Abstract
Background:
Simian varicella virus (SVV) is a primate herpesvirus that causes a natural varicella-like disease in Old World monkeys and may cause epizootics in facilities housing nonhuman primates. SVV infection of nonhuman primates is used as an experimental model to investigate varicella pathogenesis and to develop antiviral strategies.
Methods:
An indirect enzyme-linked immunosorbent assay (ELISA) was developed to detect SVV antibodies in infected rhesus macaque monkeys.
Results:
An ELISA determined SVV antibody titers following experimental infection. SVV IgG was detected by day 14 postinfection and remained elevated for at least 84 days.
Conclusions:
The SVV ELISA is a safe and rapid approach to confirm SVV seropositivity and to determine SVV antibody titers in naturally and experimentally SVV-infected monkeys. In addition to being a useful diagnostic assay to rapidly confirm acute disease or past SVV infection, the SVV ELISA is a valuable epidemiological tool to determine the incidence of SVV in nonhuman primate facilities.
Keywords: SVV, simian varicella, macaque, monkey, ELISA
1. Introduction
Simian varicella virus (SVV, Cercopithecine herpesvirus 9) causes a natural disease in Old World monkeys that clinically resembles human varicella zoster virus (VZV) infection.1 After a 10 to 12 day incubation period, infected monkeys develop fever and a vesicular skin rash over the entire body.2 Clinical disease may range from mild to severe and life-threatening. Infected animals are confirmed to be SVV seropositive, although SVV antibody titer has not been correlated to disease severity. Following resolution of the primary disease, SVV, like VZV, establishes life-long latent infection within neural ganglia. Viral reactivation, induced by stress or immunosuppression, may cause a secondary disease similar to herpes zoster in humans.3,4
Epizootics of simian varicella have occurred in facilities housing nonhuman primates (NHP), including populations of African green (Chlorocebus aethiops), patas (Erythrocebus patas) and various species of macaque (Macaca) monkeys.5 Some of these outbreaks have been associated with high morbidity and mortality while others have involved milder disease.
SVV infection of NHP is a useful experimental model since VZV infection of other laboratory animals does not simulate a varicella-like disease or induce viral latency and reactivation. Therefore, the simian varicella NHP model is valuable to gain a better understanding of the molecular basis of varicella pathogenesis and latency and to develop and assess antiviral therapies.1 The model has also been employed to evaluate vaccines including recombinant varicella vaccines against other viral pathogens such as simian immunodeficiency virus (SIV) and respiratory syncytial virus (RSV).6–9
The importance of simian varicella as a veterinary disease and as an experimental animal model requires a reliable and convenient method to detect and measure antibody responses in SVV-infected animals. The plaque reduction neutralization test (PRNT) has most commonly been used to confirm SVV seropositivity6,8,10,11. While useful to determine antibody neutralization titers, PRNT has some disadvantages, including safety considerations as infectious virus must be propagated in cell culture. At least five days are required for results until viral plaques appear on cell monolayers. In addition, the highly cell-associated nature of SVV requires that neutralizing antibody inactivate SVV-infected cells rather than infectious cell-free viruses.
In this study, an enzyme-linked immunosorbent assay (ELISA) is used to detect SVV antibodies in sera of infected rhesus macaque monkeys and to determine viral antibody titers following experimental SVV infection. The results are compared to PRNT as a means to evaluate humoral immune responses in SVV-infected monkeys.
2. Materials and Methods
2.1. Humane Care Guidelines
SVV infections and procedures involving NHP were performed at Tulane National Primate Research Center following the recommendations of the U.S. Department of Agriculture Animal Welfare Act regulations, the Guide for the Care and Use of Laboratory Animals, and regulations of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). All experiments were approved by the Tulane University Institutional Animal Care and Use Committee (IACUC), prior to the start of the study (protocol number P0177).
