Publications by authors named "Udeni B R Balasuriya"

97 Publications

TOP1 inhibition therapy protects against SARS-CoV-2-induced lethal inflammation.

Cell 2021 Mar 30. Epub 2021 Mar 30.

Department of Genetics and Genomics, Icahn School of Medicine at Mount Sinai, New York, NY, USA; Icahn Institute of Genomics and Multiscale Biology, Icahn School of Medicine at Mount Sinai, New York, NY, USA.

The ongoing pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is currently affecting millions of lives worldwide. Large retrospective studies indicate that an elevated level of inflammatory cytokines and pro-inflammatory factors are associated with both increased disease severity and mortality. Here, using multidimensional epigenetic, transcriptional, in vitro, and in vivo analyses, we report that topoisomerase 1 (TOP1) inhibition suppresses lethal inflammation induced by SARS-CoV-2. Therapeutic treatment with two doses of topotecan (TPT), an FDA-approved TOP1 inhibitor, suppresses infection-induced inflammation in hamsters. TPT treatment as late as 4 days post-infection reduces morbidity and rescues mortality in a transgenic mouse model. These results support the potential of TOP1 inhibition as an effective host-directed therapy against severe SARS-CoV-2 infection. TPT and its derivatives are inexpensive clinical-grade inhibitors available in most countries. Clinical trials are needed to evaluate the efficacy of repurposing TOP1 inhibitors for severe coronavirus disease 2019 (COVID-19) in humans.
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http://dx.doi.org/10.1016/j.cell.2021.03.051DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8008343PMC
March 2021

Pathologic and immunohistochemical findings in an outbreak of systemic toxoplasmosis in a mob of red kangaroos.

J Vet Diagn Invest 2021 May 19;33(3):554-565. Epub 2021 Mar 19.

Louisiana Animal Disease Diagnostic Laboratory and Department of Pathobiological Sciences.

is a zoonotic protozoan pathogen that infects many endothermic vertebrates, including humans; the domestic cat and other felids serve as the definitive host. Macropodids are considered highly susceptible to toxoplasmosis. Here, we describe the clinical, pathologic, and immunohistochemical findings of an outbreak of systemic toxoplasmosis in a mob of 11 red kangaroos (), with high morbidity (73%) and mortality (100%) rates. Affected animals had either severe and rapidly deteriorating clinical conditions or sudden death, which was correlated with widespread necrotizing lesions in multiple organs and intralesional organisms identified via MIC3-specific immunohistochemistry and confirmed by REP529-specific rtPCR. Quantification of parasites demonstrated the highest parasite density in pulmonary parenchyma compared with other tissues. Our study highlights the continued importance of this severe condition in Australian marsupials.
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http://dx.doi.org/10.1177/10406387211001869DOI Listing
May 2021

Experimental re-infected cats do not transmit SARS-CoV-2.

Emerg Microbes Infect 2021 Dec;10(1):638-650

Department of Diagnostic Medicine/Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, KS, USA.

SARS-CoV-2 is the causative agent of COVID-19 and responsible for the current global pandemic. We and others have previously demonstrated that cats are susceptible to SARS-CoV-2 infection and can efficiently transmit the virus to naïve cats. Here, we address whether cats previously exposed to SARS-CoV-2 can be re-infected with SARS-CoV-2. In two independent studies, SARS-CoV-2-infected cats were re-challenged with SARS-CoV-2 at 21 days post primary challenge (DPC) and necropsies performed at 4, 7 and 14 days post-secondary challenge (DP2C). Sentinels were co-mingled with the re-challenged cats at 1 DP2C. Clinical signs were recorded, and nasal, oropharyngeal, and rectal swabs, blood, and serum were collected and tissues examined for histologic lesions. Viral RNA was transiently shed via the nasal, oropharyngeal and rectal cavities of the re-challenged cats. Viral RNA was detected in various tissues of re-challenged cats euthanized at 4 DP2C, mainly in the upper respiratory tract and lymphoid tissues, but less frequently and at lower levels in the lower respiratory tract when compared to primary SARS-CoV-2 challenged cats at 4 DPC. Viral RNA and antigen detected in the respiratory tract of the primary SARS-CoV-2 infected cats at early DPCs were absent in the re-challenged cats. Naïve sentinels co-housed with the re-challenged cats did not shed virus or seroconvert. Together, our results indicate that cats previously infected with SARS-CoV-2 can be experimentally re-infected with SARS-CoV-2; however, the levels of virus shed was insufficient for transmission to co-housed naïve sentinels. We conclude that SARS-CoV-2 infection in cats induces immune responses that provide partial, non-sterilizing immune protection against re-infection.
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http://dx.doi.org/10.1080/22221751.2021.1902753DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8023599PMC
December 2021

Paternally expressed retrotransposon Gag like 1 gene, RTL1, is one of the crucial elements for placental angiogenesis in horses.

Biol Reprod 2021 Mar 8. Epub 2021 Mar 8.

Gluck Equine Research Center, Department of Veterinary Science, University of Kentucky, Lexington, KY, USA, 40503.

RTL1 (retrotransposon Gag like 1) is an essential gene in the development of the human and murine placenta. Several fetal and placental abnormalities such as intrauterine growth restriction (IUGR) and hydrops conditions have been associated with altered expression of this gene. However, the function of RTL1 has not been identified. RTL1 is located on a highly conserved region in eutherian mammals. Therefore, the genetic and molecular analysis in horses could hold important implications for other species, including humans. Here we demonstrated that RTL1 is paternally expressed and is localized within the endothelial cells of the equine (Equus caballus) chorioallantois. We developed an equine placental microvasculature primary cell culture and demonstrated that RTL1 knock-down leads to loss of the sprouting ability of these endothelial cells. We further demonstrated an association between abnormal expression of RTL1 and development of hydrallantois. Our data suggest that RTL1 may be essential for placental angiogenesis, and its abnormal expression can lead to placental insufficiency. This placental insufficiency could be the reason for IUGR and hydrops conditions reported in other species, including humans.
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http://dx.doi.org/10.1093/biolre/ioab039DOI Listing
March 2021

Development and validation of a one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) for rapid detection of ZIKV in patient samples from Brazil.

Sci Rep 2021 Feb 18;11(1):4111. Epub 2021 Feb 18.

Department of Virology, Aggeu Magalhães Institute (IAM), Oswaldo Cruz Foundation (Fiocruz), Avenida Professor Moraes Rego, Recife, Pernambuco, 50670-420, Brazil.

