Abstract
Equid alphaherpesvirus 1 (EqAHV1; Orthoherpesviridae, Varicellovirus equidalpha1) spreads by viremia to susceptible organs. Because EqAHV1 circulates in the bloodstream in a cell-associated manner, serum samples are not considered valuable for detecting EqAHV1 and have therefore not been tested by highly sensitive detection methods such as real-time PCR (rtPCR). We investigated whether EqAHV1 could be detected by this method in equine serum samples. We performed rtPCR on archived sera and peripheral blood mononuclear cells (PBMCs) collected from 3 horses experimentally inoculated with EqAHV1. Acute-phase field sera from 40 febrile horses, including 11 positive for EqAHV1 on antibody ELISA, were also tested by both standard rtPCR and direct rtPCR without nucleic acid purification. EqAHV1 was detected by standard rtPCR in the PBMCs of the experimentally infected horses for 3–6 d and in the serum of these horses for 5–7 d. Six of the 11 ELISA-positive acute-phase field sera were positive on standard rtPCR, whereas the remaining were negative. All 6 of these samples were positive on direct rtPCR without nucleic acid purification. These results suggest that serum samples can be used to detect EqAHV1; however, false-negatives may result from low viral gene copy numbers.
The cycle of latent infection and reactivation with viral shedding that is characteristic of herpesviruses allows equid alphaherpesvirus 1 (EqAHV1; Orthoherpesviridae, Varicellovirus equidalpha1; syn. equine herpesvirus 1, equine abortion virus) to become endemic in horse populations worldwide. 3 EqAHV1 is one of the most important equine viral pathogens and leads to severe economic losses in the equine industry. 16 EqAHV1 causes respiratory disease, abortion, neonatal death, and equine herpesvirus myeloencephalopathy (EHM).2,4,17 The virus enters the body through the nasal cavity and replicates in the nasopharyngeal mucosa. 15 Peripheral blood mononuclear cells (PBMCs) in the lymph nodes draining the respiratory tract, which are the secondary multiplication sites of the virus, are infected with EqAHV1, and carry the virus to susceptible organs via cell-associated viremia. 14 Viral infection of the pregnant uterus can cause abortion, and infection of the CNS can cause EHM.9,10,20
Detection of EqAHV1 viremia is essential for confirming infection and estimating the prognosis of the disease. 7 Anticoagulated blood, such as EDTA-treated blood, is needed to collect PBMCs for the diagnosis of EqAHV1 cell-associated viremia. However, when a respiratory infection other than EqAHV1 infection—such as equine influenza—is suspected as the cause of a fever, anticoagulated blood may not be collected for diagnostic purposes on the first sampling occasion; instead, acute-phase sera are usually collected initially for serum biochemistry tests, and convalescent sera are collected later for subsequent serologic testing. Arthropod-borne virus infections, such as equine infectious anemia, Japanese encephalitis, and Getah virus infection, cause viremia, and in these diseases, both serum and plasma samples are used for virus detection.13,18,19 In contrast, in EqAHV1 infection, serum samples do not have enough cells, such as PBMCs, to detect viremia, and these samples are therefore considered of no value for detecting EqAHV1. However, the EqAHV1 viral load in serum samples has not been thoroughly investigated by using a highly sensitive detection method such as real-time PCR (rtPCR). Successful direct detection of pathogens by using untreated serum samples as PCR templates has been reported in hepatitis B, C, and G and bovine viral diarrhea.1,6 Here, we evaluated the usefulness of equine serum samples with or without nucleic acid purification for the detection of viremia during EqAHV1 infection.
