reference to the World Organisation for Animal Health (OIE) guidelines for validation of nucleic acid detection assays used in veterinary diagnostic laboratories. Additionally, our proposed practices are compared to the guidelines from the Foods Program Regulatory Subdivision of the U.S. Food and Drug Administration (FDA) and from the American Society for Veterinary Clinical Pathology (ASVCP). The LTC suggestions are closely aligned with those from the OIE and comply with version 2021-01 of the AAVLD Requirements for an Accredited Veterinary Medical Diagnostic Laboratory, although some LTC recommendations are more stringent and extend beyond the AAVLD requirements. LTC suggested guidelines are substantially different than the guidelines recently published by the U.S. FDA for validation and modification of regulated tests used for detection of pathogens in pet food and animal-derived products, such as dairy. Veterinary diagnostic laboratories that perform assays from the FDA Bacteriological Analytical Method (BAM) manual must be aware of the different standard.

Active surveillance of pathogens from ticks collected in New York State suburban parks and schoolyards.

Schoolyards and suburban parks are two environments where active tick surveillance may inform local management approaches. Even in a state such as New York with a robust active tick surveillance programme operated by the state Department of Health, these settings are not routinely covered. The goal of this study was to highlight the importance of active surveillance for tick-borne pathogens by describing their prevalence in ticks collected from schoolyards and suburban parks and to guide the use of integrated pest management in these settings. Tick dragging was performed in three regions of New York State: Long Island, the Lower Hudson Valley and the Capital Region. A total of 19 schoolyards and 32 parks were sampled. The location, habitat and weather at the time of tick collection were recorded. Ticks were speciated and tested for the presence of 17 pathogens with a novel application of nanoscale real-time PCR. The causative agents of Lyme disease, anaplasmosis, babesiosis and Powassan virus disease were all detected from Ixodes scapularis in various sites throughout the capital region and south-eastern counties of New York state. The most common agent detected was Borrelia burgdorferi, and coinfection rates were as high as 36%. This surveillance study also captured the first of the invasive Asian longhorned tick species, Haemaphysalis longicornis, in New York state (collected 2 June 2017). Results from this study highlight the importance of collaborative efforts and data sharing for improvement of surveillance for tick-borne disease agents.

Common variable immunodeficiency in horses is characterized by B cell depletion in primary and secondary lymphoid tissues.

INTRODUCTION: Common variable immunodeficiency (CVID) in horse patients is characterized by late-onset B cell lymphopenia or depletion, hypo- or agammaglobulinemia, impaired humoral response to tetanus toxoid vaccination, and recurrent fevers and bacterial infections. DISCUSSION: This study describes the clinical and immunologic findings of 14 affected horses (average age 10.7 +/- 4.4 years) of both genders (six females, eight males) and different breeds (eight Thoroughbreds, four Quarter Horses, one Warmblood, one Pony). Serial immunological testing in peripheral blood revealed persistent, severe B cell lymphopenia (mean 1.3 +/- 2.3% positive cells) in all patients. Serum IgG (range <200 to 800 mg/dL) and IgM (

Equine infectious anemia virus resists the antiretroviral activity of equine APOBEC3 proteins through a packaging-independent mechanism.

Equine infectious anemia virus (EIAV), uniquely among lentiviruses, does not encode a vif gene product. Other lentiviruses, including human immunodeficiency virus type 1 (HIV-1), use Vif to neutralize members of the APOBEC3 (A3) family of intrinsic immunity factors that would otherwise inhibit viral infectivity. This suggests either that equine cells infected by EIAV in vivo do not express active A3 proteins or that EIAV has developed a novel mechanism to avoid inhibition by equine A3 (eA3). Here, we demonstrate that horses encode six distinct A3 proteins, four of which contain a single copy of the cytidine deaminase (CDA) consensus active site and two of which contain two CDA motifs. This represents a level of complexity previously seen only in primates. Phylogenetic analysis of equine single-CDA A3 proteins revealed two proteins related to human A3A (hA3A), one related to hA3C, and one related to hA3H. Both equine double-CDA proteins are similar to hA3F and were named eA3F1 and eA3F2. Analysis of eA3F1 and eA3F2 expression in vivo shows that the mRNAs encoding these proteins are widely expressed, including in cells that are natural EIAV targets. Both eA3F1 and eA3F2 inhibit retrotransposon mobility, while eA3F1 is a potent inhibitor of a Vif-deficient HIV-1 mutant and induces extensive editing of HIV-1 reverse transcripts. However, both eA3F1 and eA3F2 are weak inhibitors of EIAV. Surprisingly, eA3F1 and eA3F2 were packaged into EIAV and HIV-1 virions as effectively as hA3G, although only the latter inhibited EIAV infectivity. Moreover, all three proteins bound both the HIV-1 and EIAV nucleocapsid protein specifically in vitro. It therefore appears that EIAV has evolved a novel mechanism to specifically neutralize the biological activities of the cognate eA3F1 and eA3F2 proteins at a step subsequent to virion incorporation.