We have previously developed and validated a one-step assay based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) for rapid detection of the Zika virus (ZIKV) from mosquito samples. Patient diagnosis of ZIKV is currently carried out in centralized laboratories using the reverse transcription-quantitative polymerase chain reaction (RT-qPCR), which, while the gold standard molecular method, has several drawbacks for use in remote and low-resource settings, such as high cost and the need of specialized equipment. Point-of-care (POC) diagnostic platforms have the potential to overcome these limitations, especially in low-resource countries where ZIKV is endemic. With this in mind, here we optimized and validated our RT-LAMP assay for rapid detection of ZIKV from patient samples. We found that the assay detected ZIKV from diverse sample types (serum, urine, saliva, and semen) in as little as 20 min, without RNA extraction. The RT-LAMP assay was highly specific and up to 100 times more sensitive than RT-qPCR. We then validated the assay using 100 patient serum samples collected from suspected cases of arbovirus infection in the state of Pernambuco, which was at the epicenter of the last Zika epidemic. Analysis of the results, in comparison to RT-qPCR, found that the ZIKV RT-LAMP assay provided sensitivity of 100%, specificity of 93.75%, and an overall accuracy of 95.00%. Taken together, the RT-LAMP assay provides a straightforward and inexpensive alternative for the diagnosis of ZIKV from patients and has the potential to increase diagnostic capacity in ZIKV-affected areas, particularly in low and middle-income countries.
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http://dx.doi.org/10.1038/s41598-021-83371-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7893177PMC
February 2021

Fatal neuroinvasion of SARS-CoV-2 in K18-hACE2 mice is partially dependent on hACE2 expression.

bioRxiv 2021 Jan 15. Epub 2021 Jan 15.

Animal models recapitulating the distinctive features of severe COVID-19 are critical to enhance our understanding of SARS-CoV-2 pathogenesis. Transgenic mice expressing human angiotensin-converting enzyme 2 (hACE2) under the cytokeratin 18 promoter (K18-hACE2) represent a lethal model of SARS-CoV-2 infection. However, the cause(s) and mechanisms of lethality in this mouse model remain unclear. Here, we evaluated the spatiotemporal dynamics of SARS-CoV-2 infection for up to 14 days post-infection. Despite infection and moderate inflammation in the lungs, lethality was invariably associated with viral neuroinvasion and neuronal damage (including spinal motor neurons). Neuroinvasion occurred following virus transport through the olfactory neuroepithelium in a manner that was only partially dependent on hACE2. Interestingly, SARS-CoV-2 tropism was overall neither widespread among nor restricted to only ACE2-expressing cells. Although our work incites caution in the utility of the K18-hACE2 model to study global aspects of SARS-CoV-2 pathogenesis, it underscores this model as a unique platform for exploring the mechanisms of SARS-CoV-2 neuropathogenesis.

Summary: COVID-19 is a respiratory disease caused by SARS-CoV-2, a betacoronavirus. Here, we show that in a widely used transgenic mouse model of COVID-19, lethality is invariably associated with viral neuroinvasion and the ensuing neuronal disease, while lung inflammation remains moderate.
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http://dx.doi.org/10.1101/2021.01.13.425144DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7814818PMC
January 2021

Topoisomerase 1 inhibition therapy protects against SARS-CoV-2-induced inflammation and death in animal models.

bioRxiv 2020 Dec 1. Epub 2020 Dec 1.

The ongoing pandemic caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is currently affecting millions of lives worldwide. Large retrospective studies indicate that an elevated level of inflammatory cytokines and pro-inflammatory factors are associated with both increased disease severity and mortality. Here, using multidimensional epigenetic, transcriptional, and analyses, we report that Topoisomerase 1 (Top1) inhibition suppresses lethal inflammation induced by SARS-CoV-2. Therapeutic treatment with two doses of Topotecan (TPT), a FDA-approved Top1 inhibitor, suppresses infection-induced inflammation in hamsters. TPT treatment as late as four days post-infection reduces morbidity and rescues mortality in a transgenic mouse model. These results support the potential of Top1 inhibition as an effective host-directed therapy against severe SARS-CoV-2 infection. TPT and its derivatives are inexpensive clinical-grade inhibitors available in most countries. Clinical trials are needed to evaluate the efficacy of repurposing Top1 inhibitors for COVID-19 in humans.
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http://dx.doi.org/10.1101/2020.12.01.404483DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7724667PMC
December 2020

Viral Diseases that Affect Donkeys and Mules.

Animals (Basel) 2020 Nov 25;10(12). Epub 2020 Nov 25.

Laboratório de Retroviroses, Departamento de Medicina Veterinária Preventiva, Escola de Veterinária, Universidade Federal de Minas Gerais, Belo Horizonte 31270-901, Brazil.

Donkeys () and mules represent approximately 50% of the entire domestic equine herd in the world and play an essential role in the lives of thousands of people, primarily in developing countries. Despite their importance, donkeys are currently a neglected and threatened species due to abandonment, indiscriminate slaughter, and a lack of proper sanitary management. Specific knowledge about infectious viral diseases that affect this group of is still limited. In many cases, donkeys and mules are treated like horses, with the physiological differences between these species usually not taken into account. Most infectious diseases that affect the family are exclusive to the family, and they have a tremendous economic impact on the equine industry. However, some viruses may cross the species barrier and affect humans, representing an imminent risk to public health. Nevertheless, even with such importance, most studies are conducted on horses (), and there is little comparative information on infection in donkeys and mules. Therefore, the objective of this article is to provide a brief update on viruses that affect donkeys and mules, thereby compromising their performance and well-being. These diseases may put them at risk of extinction in some parts of the world due to neglect and the precarious conditions they live in and may ultimately endanger other species' health and humans.
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http://dx.doi.org/10.3390/ani10122203DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7760297PMC
November 2020

SARS-CoV-2 infection, disease and transmission in domestic cats.

Emerg Microbes Infect 2020 Dec;9(1):2322-2332

Department of Diagnostic Medicine/Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, KS, USA.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is the cause of Coronavirus Disease 2019 (COVID-19) and responsible for the current pandemic. Recent SARS-CoV-2 susceptibility studies in cats show that the virus can replicate in these companion animals and transmit to other cats. Here, we present an in-depth study of SARS-CoV-2 infection, disease and transmission in domestic cats. Cats were challenged with SARS-CoV-2 via intranasal and oral routes. One day post challenge (DPC), two sentinel cats were introduced. Animals were monitored for clinical signs, clinicopathological abnormalities and viral shedding. examinations were performed at 4, 7 and 21 DPC. Viral RNA was not detected in blood but transiently in nasal, oropharyngeal and rectal swabs and bronchoalveolar lavage fluid as well as various tissues. Tracheobronchoadenitis of submucosal glands with the presence of viral RNA and antigen was observed in airways of the infected cats. Serology showed that both, principals and sentinels, developed antibodies to SARS-CoV-2. All animals were clinically asymptomatic during the course of the study and capable of transmitting SARS-CoV-2 to sentinels. The results of this study are critical for understanding the clinical course of SARS-CoV-2 in a naturally susceptible host species, and for risk assessment.
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http://dx.doi.org/10.1080/22221751.2020.1833687DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7594869PMC
December 2020

Susceptibility of swine cells and domestic pigs to SARS-CoV-2.