The experimental serum and PBMC samples originated from our horse study reported in 2018. 8 Three female Thoroughbred horses (20-, 22-, and 23-mo-old) were inoculated intranasally with EqAHV1 strain 10-I-224 (4 × 106 plaque-forming units/head) and sampled daily during the 2-wk observation period. Blood samples were collected by jugular venipuncture into plain and EDTA-treated tubes. Serum samples were obtained by centrifugal separation of plain blood at 1,811 × g for 10 min. The EDTA-treated blood samples were processed for PBMC purification by using porous barrier–incorporated centrifugal tubes (Leucosep; Greiner Bio-One) and density gradient medium (Lymphoprep; Alere Technologies) following the manufacturers’ instructions. The purified PBMCs were suspended in cell freezing medium (TC-Protector; KAC) at ~1 × 107 cells/mL. The serum and blood samples were stored at −80°C until use. Acute-phase field serum samples submitted to our laboratory for diagnosis in 2020 were also used. The samples had been collected from 40 febrile (≥38.5°C) horses reared at the Miho Training Center of the Japan Racing Association, in Ibaraki Prefecture. Eleven of the horses had been diagnosed with EqAHV1 infection by the confirmation of a 4-fold or greater increase in antibody titer, as measured by our in-house EqAHV1 glycoprotein E–specific ELISA, between their acute- and convalescent-phase sera (Suppl. Table 1). 5
Nucleic acid was purified from 400 μL of each of the serum and the PBMC (4 × 106 cells) samples by using a magnetic beads–based commercial kit and an automated nucleic acid extraction system (MagDEA Dx SV, magLEAD 12gC; Precision System Science), with an elution volume of 50 μL. Real-time PCR targeting the IR6 gene of EqAHV1 was conducted with the primers and probe described elsewhere. 12 The total reaction mixture (20 μL) consisted of the rtPCR master mix (TaqPath 1-step RT-qPCR master mix; Thermo Fisher), 18 pmol of the primers, 5 pmol of the probe, and 4 μL of the template. For direct detection without nucleic acid purification, 4 μL of untreated serum was added to the reaction mixture. The PCR cycling conditions followed the manufacturer’s protocol. The gene copy number was determined by absolute quantification against a standard curve generated by 10-fold serially diluted (101–107 copies/reaction) plasmids containing the target sequences. All PCR reactions were run in triplicate (StepOnePlus real-time PCR system; Thermo Fisher). A sample was considered positive if the amplification curve above the threshold line fixed at 0.02 was observed in all triplicates within the 40-cycle cutoff. Using JMP 17 software (JMP Statistical Discovery), the number of copies per reaction of the positive control plasmids required to achieve a 95% detection rate by rtPCR targeting the IR6 gene was predicted to be 4.3. This was calculated using a logistic fit of the detection rates of plasmids serially diluted 2-fold from 16 to 0.25 copies/reaction, followed by reverse prediction (data not shown). Additionally, the detection rate of the positive control plasmid at 1 copy/reaction was 50%, with a mean Ct value of 34.8 ± 0.8 (12 positives of a total of 24 replicates across 3 experiments).
EqAHV1 was detected in the PBMC samples of horse 1 at 7–9 days post-infection (dpi) and in the serum samples at 7–11 dpi (Table 1). The duration of virus detection in the serum samples of horse 2 was 5–11 dpi; this coincided with that in the PBMC samples, although there was no detection in the PBMCs at 10 dpi. Virus detection in the serum samples of horse 3 began 1 d later (7 dpi) than detection in the PBMCs but ended simultaneously (11 dpi). EqAHV1 was detected on all days of fever (≥38.5°C), with the exception of 2 dpi in horse 1. Six of the 11 acute-phase serum samples collected from the ELISA-positive horses were positive on standard PCR testing (Table 2). No EqAHV1 was detected in the ELISA-seronegative horses. The 6 acute-phase field sera that were PCR-positive were then used to evaluate direct PCR detection without nucleic acid purification. We detected 15–199 viral gene copy numbers/reaction in the purified serum samples, and 1–12 copies in the untreated serum samples (Table 3).
Body temperatures and equid alphaherpesvirus 1 (EqAHV1) IR6 gene copy numbers detected by real-time PCR in peripheral blood mononuclear cells (PBMCs) and serum samples after experimental EqAHV1 infection of 3 horses.
BT = body temperature; CN = copy number; – = negative for detectable EqAHV1 IR6 gene.
Body temperatures (°C) are quoted from our previous study. 8
Copy numbers per reaction are shown as the mean of triplicates rounded to decimals.