2.2. Experimental SVV infection
Rhesus macaque monkeys (Macaca mulatta), four to five years old, were confirmed to be seronegative for SVV by PRNT prior to experimental infection. The animals were infected with 5 × 104 plaque forming units (pfu) of SVV Delta herpesvirus strain by intratracheal inoculation.12 Clinical and virological parameters of simian varicella were evaluated as previously described.12 Viremia was monitored by harvesting peripheral blood mononuclear cells (PBMCs) from EDTA-anticoagulated blood using Ficoll-Hypaque gradients, and determining infectious SVV titers by co-culture of PBMCs on Vero cells. Skin rash was scored on an established scale of 0 to 4+, with 0 for no rash and +4 for severe rash. Hepatitis was assessed by aspartate transaminase (AST) assays of blood specimens. Serum was prepared from 3 to 5 ml blood samples collected prior to experimental infection and on days 14, 21 and 84 postinfection (pi).
2.3. PRNT to detect antibodies against SVV
Dilutions of monkey sera in 0.5 milliliter (ml) minimal essential media (MEM, Gibco, Life Technologies) were mixed with 100- 200 plaque forming units (pfu) of SVV-infected CV-1 (African green monkey kidney) cells or with uninfected CV-1 cells in 0.5 ml MEM and incubated for one hour at 25°C. Controls included 100 - 200 pfu SVV-infected cells incubated with dilutions of sera from uninfected monkeys or with MEM. The mixture was added to wells of six well plates containing confluent CV-1 cell monolayers. After one hour, two ml of MEM + 2% normal calf serum (NCS) were added to the monolayers and the plates were incubated for five days at 37°C. Following the five-day incubation, cells were fixed with methanol, stained with methylene blue, and viral plaques were counted. Viral titer was calculated as the reciprocal of the serum dilution that inhibited greater than or equal to 50% of the viral plaques compared to the control plates.
2.4. Preparation of SVV ELISA plates
SVV-infected Vero cells were mixed with uninfected Vero cells at a 1:3 ratio in 175 cm2 flasks. When cytopathic effect was observed, monolayers were scraped off, centrifuged at 1000g for ten minutes, washed twice in phosphate buffered saline (PBS), and resuspended in two ml of 0.1 M glycine buffer containing 1% Triton-X. Uninfected Vero cells were treated in a similar manner. After 30 minutes incubation on ice the cells were sonicated with two 15 second bursts using a Fisher Scientific Sonic Dismembrator. The lysate was clarified by centrifugation at 41,000g for 30 minutes and the supernatant was frozen in 0.5 ml aliquots at −80 C. The total antigen concentration was determined by Bradford protein assay (Biorad Laboratories). Cell lysates (100 microliters [ul] of 5 μg antigen/ml) were added to individual wells of 96 well, polystyrene, flat-bottom, tissue culture plates (Falcon) and coated at 4°C until ready for use.
2.5. SVV ELISA
SVV antigen or uninfected Vero cell antigen coated onto wells was washed twice in PBS containing Tween 20 (0.05%, PBS-T) and then incubated overnight at 4°C with blocking buffer (3% bovine serum albumen [BSA], 0.2% sodium azide in PBS). Antigen was washed twice with PBS-T and dilutions of monkey antiserum in 100 μl sample diluent buffer (2% BSA, 0.1% sodium azide, 0.05% Tween 20 in PBS) were added and incubated for one hour at 25°C. After washing three times with PBS-T, 100 μl of secondary antibody (mouse anti-monkey IgG horseradish peroxidase [HRP] conjugate, Southern Biotech) diluted in sample diluent buffer was added and incubated for 30 minutes at 25°C. After washing thrice with PBS-T, 100 μl per well of tetramethylbenzidene (TMB, MP Biomedical) substrate was added for 15 minutes followed by addition of 100 μl/well of stop solution (0.16 M sulfuric acid). Fresh buffers were important for consistent results. Color intensities were read at 450 nm absorbance (A450) using a Biotek ELISA reader. SVV antibody titers were expressed as the reciprocal of the serum dilution that resulted in A450 absorbance readings above the determined cut-off value.