Emerg Microbes Infect 2020 Dec;9(1):2278-2288

Center of Excellence for Emerging and Zoonotic Animal Diseases, Department of Diagnostic Medicine/Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, KS, USA.

The emergence of SARS-CoV-2 has resulted in an ongoing global pandemic with significant morbidity, mortality, and economic consequences. The susceptibility of different animal species to SARS-CoV-2 is of concern due to the potential for interspecies transmission, and the requirement for pre-clinical animal models to develop effective countermeasures. In the current study, we determined the ability of SARS-CoV-2 to (i) replicate in porcine cell lines, (ii) establish infection in domestic pigs via experimental oral/intranasal/intratracheal inoculation, and (iii) transmit to co-housed naïve sentinel pigs. SARS-CoV-2 was able to replicate in two different porcine cell lines with cytopathic effects. Interestingly, none of the SARS-CoV-2-inoculated pigs showed evidence of clinical signs, viral replication or SARS-CoV-2-specific antibody responses. Moreover, none of the sentinel pigs displayed markers of SARS-CoV-2 infection. These data indicate that although different porcine cell lines are permissive to SARS-CoV-2, five-week old pigs are not susceptible to infection via oral/intranasal/intratracheal challenge. Pigs are therefore unlikely to be significant carriers of SARS-CoV-2 and are not a suitable pre-clinical animal model to study SARS-CoV-2 pathogenesis or efficacy of respective vaccines or therapeutics.
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http://dx.doi.org/10.1080/22221751.2020.1831405DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7594707PMC
December 2020

Susceptibility of swine cells and domestic pigs to SARS-CoV-2.

bioRxiv 2020 Aug 16. Epub 2020 Aug 16.

The emergence of SARS-CoV-2 has resulted in an ongoing global pandemic with significant morbidity, mortality, and economic consequences. The susceptibility of different animal species to SARS-CoV-2 is of concern due to the potential for interspecies transmission, and the requirement for pre-clinical animal models to develop effective countermeasures. In the current study, we determined the ability of SARS-CoV-2 to (i) replicate in porcine cell lines, (ii) establish infection in domestic pigs via experimental oral/intranasal/intratracheal inoculation, and (iii) transmit to co-housed naive sentinel pigs. SARS-CoV-2 was able to replicate in two different porcine cell lines with cytopathic effects. Interestingly, none of the SARS-CoV-2-inoculated pigs showed evidence of clinical signs, viral replication or SARS-CoV-2-specific antibody responses. Moreover, none of the sentinel pigs displayed markers of SARS-CoV-2 infection. These data indicate that although different porcine cell lines are permissive to SARS-CoV-2, five-week old pigs are not susceptible to infection via oral/intranasal/intratracheal challenge. Pigs are therefore unlikely to be significant carriers of SARS-CoV-2 and are not a suitable pre-clinical animal model to study SARS-CoV-2 pathogenesis or efficacy of respective vaccines or therapeutics.
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http://dx.doi.org/10.1101/2020.08.15.252395DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7430576PMC
August 2020

Detection of SARS-CoV-2 by RNAscope in situ hybridization and immunohistochemistry techniques.

Arch Virol 2020 Oct 5;165(10):2373-2377. Epub 2020 Aug 5.

Louisiana Animal Disease Diagnostic Laboratory, Department of Pathobiological Sciences, School of Veterinary Medicine, Louisiana State University, River Rd, Room 1043, Baton Rouge, LA, 70803, USA.

In situ hybridization (ISH) and immunohistochemistry (IHC) are essential tools to characterize SARS-CoV-2 infection and tropism in naturally and experimentally infected animals and also for diagnostic purposes. Here, we describe three RNAscope-based ISH assays targeting the ORF1ab, spike, and nucleocapsid genes and IHC assays targeting the spike and nucleocapsid proteins of SARS-CoV-2.
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http://dx.doi.org/10.1007/s00705-020-04737-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7406679PMC
October 2020

Type A Influenza Virus Detection from Horses by Real-Time RT-qPCR and Insulated Isothermal RT-PCR.

Methods Mol Biol 2020 ;2123:383-392

Louisiana Animal Disease Diagnostic Laboratory and Department of Pathobiological Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA, USA.

Equine influenza (EI) is a highly contagious disease of horses caused by the equine influenza virus (EIV) H3N8 subtype. EI is the most important respiratory virus infection of horses and can disrupt major equestrian events and cause significant economic losses to the equine industry worldwide. Influenza H3N8 virus spreads rapidly in susceptible horses and can result in very high morbidity within 24-48 h after exposure to the virus. Therefore, rapid and accurate diagnosis of EI is critical for implementation of prevention and control measures to avoid the spread of EIV and to reduce the economic impact of the disease. The probe-based real-time reverse transcriptase polymerase chain reaction (RT-qPCR) assays targeting various EIV genes are reported to be highly sensitive and specific compared to the Directigen Flu-A test and virus isolation in embryonated hens' eggs. Recently, a TaqMan probe-based insulated isothermal RT-PCR (iiRT-PCR) assay for the detection of EIV H3N8 subtype has been described. These molecular-based diagnostic assays provide a fast and reliable means of EIV detection and disease surveillance.
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http://dx.doi.org/10.1007/978-1-0716-0346-8_29DOI Listing
February 2021

RNA Extraction from Equine Samples for Equine Influenza Virus.

Methods Mol Biol 2020 ;2123:369-382

Louisiana Animal Disease Diagnostic Laboratory and Department of Pathobiological Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA, USA.

The primary goals of this chapter are to discuss common viral RNA isolation and purification methods that are routinely used by various diagnostic laboratories and to highlight the advantages and drawbacks of each method and to identify the most suitable and reliable method to increase the sensitivity and specificity of RT-PCR assays for the detection of equine influenza virus (EIV) in clinical specimens. Our experiences and review of literature show that magnetic bead-based nucleic extraction methods (manual and automatic) work well for isolation and purification of EIV RNA from nasal swab specimens. Furthermore, most of the information presented in this chapter could be directly applicable to isolation and purification of nucleic acids (both DNA and RNA) from other equine clinical samples.
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http://dx.doi.org/10.1007/978-1-0716-0346-8_28DOI Listing
February 2021

Genome-wide association study for host genetic factors associated with equine herpesvirus type-1 induced myeloencephalopathy.