Whole-Genome Sequence of the Mycoplasma mucosicanis Type Strain.

Whole-genome sequencing of Mycoplasma mucosicanis type strain 1642 was performed to support efforts to better understand the clinical significance of Mycoplasma infection in canine health. The availability of this sequence will also further the development of highly specific diagnostic tests.

Transcriptome analysis of immune genes in peripheral blood mononuclear cells of young foals and adult horses.

During the neonatal period, the ability to generate immune effector and memory responses to vaccines or pathogens is often questioned. This study was undertaken to obtain a global view of the natural differences in the expression of immune genes early in life. Our hypothesis was that transcriptome analyses of peripheral blood mononuclear cells (PBMCs) of foals (on day 1 and day 42 after birth) and adult horses would show differential gene expression profiles that characterize natural immune processes. Gene ontology enrichment analysis provided assessment of biological processes affected by age, and a list of 897 genes with ≥2 fold higher (p<0.01) expression in day 42 when compared to day 1 foal samples. Up-regulated genes included B cell and T cell receptor diversity genes; DNA replication enzymes; natural killer cell receptors; granzyme B and perforin; complement receptors; immunomodulatory receptors; cell adhesion molecules; and cytokines/chemokines and their receptors. The list of 1,383 genes that had higher (p<0.01) expression on day 1 when compared to day 42 foal samples was populated by genes with roles in innate immunity such as antimicrobial proteins; pathogen recognition receptors; cytokines/chemokines and their receptors; cell adhesion molecules; co-stimulatory molecules; and T cell receptor delta chain. Within the 742 genes with increased expression between day 42 foal and adult samples, B cell immunity was the main biological process (p = 2.4E-04). Novel data on markedly low (p<0.0001) TLR3 gene expression, and high (p≤0.01) expression of IL27, IL13RA1, IREM-1, SIRL-1, and SIRPα on day 1 compared to day 42 foal samples point out potential mechanisms of increased susceptibility to pathogens in early life. The results portray a progression from innate immune gene expression predominance early in life to adaptive immune gene expression increasing with age with a putative overlay of immune suppressing genes in the neonatal phase. These results provide insight to the unique attributes of the equine neonatal and young immune system, and offer many avenues of future investigation.

Effect of a Histone Demethylase Inhibitor on Equine Herpesvirus-1 Activity In Vitro.

Equine herpesvirus type 1 (EHV-1) is a ubiquitous and highly contagious pathogen that causes a range of disease severities with outbreaks of notable economic impact. Given the limitations in immune protection of current vaccines and the limited effectiveness of antiviral drugs on EHV-1 infections in vivo, improved treatment measures are needed to control disease. The use of drugs that alter the epigenetic state of herpes simplex virus genome has been shown to limit viral primary infection and reactivation both in vitro and in vivo. Therefore, we tested the hypothesis that maintaining a repressive epigenetic state on the EHV-1 genome in the host equine cell would decrease viral load during lytic infection. Equine fetal kidney cells (EFKCs) or isolated peripheral blood leukocytes were treated in vitro with (a) the nucleoside analog ganciclovir; (b) the histone demethylase inhibitor OG-L002; (c) both ganciclovir and OG-L002; or (d) dimethyl sulfoxide (DMSO, vehicle control); and then infected with a clinical EHV-1 isolate. Treatment of EFKCs with ganciclovir (mean 22.3 DNA copies per cell, p = 0.0005), OG-L002 (mean 25.6, p = 0.005) or both ganciclovir and OG-L002 (mean 7.1, p = 0.0001) resulted in decreased EHV-1 viral load at 24 h post-infection (hpi) in comparison with DMSO (mean 42.0), with greater impact using the combined treatment. Further, EHV-1 gene expression at 3 hpi decreased when EFKCs were infected in the presence of ganciclovir (p = 0.04) and combined treatment of ganciclovir and OG-L002 (p = 0.0003). In contrast, under similar conditions, neither ganciclovir nor OG-L002 suppressed EHV-1 infection in leukocytes. Differences between cell types, drug penetrance, or drug turnover, may have contributed to the distinct effects observed in this study.