Ct values are shown as the mean of triplicates rounded to one decimal place.
Standard real-time PCR detection of equid alphaherpesvirus 1 (EqAHV1) in acute-phase field sera from febrile horses positive or negative for infection by EqAHV1 glycoprotein E–specific ELISA.
Comparison of equid alphaherpesvirus 1 (EqAHV1) IR6 gene copy numbers detected by real-time PCR using standard nucleic acid–purified serum samples and untreated serum samples.
Copy numbers per reaction are shown as the mean of triplicates rounded to whole numbers.
Ct values are shown as the mean of triplicates rounded to one decimal place.
EqAHV1 was detected by rtPCR on almost the same days post-infection in the PBMC and serum samples from the experimentally infected horses. The total number of sampling days when EqAHV1 was detected in the PBMC or serum samples, or both, was 18, consisting of 14 days with positive results in both samples, 1 day on which only the PBMC sample was positive, and 3 days on which only the serum sample was positive. In horse 3, more EqAHV1 was detected in the PBMC sample than in the serum sample on every positive sampling day. In contrast, more EqAHV1 was detected in the serum sample than in the PBMC sample at 9–11 dpi in horse 1, as well as 7, 9, and 10 dpi in horse 2. The results suggest that the probability of detecting EqAHV1 viremia can be increased by testing both PBMC samples and serum samples at the same time.
EqAHV1 could not be isolated even from serum samples containing >100 copies/reaction of EqAHV1 genes after virus inoculation of RK-13 cells (data not shown). EqAHV1 does not form infectious particles in PBMCs, and complete assembly of the EqAHV1 virion has been achieved only after viral glycoprotein-mediated fusion of PBMCs with permissive cells. 3 Therefore, it is possible that the PCR-positive serum samples contained noninfectious incomplete EqAHV1 virions released from lysed PBMCs. EqAHV1 was also detected by standard rtPCR in more than half of the acute-phase field sera collected from febrile horses that had been serologically determined by ELISA to be infected with EqAHV1. This result suggests that testing acute-phase sera for viremia is helpful in the detection of EqAHV1 infection. However, it should be noted that, as observed in experimental infections, infected horses usually are not viremic in the early stages of EqAHV1 infection. 11 In addition, we successfully detected EqAHV1 by direct rtPCR using untreated acute-phase field serum samples. Although the viral gene copy numbers of the untreated samples were lower than those of the purified samples, all 6 of the sera that tested positive when purified were also positive when untreated. Using untreated sera as a template instead of purified nucleic acid can reduce the turnaround time and the cost of rtPCR detection.
Our results indicate that serum samples could be useful for detecting EqAHV1, especially when PBMC samples are not available. However, because the copy number of the EqAHV1 gene in serum samples is generally low, there is a risk of false-negative results when testing serum alone. Therefore, we strongly recommend that negative rtPCR results for EqAHV1 in serum samples be verified using PBMC samples collected from the same horses. Additionally, further verification in a larger field population of horses would be beneficial for clarifying the clinical significance of using serum as a sample for EqAHV1 detection.
Supplemental Material
sj-pdf-1-vdi-10.1177_10406387251323272 – Supplemental material for Detection of equid alphaherpesvirus 1 in serum samples collected from infected horses
Supplemental material, sj-pdf-1-vdi-10.1177_10406387251323272 for Detection of equid alphaherpesvirus 1 in serum samples collected from infected horses by Koji Tsujimura, Hiroshi Bannai, Yoshinori Kambayashi, Manabu Nemoto and Minoru Ohta in Journal of Veterinary Diagnostic Investigation
Footnotes
Acknowledgements
We thank the clinical veterinarians at Miho Training Center (JRA) for collecting serum samples. We also thank Akiko Kasagawa, Miwa Tanaka, Akiko Suganuma, Kaoru Watanabe, Akira Kokubun, and Kayo Iino (Equine Research Institute, JRA) for their technical assistance.
Declaration of conflicting interests
The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding
The authors received no financial support for the research, authorship, and/or publication of this article.
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References
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