Equine Vet J 2020 Nov 6;52(6):794-798. Epub 2020 Apr 6.

Maxwell H. Gluck Equine Research Center, Department of Veterinary Science, University of Kentucky, Lexington, KY, USA.

Background: Equid herpesvirus (EHV-1) infections in horses can lead to equine herpesvirus myeloencephalopathy (EHM), characterised by neurological clinical signs. The sporadic occurrence of the disease in horse herds suggests a host genetic component. A recent study reported an association between the occurrence of EHM and genetic markers on horse chromosome 6 (ECA6).

Objectives: To investigate the association of EHM with genetic host factors, especially with reference to the association reported for ECA6.

Study Design: Genome-wide association study (GWAS) was conducted based on 94 horses that had EHV-1 infections and comparing the 27 developing clinical EHM to the 67 which did not.

Methods: DNA samples were tested from 94 horses for 382,529 single nucleotide polymorphisms (SNPs) with the Affymetrix Axiom 670K SNP array to identify possible associations with EHM. The data analysis included tests for basic, additive, dominant and recessive modes of inheritance, haplotype associations and runs of homozygosity (ROH).

Results: Results from this study did not identify significant SNPs, haplotypes or ROH associations with the development of EHM following EHV-1 infections and excluded the involvement of a recessive genetic factor in the susceptibility to develop EHM.

Main Limitations: Sample size and complex phenotype.

Conclusions: The results exclude the involvement of a recessive genetic factor in the susceptibility to develop clinically apparent EHM but do not have the power to exclude the involvement of other, complex host genetic factors. Furthermore, there was no association between development of EHM and genes on equine chromosome 6, as previously reported.
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http://dx.doi.org/10.1111/evj.13261DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7586946PMC
November 2020

The effect of equine herpesvirus type 4 on type-I interferon signaling molecules.

Vet Immunol Immunopathol 2020 Jan 2;219:109971. Epub 2019 Nov 2.

Maxwell H. Gluck Equine Research Center, Department of Veterinary Science, University of Kentucky, Lexington, KY 40546, USA.

Equine herpesvirus type 4 (EHV-4) is mildly pathogenic but is a common cause of respiratory disease in horses worldwide. We previously demonstrated that unlike EHV-1, EHV-4 is not a potent inducer of type-I IFN and does not suppress that IFN response, especially during late infection, when compared to EHV-1 infection in equine endothelial cells (EECs). Here, we investigated the impact of EHV-4 infection in EECs on type-I IFN signaling molecules at 3, 6, and 12 hpi. Findings from our study revealed that EHV-4 did not induce nor suppress TLR3 and TLR4 expression in EECs at all the studied time points. EHV-4 was able to induce variable amounts of IRF7 and IRF9 in EECs with no evidence of suppressive effect on these important transcription factors of IFN-α/β induction. Intriguingly, EHV-4 did interfere with the phosphorylation of STAT1/STAT2 at 3 hpi and 6 hpi, less so at 12 hpi. An active EHV-4 viral gene expression was required for the suppressive effect of EHV-4 on STAT1/STAT2 phosphorylation during early infection. One or more early viral genes of EHV-4 are involved in the suppression of STAT1/STAT2 phosphorylation observed during early time points in EHV-4-infected EECs. The inability of EHV-4 to significantly down-regulate key molecules of type-I IFN signaling may be related to the lower severity of pathogenesis when compared with EHV-1. Harnessing this knowledge may prove useful in controlling future outbreaks of the disease.
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http://dx.doi.org/10.1016/j.vetimm.2019.109971DOI Listing
January 2020

Equid Herpesvirus 1 Targets the Sensitization and Induction Steps To Inhibit the Type I Interferon Response in Equine Endothelial Cells.

J Virol 2019 12 13;93(23). Epub 2019 Nov 13.

Maxwell H. Gluck Equine Research Center, Department of Veterinary Science, University of Kentucky, Lexington, Kentucky, USA.

Equid herpesvirus 1 (EHV-1) is a viral pathogen of horse populations worldwide spread by the respiratory route and is known for causing outbreaks of neurologic syndromes and abortion storms. Previously, we demonstrated that an EHV-1 strain of the neuropathogenic genotype, T953, downregulates the beta interferon (IFN-β) response in equine endothelial cells (EECs) at 12 h postinfection (hpi). In the present study, we explored the molecular correlates of this inhibition as clues toward an understanding of the mechanism. Data from our study revealed that EHV-1 infection of EECs significantly reduced both Toll-like receptor 3 (TLR3) and TLR4 mRNA expression at 6 hpi and 12 hpi. While EHV-1 was able to significantly reduce IRF9 mRNA at both 6 hpi and 12 hpi, the virus significantly reduced IFN regulatory factor 7 (IRF7) mRNA only at 12 hpi. EHV-1 did not alter the cellular level of Janus-activated kinase 1 (JAK1) at any time point. However, EHV-1 reduced the cellular level of expression of tyrosine kinase 2 (TYK2) at 12 hpi. Downstream of JAK1-TYK2 signaling, EHV-1 blocked the phosphorylation and activation of signal transducer and activator of transcription 2 (STAT2) when coincubated with exogenous IFN, at 12 hpi, although not at 3 or 6 hpi. Immunofluorescence staining revealed that the virus prevented the nuclear translocation of STAT2 molecules, confirming the virus-mediated inhibition of STAT2 activation. The pattern of suppression of phosphorylation of STAT2 by EHV-1 implicated viral late gene expression. These data help illuminate how EHV-1 strategically inhibits the host innate immune defense by limiting steps required for type I IFN sensitization and induction. To date, no commercial vaccine label has a claim to be fully protective against the diseases caused by equid herpesvirus 1 (EHV-1), especially the neurologic form. The interferon (IFN) system, of which type I IFN is of great importance, still remains a viable immunotherapeutic option against EHV-1 infection. The type I IFN system has been exploited successfully to treat other viral infections, such as chronic hepatitis B and C in humans. The current state of research on how EHV-1 interferes with the protective effect of type I IFN has indicated transient induction of type I IFN production followed by a rapid shutdown in equine endothelial cells (EECs). The significance of our study is the identification of certain steps in the type I IFN signaling pathway targeted for inhibition by EHV-1. Understanding this pathogen-host relationship is essential for the long-term goal of developing effective immunotherapy against EHV-1.
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http://dx.doi.org/10.1128/JVI.01342-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6854505PMC
December 2019

Equine arteritis virus long-term persistence is orchestrated by CD8+ T lymphocyte transcription factors, inhibitory receptors, and the CXCL16/CXCR6 axis.