The ELISA sensitivity (true positive rate) was calculated as the number of SVV infected animals determined to be SVV seropositive by ELISA (titer greater than or equal to 4000) divided by the total number of SVV experimentally infected animals tested. Specificity (true negative rate) was calculated as number of uninfected animals determined to be seronegative by ELISA (titer equal to or less than 500) divided by the number of uninfected animals tested.
3. Results
3.1. Optimization of ELISA conditions
An absorbance cut-off value for the SVV ELISA was determined in order to discriminate between positive and negative A450 absorbance results. Serum samples were derived from 21 uninfected rhesus monkeys which were confirmed to be SVV seronegative by PRNT. The serum samples at a 1:1000 dilution were evaluated in the ELISA assay using antigen derived from SVV infected Vero cells. The mean A450 absorbance reading was 0.057 ± 0.020 (range 0.040 to 0.081) with a standard deviation of 0.011. An optimal cut-off absorbance value of 0.090 for the SVV ELISA was determined as the mean absorbance plus three times the standard deviation.13 Based on this cut-off value, each of the 21 serum samples derived from uninfected, PRNT seronegative monkeys were also determined to be seronegative by ELISA. In contrast, 21 of 21 serum samples derived from SVV experimentally infected monkeys were confirmed to be seropositive by both PRNT and ELISA (Table 1). The results indicate a 100% sensitivity and 100% specificity for the SVV ELISA.
Table 1.
SVV ELISA and PRNT antibody titers
| SVV Positive by PRNT@ |
SVV Positive by ELISA# |
|
|---|---|---|
| Uninfected monkeys | 0/21 (0%)a | 0/21 (0%)c |
| Experimentally SVV infected monkeys | 13/13 (100%)b | 13/13 (100%)d |
serum PRNT titer of 20 or less
serum neutralization titer of 80 or greater
ELISA titer of 500 or less
ELISA titer of 4000 or greater
The optimal concentration of SVV-infected Vero cell antigen distributed onto ELISA plate wells was determined by reacting serum derived from SVV infected monkeys with various concentrations of SVV-infected Vero cell protein. Concentrations of SVV-infected or uninfected Vero cell antigen in 100 ul were added to wells of a 96-well plate and reacted with serum (1:1000 dilution) derived from SVV-infected or uninfected rhesus monkeys by ELISA. Figure 1 shows that SVV-seropositive monkey serum reacted with concentrations of SVV-infected cell antigen ranging from 10 μg/ml to less than 1 μg/ml. In contrast, the SVV-positive monkey serum did not react with similar concentrations of uninfected Vero cell antigen. Also, sera derived from uninfected monkeys did not react with SVV-infected Vero cell antigen. The results demonstrated that addition of 100 μl of SVV-infected Vero cell antigen at 5 μg/ml was optimal for addition to each well of the SVV ELISA plate.
Figure 1-.
Titration of SVV antigen using SVV seropositive serum. Various concentrations of SVV-infected Vero cell antigen or uninfected Vero cell antigen were coated onto wells. Reaction of SVV monkey serum with concentrations of SVV-infected Vero cell antigen (closed circles). Reaction of SVV monkey serum with concentration of uninfected Vero cell antigen (closed triangles). Reaction of uninfected monkey serum with SVV-infected Vero cell antigen (closed blocks).
3.2. Determination of SVV antibody titers in monkey sera by ELISA
The SVV antibody titer in serum of SVV-infected and uninfected rhesus macaques was determined by ELISA. Dilutions of serum derived from three SVV-infected monkeys (KG58, KI87, and KI92) were reacted with SVV-infected Vero cell antigen by ELISA. Figure 2 demonstrates that the SVV ELISA A450 values were directly related to SVV antibody concentration. SVV antibody titers were determined as the reciprocal of the antibody dilution that resulted in a positive A450 absorbance value above the cut-off value of 0.090. Monkey KG58 was SVV seropositive with a titer of 16,000, while monkeys KI87 and KI92 had SVV antibody titers of 8,000. In contrast, uninfected monkey KT89 had a SVV antibody titer of <250 and was confirmed to be SVV seronegative.
Figure 2-.