PLoS Pathog 2019 07 29;15(7):e1007950. Epub 2019 Jul 29.

Louisiana Animal Disease Diagnostic Laboratory and Department of Pathobiological Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA, United States of America.

Equine arteritis virus (EAV) has the unique ability to establish long-term persistent infection in the reproductive tract of stallions and be sexually transmitted. Previous studies showed that long-term persistent infection is associated with a specific allele of the CXCL16 gene (CXCL16S) and that persistence is maintained despite the presence of local inflammatory and humoral and mucosal antibody responses. Here, we performed transcriptomic analysis of the ampullae, the primary site of EAV persistence in long-term EAV carrier stallions, to understand the molecular signatures of viral persistence. We demonstrated that the local CD8+ T lymphocyte response is predominantly orchestrated by the transcription factors eomesodermin (EOMES) and nuclear factor of activated T-cells cytoplasmic 2 (NFATC2), which is likely modulated by the upregulation of inhibitory receptors. Most importantly, EAV persistence is associated with an enhanced expression of CXCL16 and CXCR6 by infiltrating lymphocytes, providing evidence of the implication of this chemokine axis in the pathogenesis of persistent EAV infection in the stallion reproductive tract. Furthermore, we have established a link between the CXCL16 genotype and the gene expression profile in the ampullae of the stallion reproductive tract. Specifically, CXCL16 acts as a "hub" gene likely driving a specific transcriptional network. The findings herein are novel and strongly suggest that RNA viruses such as EAV could exploit the CXCL16/CXCR6 axis in order to modulate local inflammatory and immune responses in the male reproductive tract by inducing a dysfunctional CD8+ T lymphocyte response and unique lymphocyte homing in the reproductive tract.
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http://dx.doi.org/10.1371/journal.ppat.1007950DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6692045PMC
July 2019

Development and evaluation of a one-step multiplex real-time TaqMan RT-qPCR assay for the detection and genotyping of equine G3 and G14 rotaviruses in fecal samples.

Virol J 2019 04 25;16(1):49. Epub 2019 Apr 25.

Louisiana Animal Disease Diagnostic Laboratory and Department of Pathobiological Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA, USA.

Background: Equine rotavirus A (ERVA) is the leading cause of diarrhea in neonatal foals and has a negative impact on equine breeding enterprises worldwide. Among ERVA strains infecting foals, the genotypes G3P[12] and G14P[12] are the most prevalent, while infections by strains with other genomic arrangements are infrequent. The identification of circulating strains of ERVA is critical for diagnostic and surveillance purposes, as well as to understand their molecular epidemiology. Current genotyping methods available for ERVA and rotaviruses affecting other animal species rely on Sanger sequencing and are significantly time-consuming, costly and labor intensive. Here, we developed the first one-step multiplex TaqMan real-time reverse transcription polymerase chain reaction (RT-qPCR) assay targeting the NSP3 and VP7 genes of ERVA G3 and G14 genotypes for the rapid detection and G-typing directly from fecal specimens.

Methods: A one-step multiplex TaqMan RT-qPCR assay targeting the NSP3 and VP7 genes of ERVA G3 and G14 genotypes was designed. The analytical sensitivity was assessed using serial dilutions of in vitro transcribed RNA containing the target sequences while the analytical specificity was determined using RNA and DNA derived from a panel of group A rotaviruses along with other equine viruses and bacteria. The clinical performance of this multiplex assay was evaluated using a panel of 177 fecal samples and compared to a VP7-specific standard RT-PCR assay and Sanger sequencing. Limits of detection (LOD), sensitivity, specificity, and agreement were determined.

Results: The multiplex G3 and G14 VP7 assays demonstrated high specificity and efficiency, with perfect linearity. A 100-fold difference in their analytical sensitivity was observed when compared to the singleplex assays; however, this difference did not have an impact on the clinical performance. Clinical performance of the multiplex RT-qPCR assay demonstrated that this assay had a high sensitivity/specificity for every target (100% for NSP3, > 90% for G3 VP7 and > 99% for G14 VP7, respectively) and high overall agreement (> 98%) compared to conventional RT-PCR and sequencing.

Conclusions: This new multiplex RT-qPCR assay constitutes a useful, very reliable tool that could significantly aid in the rapid detection and G-typing of ERVA strains circulating in the field.
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http://dx.doi.org/10.1186/s12985-019-1149-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6482509PMC
April 2019

Intrahost Selection Pressure Drives Equine Arteritis Virus Evolution during Persistent Infection in the Stallion Reproductive Tract.

J Virol 2019 06 29;93(12). Epub 2019 May 29.

Louisiana Animal Disease Diagnostic Laboratory and Department of Pathobiological Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, Louisiana, USA

Equine arteritis virus (EAV) is the causative agent of equine viral arteritis (EVA), a reproductive and respiratory disease of horses. Following natural infection, 10 to 70% of infected stallions can become carriers of EAV and continue to shed virus in the semen. In this study, sequential viruses isolated from nasal secretions, buffy coat cells, and semen of seven experimentally infected and two naturally infected EAV carrier stallions were deep sequenced to elucidate the intrahost microevolutionary process after a single transmission event. Analysis of variants from nasal secretions and buffy coat cells lacked extensive positive selection; however, characteristics of the mutant spectra were different in the two sample types. In contrast, the initial semen virus populations during acute infection have undergone a selective bottleneck, as reflected by the reduction in population size and diversifying selection at multiple sites in the viral genome. Furthermore, during persistent infection, extensive genome-wide purifying selection shaped variant diversity in the stallion reproductive tract. Overall, the nonstochastic nature of EAV evolution during persistent infection was driven by active intrahost selection pressure. Among the open reading frames within the viral genome, ORF3, ORF5, and the nsp2-coding region of ORF1a accumulated the majority of nucleotide substitutions during persistence, with ORF3 and ORF5 having the highest intrahost evolutionary rates. The findings presented here provide a novel insight into the evolutionary mechanisms of EAV and identified critical regions of the viral genome likely associated with the establishment and maintenance of persistent infection in the stallion reproductive tract. EAV can persist in the reproductive tract of infected stallions, and consequently, long-term carrier stallions constitute its sole natural reservoir. Previous studies demonstrated that the ampullae of the vas deferens are the primary site of viral persistence in the stallion reproductive tract and the persistence is associated with a significant inflammatory response that is unable to clear the infection. This is the first study that describes EAV full-length genomic evolution during acute and long-term persistent infection in the stallion reproductive tract using next-generation sequencing and contemporary sequence analysis techniques. The data provide novel insight into the intrahost evolution of EAV during acute and persistent infection and demonstrate that persistent infection is characterized by extensive genome-wide purifying selection and a nonstochastic evolutionary pattern mediated by intrahost selective pressure, with important nucleotide substitutions occurring in ORF1a (region encoding nsp2), ORF3, and ORF5.
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http://dx.doi.org/10.1128/JVI.00045-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6613756PMC
June 2019

Detection, molecular characterization and phylogenetic analysis of G3P[12] and G14P[12] equine rotavirus strains co-circulating in central Kentucky.

Virus Res 2018 08 1;255:39-54. Epub 2018 Jun 1.

Maxwell H. Gluck Equine Research Center, Department of Veterinary Science, University of Kentucky, Lexington, KY, USA. Electronic address:

Equine rotavirus A (ERVA) is the leading cause of diarrhea in neonatal foals and a major health problem to the equine breeding industry worldwide. The G3P[12] and G14P[12] ERVA genotypes are the most prevalent in foals with diarrhea. Control and prevention strategies include vaccination of pregnant mares with an inactivated vaccine containing a prototype ERVA G3P[12] strain with limited and controversial field efficacy. Here, we performed the molecular characterization of ERVA strains circulating in central Kentucky using fecal samples collected during the 2017 foaling season. The data indicated for the first time that the G14P[12] genotype is predominant in this region in contrast to a previous serotyping study where only G3 genotype strains were reported. Overall, analysis of antigenic sites in the VP7 protein demonstrated the presence of several amino acid substitutions in the epitopes exposed on the surface including a non-conserved N-linked glycosylation site (D123N) in G14P[12] strains, while changes in antigenic sites of VP8* were minor. Also, we report the successful isolation of three ERVA G14P[12] strains which presented a high identity with other G14 strains from around the world. These may constitute ideal reference strains to comparatively study the molecular biology of G3 and G14 strains and perform vaccine efficacy studies following heterologous challenge in the future.
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http://dx.doi.org/10.1016/j.virusres.2018.05.025DOI Listing
August 2018

Systemic equid alphaherpesvirus 9 in a Grant's zebra.

J Vet Diagn Invest 2018 Jul 12;30(4):580-583. Epub 2018 Apr 12.

California Animal Health and Food Safety Laboratory, Tulare Laboratory (Moeller), School of Veterinary Medicine, University of California, Davis, CA.

A 2-y-old female Grant's zebra ( Equus quagga [ burchellii] boehmi) was presented with a clinical history of depression, anorexia, and weakness of 1-wk duration. Postmortem examination identified ulcers on the tongue and palate; a large abscess adjacent to the larynx; left lung consolidation; mild swelling, darkening, and congestion of the liver with accentuation of the lobular pattern; and edema and congestion of the distal small and large intestines. Histologic examination identified necrotizing bronchopneumonia, necrotizing hepatitis, nephritis, and enterocolitis. Eosinophilic intranuclear inclusions were detected in syncytial cells and degenerate bronchial epithelium in the lungs and in some hepatocytes associated with necrotic foci. Bacterial cultures of the lung, liver, and laryngeal abscess failed to detect any significant pathogen. Lung and liver tested positive for equine herpesvirus with neuropathogenic marker by real-time PCR. Subsequently, equine herpesvirus was isolated in tissue culture, and the entire viral DNA polymerase gene (ORF30) was sequenced. The zebra lung isolate had a very close nucleotide and amino acid sequence identity to equid alphaherpesvirus 9 (EHV-9; 99.6% and 99.8%, respectively) in contrast to the neuropathogenic T953 strain of EHV-1 (94.7% and 96.6%, respectively). Although zebras are considered the natural host for EHV-9, we document an unusual acute systemic, fatal EHV-9 infection in a 2-y-old Grant's zebra.
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http://dx.doi.org/10.1177/1040638718767722DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6505909PMC
July 2018

Absence of relationship between type-I interferon suppression and neuropathogenicity of EHV-1.

Vet Immunol Immunopathol 2018 Mar;197:24-30

Maxwell H. Gluck Equine Research Center, Department of Veterinary Science, University of Kentucky, Lexington, KY 40546-0099, USA.

Equine herpesvirus-1 (EHV-1) infection is an important and highly prevalent disease in equine populations worldwide. Previously we have demonstrated that a neuropathogenic strain of EHV-1, T953, suppresses the host cell's antiviral type-I interferon (IFN) response in vitro. Whether or not this is unique to EHV-1 strains possessing the neuropathogenic genotype has been undetermined. Here, we examined whether there is any direct relationship between neuropathogenic genotype and the induced IFN-β response in equine endothelial cells (EECs) infected with 10 different strains of EHV-1. The extent of virus cell-to-cell spread following infection in EECs was also compared between the neuropathogenic and the non-neuropathogenic genotype of EHV-1. We then compared IFN-β and the total type-I IFN protein suppression between T953, an EHV-1 strain that is neuropathogenic and T445, an EHV-4 strain mainly associated only with respiratory disease. Data from our study revealed no relationship between the neuropathogenic genotype of EHV-1 and the induced IFN-β mRNA by the host cell. Results also indicate no statistically significant difference in plaque sizes of both genotypes of EHV-1 produced in EECs. However, while the T953 strain of EHV-1 was able to suppress IFN-β mRNA and type-I IFN biological activity at 12 h post-infection (hpi), EHV-4 weakly induces both IFN-β mRNA and type-I IFN biological activity. This finding correlated with a statistically significant difference in the mean plaque sizes produced by the two EHV subtypes in EECs. Our data help illuminate how EHV-1, irrespective of its genotype, evades the host cell's innate immune response thereby enabling viral spread to susceptible cells.
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http://dx.doi.org/10.1016/j.vetimm.2018.01.007DOI Listing
March 2018

Downregulation of MicroRNA eca-mir-128 in Seminal Exosomes and Enhanced Expression of CXCL16 in the Stallion Reproductive Tract Are Associated with Long-Term Persistence of Equine Arteritis Virus.

J Virol 2018 05 13;92(9). Epub 2018 Apr 13.

Maxwell H. Gluck Equine Research Center, Department of Veterinary Science, University of Kentucky, Lexington, Kentucky, USA

Equine arteritis virus (EAV) can establish long-term persistent infection in the reproductive tract of stallions and is shed in the semen. Previous studies showed that long-term persistence is associated with a specific allele of the gene () and that persistent infection is maintained despite the presence of a local inflammatory and humoral and mucosal antibody responses. In this study, we demonstrated that equine seminal exosomes (SEs) are enriched in a small subset of microRNAs (miRNAs). Most importantly, we demonstrated that long-term EAV persistence is associated with the downregulation of an SE-associated miRNA (eca-mir-128) and with an enhanced expression of CXCL16 in the reproductive tract, a putative target of eca-mir-128. The findings presented here suggest that SE eca-mir-128 is implicated in the regulation of the CXCL16/CXCR6 axis in the reproductive tract of persistently infected stallions, a chemokine axis strongly implicated in EAV persistence. This is a novel finding and warrants further investigation to identify its specific mechanism in modulating the CXCL16/CXCR6 axis in the reproductive tract of the EAV long-term carrier stallion. Equine arteritis virus (EAV) has the ability to establish long-term persistent infection in the stallion reproductive tract and to be shed in semen, which jeopardizes its worldwide control. Currently, the molecular mechanisms of viral persistence are being unraveled, and these are essential for the development of effective therapeutics to eliminate persistent infection. Recently, it has been determined that long-term persistence is associated with a specific allele of the gene () and is maintained despite induction of local inflammatory, humoral, and mucosal antibody responses. This study demonstrated that long-term persistence is associated with the downregulation of seminal exosome miRNA eca-mir-128 and enhanced expression of its putative target, CXCL16, in the reproductive tract. For the first time, this study suggests complex interactions between eca-mir-128 and cellular elements at the site of EAV persistence and implicates this miRNA in the regulation of the CXCL16/CXCR6 axis in the reproductive tract during long-term persistence.
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http://dx.doi.org/10.1128/JVI.00015-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5899189PMC
May 2018

Evaluation of a field-deployable reverse transcription-insulated isothermal PCR for rapid and sensitive on-site detection of Zika virus.

BMC Infect Dis 2017 12 19;17(1):778. Epub 2017 Dec 19.

Maxwell H. Gluck Equine Research Center, Department of Veterinary Science, College of Agriculture, Food and Environment, University of Kentucky, Lexington, KY, USA.

Background: The recent emergence of Zika virus (ZIKV) in Brazil and its precipitous expansion throughout the Americas has highlighted the urgent need for a rapid and reliable on-site diagnostic assay suitable for viral detection. Such point-of-need (PON), low-cost diagnostics are essential for ZIKV control in vulnerable areas with limited resources.

Methods: We developed and evaluated a ZIKV-specific field-deployable RT-iiPCR reagent set targeting the E gene for rapid detection of ZIKV in ZIKV-spiked human and mosquito specimens, and compared its performance to the Center for Disease Control and Prevention (CDC) and Pan American Health Organization (PAHO) RT-qPCR assays targeting the E and NS2B genes, respectively.

Results: These assays demonstrated exclusive specificity for ZIKV (African and Asian lineages), had limits of detection ranging from 10 to 100 in vitro transcribed RNA copies/μl and detection endpoints at 10 plaque forming units/ml of infectious tissue culture fluid. Analysis of human whole blood, plasma, serum, semen, urine, and mosquito pool samples spiked with ZIKV showed an agreement of 90% (k = 0.80), 92% (k = 0.82), 95% (k = 0.86), 92% (k = 0.81), 90% (k = 0.79), and 100% (k = 1), respectively, between the RT-iiPCR assay and composite results from the reference RT-qPCR assays. Overall, the concurrence between the ZIKV RT-iiPCR and the reference RT-qPCR assays was 92% (k = 0.83).

Conclusions: The ZIKV RT-iiPCR has a performance comparable to the reference CDC and PAHO RT-qPCR assays but provides much faster results (~1.5 h) with a field-deployable system that can be utilized as a PON diagnostic with the potential to significantly improve the quality of the health care system in vulnerable areas.
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http://dx.doi.org/10.1186/s12879-017-2852-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5735522PMC
December 2017

Equine Arteritis Virus Elicits a Mucosal Antibody Response in the Reproductive Tract of Persistently Infected Stallions.

Clin Vaccine Immunol 2017 Oct 5;24(10). Epub 2017 Oct 5.

Maxwell H. Gluck Equine Research Center, Department of Veterinary Science, University of Kentucky, Lexington, Kentucky, USA

Equine arteritis virus (EAV) has the ability to establish persistent infection in the reproductive tract of the stallion (carrier) and is continuously shed in its semen. We have recently demonstrated that EAV persists within stromal cells and a subset of lymphocytes in the stallion accessory sex glands in the presence of a significant local inflammatory response. In the present study, we demonstrated that EAV elicits a mucosal antibody response in the reproductive tract during persistent infection with homing of plasma cells into accessory sex glands. The EAV-specific immunoglobulin isotypes in seminal plasma included IgA, IgG1, IgG3/5, and IgG4/7. Interestingly, seminal plasma IgG1 and IgG4/7 possessed virus-neutralizing activity, while seminal plasma IgA and IgG3/5 did not. However, virus-neutralizing IgG1 and IgG4/7 in seminal plasma were not effective in preventing viral infectivity. In addition, the serological response was primarily mediated by virus-specific IgM and IgG1, while virus-specific serum IgA, IgG3/5, IgG4/7, and IgG6 isotype responses were not detected. This is the first report characterizing the immunoglobulin isotypes in equine serum and seminal plasma in response to EAV infection. The findings presented herein suggest that while a broader immunoglobulin isotype diversity is elicited in seminal plasma, EAV has the ability to persist in the reproductive tract, in spite of local mucosal antibody and inflammatory responses. This study provides further evidence that EAV employs complex immune evasion mechanisms during persistence in the reproductive tract that warrant further investigation.
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http://dx.doi.org/10.1128/CVI.00215-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5629664PMC
October 2017

Evaluation and Clinical Validation of Two Field-Deployable Reverse Transcription-Insulated Isothermal PCR Assays for the Detection of the Middle East Respiratory Syndrome-Coronavirus.

J Mol Diagn 2017 11 12;19(6):817-827. Epub 2017 Aug 12.

Maxwell H. Gluck Equine Research Center, Department of Veterinary Science, College of Agriculture, Food and Environment and Department of Microbiology, Immunology and Molecular Genetics, College of Medicine, University of Kentucky, Lexington, Kentucky. Electronic address:

Middle East respiratory syndrome (MERS) is an emerging zoonotic viral respiratory disease that was first identified in Saudi Arabia in 2012. In 2015, the largest MERS outbreak outside of the Middle East region occurred in the Republic of Korea. The rapid nosocomial transmission of MERS-coronavirus (MERS-CoV) in Korean health care settings highlighted the importance and urgent need for a rapid and reliable on-site diagnostic assay to implement effective control and preventive measures. Here, the evaluation and validation of two newly developed reverse transcription-insulated isothermal PCR (RT-iiPCR) methods targeting the ORF1a and upE genes of MERS-CoV are described. Compared with World Health Organization-recommended singleplex real-time quantitative RT-PCR (RT-qPCR) assays, both RT-iiPCR assays had comparable analytical sensitivity for the detection of MERS-CoV RNA in tissue culture fluid and in sputum samples spiked with infectious virus. Furthermore, clinical evaluation was performed with sputum samples collected from subjects with acute and chronic respiratory illnesses, including patients infected with MERS-CoV. The overall agreement values between the two RT-iiPCR assays and the reference RT-qPCR assays were 98.06% (95% CI, 94.43%-100%; κ = 0.96) and 99.03% (95% CI, 95.88%-100%; κ = 0.99) for ORF1a and upE assays, respectively. The ORF1a and upE MERS-CoV RT-iiPCR assays coupled with a field-deployable system provide a platform for a highly sensitive and specific on-site tool for diagnosis of MERS-CoV infections.
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http://dx.doi.org/10.1016/j.jmoldx.2017.06.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7106309PMC
November 2017

Development and Characterization of an Infectious cDNA Clone of Equine Arteritis Virus.

Methods Mol Biol 2017 ;1602:11-28

Department of Veterinary Diagnostic and Production Animal Medicine, College of Veterinary Medicine, Iowa State University, Ames, IA, 50011, USA.

Development and characterization of several infectious cDNA clones of equine arteritis virus (EAV) have been described in the literature. Here we describe the assembly of the full-length infectious cDNA clone of the virulent Bucyrus strain (VBS; ATCC VR-796) of EAV in a plasmid vector. This system allows generation of infectious in vitro-transcribed (IVT) RNA from the linearized plasmid that can be transfected or electroporated into mammalian cells to produce infectious recombinant progeny virus. This is an efficient reverse genetics system that allows easy manipulation of EAV genomes to study molecular biology of the virus and pathogenesis of equine viral arteritis.
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http://dx.doi.org/10.1007/978-1-4939-6964-7_2DOI Listing
March 2018

Equine Arteritis Virus Has Specific Tropism for Stromal Cells and CD8 T and CD21 B Lymphocytes but Not for Glandular Epithelium at the Primary Site of Persistent Infection in the Stallion Reproductive Tract.

J Virol 2017 07 9;91(13). Epub 2017 Jun 9.

Department of Veterinary Science, Maxwell H. Gluck Equine Research Center, College of Agriculture, Food and Environment, University of Kentucky, Lexington, Kentucky, USA

Equine arteritis virus (EAV) has a global impact on the equine industry as the causative agent of equine viral arteritis (EVA), a respiratory, systemic, and reproductive disease of equids. A distinctive feature of EAV infection is that it establishes long-term persistent infection in 10 to 70% of infected stallions (carriers). In these stallions, EAV is detectable only in the reproductive tract, and viral persistence occurs despite the presence of high serum neutralizing antibody titers. Carrier stallions constitute the natural reservoir of the virus as they continuously shed EAV in their semen. Although the accessory sex glands have been implicated as the primary sites of EAV persistence, the viral host cell tropism and whether viral replication in carrier stallions occurs in the presence or absence of host inflammatory responses remain unknown. In this study, dual immunohistochemical and immunofluorescence techniques were employed to unequivocally demonstrate that the ampulla is the main EAV tissue reservoir rather than immunologically privileged tissues (i.e., testes). Furthermore, we demonstrate that EAV has specific tropism for stromal cells (fibrocytes and possibly tissue macrophages) and CD8 T and CD21 B lymphocytes but not glandular epithelium. Persistent EAV infection is associated with moderate, multifocal lymphoplasmacytic ampullitis comprising clusters of B (CD21) lymphocytes and significant infiltration of T (CD3, CD4, CD8, and CD25) lymphocytes, tissue macrophages, and dendritic cells (Iba-1 and CD83), with a small number of tissue macrophages expressing CD163 and CD204 scavenger receptors. This study suggests that EAV employs complex immune evasion mechanisms that warrant further investigation. The major challenge for the worldwide control of EAV is that this virus has the distinctive ability to establish persistent infection in the stallion's reproductive tract as a mechanism to ensure its maintenance in equid populations. Therefore, the precise identification of tissue and cellular tropism of EAV is critical for understanding the molecular basis of viral persistence and for development of improved prophylactic or treatment strategies. This study significantly enhances our understanding of the EAV carrier state in stallions by unequivocally identifying the ampullae as the primary sites of viral persistence, combined with the fact that persistence involves continuous viral replication in fibrocytes (possibly including tissue macrophages) and T and B lymphocytes in the presence of detectable inflammatory responses, suggesting the involvement of complex viral mechanisms of immune evasion. Therefore, EAV persistence provides a powerful new natural animal model to study RNA virus persistence in the male reproductive tract.
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http://dx.doi.org/10.1128/JVI.00418-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5469258PMC
July 2017

Translation of a laboratory-validated equine herpesvirus-1 specific real-time PCR assay into an insulated isothermal polymerase chain reaction (iiPCR) assay for point-of-need diagnosis using POCKIT™ nucleic acid analyzer.

J Virol Methods 2017 03 16;241:58-63. Epub 2016 Dec 16.

Animal Disease Diagnostic Laboratory, Ohio Department of Agriculture, Reynoldsburg, OH, USA. Electronic address:

Equine herpesvirus myeloencephalopathy (EHM), a major problem for the equine industry in the United States, is caused by equine herpesvirus-1 (EHV-1). In addition, EHV-1 is associated with upper respiratory disease, abortion, and chorioretinal lesions in horses. Here we describe the development and evaluation of an inexpensive, user-friendly insulated isothermal PCR (iiPCR) method targeting open reading 30 (ORF30) to detect both neuropathogenic and non-neuropathogenic strains on the field-deployable POCKIT™ device for point-of-need detection of EHV-1. The analytical sensitivity of the EHV-1 iiPCR assay was 13 genome equivalents per reaction. The assay did not cross react with ten non-target equine viral pathogens. Performance of the EHV-1 iiPCR assay was compared to two previously described real-time PCR (qPCR) assays in two laboratories by using 104 archived clinical samples. All 53 qPCR-positive and 46 of the 51 qPCR-negative samples tested positive and negative, respectively, by the iiPCR. The agreement between the two assays was 95.19% (confidence interval 90.48-99.90%) with a kappa value of 0.90. In conclusion, the newly developed EHV-1 iiPCR assay is robust to provide specificity and sensitivity comparable to qPCR assays for the detection of EHV-1 nucleic acid in clinical specimens.
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http://dx.doi.org/10.1016/j.jviromet.2016.12.010DOI Listing
March 2017