Relationship between monkey serum dilution and A450 values. Sera from SVV-infected monkeys are indicated as closed circles (monkey KG58), rectangles (KI87), or triangles (KI92). Serum samples from an uninfected monkey (KT89) are indicated as open circles.
3.3. Evaluation of SVV antibody ELISA titer following experimental SVV infection of rhesus macaques
Serum samples were collected from five rhesus monkeys before experimental infection and on days 14, 21, and 84 pi and SVV antibody titers were determined by ELISA and plaque reduction neutralization test. The results are shown in Figure 3 and Table 2. Prior to SVV infection, the animals were confirmed to be SVV seronegative by ELISA (titer ≤ 250) and by serum neutralization (titer <10). By day 14 pi, SVV antibody was detected in serum samples from each of the five infected monkeys with ELISA antibody titers ranging from 2000 to 8000. SVV antibody titers remained the same at day 21 pi., and increased two to four-fold to a maximum of 16,000 by day 84 pi.
Figure 3-.
SVV antibody titers on various days following experimental SVV infection. Mean SVV antibody titers of sera derived from five monkeys prior to infection and on various days pi are indicated.
Table 2.
SVV ELISA and PRNT antibody titers in experimentally infected monkeys
| Monkey | Pre-Imm. | Day 14 pi | Day 21 pi | Day 84 pi |
|---|---|---|---|---|
| KB95 | <250 (<10) | 2000 (160) | 2000 (160) | 8000 (160) |
| KG58 | 250 (<10) | 8000 (160) | 8000 (>320) | 16000 (>320) |
| KI87 | 250 (<10) | 4000 (>320) | 4000 (>320) | 8000 (>320) |
| KI92 | <250 (<10) | 4000 (80) | 4000 (80) | 8000 (>320) |
| KT89 | 250 (<10) | 4000 (80) | 4000 (>320) | 16,000 (>320) |
SVV ELISA titers are the reciprocal of highest dilution above the cut-off absorbance value (0.090).
SVV PRNT titers are indicated in parentheses.
The SVV antibody titers as determined by SVV ELISA or by PRNT were compared (Figure 4, Table 2). Prior to SVV infection, each of the animals was confirmed to be seronegative by ELISA (titer ≤ 250) and by PRNT (titer <10). Following SVV infection, SVV ELISA antibody titers and SVV serum neutralization titers increased over time up to day 84 pi. Linear regression analysis revealed that SVV ELISA titers and SVV PRNT values were directly correlated (0.704 linear regression co-efficient).
Figure 4-.
Relationship of SVV antibody titers as determined by ELISA or by PRNT. Serum samples were derived from five monkeys prior to SVV infection and from the same monkeys at 14, 21, and 84 days pi. Each dot indicates a serum sample with ELISA and corresponding PRNT values. The line of best fit as determined by linear regression analysis is indicated.
4. Discussion
An indirect ELISA was developed to detect SVV antibody in sera derived from infected rhesus monkeys and SVV antibody titers determined by ELISA and PRNT were compared. An indirect ELISA approach offers advantages over a direct ELISA including the use of commercially available conjugated anti-monkey IgG or IgM secondary antibody rather than having to prepare conjugated primary monkey serum. In addition, an indirect ELISA has high sensitivity since more than one labeled secondary antibody can bind to the primary antibody.
PRNT has been the most common and standard means to measure SVV antibody titer in studies of SVV pathogenesis and antiviral strategies.10–12,14 In addition, PRNT is a valuable approach to assess the ability of functional antibodies in monkey sera to block virus infection and to potentially protect against disease. Indirect immunofluorescence, complement fixation, immunoprecipitation, and radioimmunoassays have also been used to detect SVV antibodies in sera derived from SVV-infected monkeys.15–17
The SVV ELISA offers several advantages as a means to analyze SVV antibody titers in sera of SVV-infected monkeys. The SVV ELISA is safer than the PRNT because working with infectious virus is not needed after the initial preparation of SVV antigen from SVV-infected Vero cells. The SVV antigen stocks are stable upon freezing at −80°C and may be used as an antigen source for at least one year. The SVV ELISA is rapid and can determine SVV antibody titers from multiple serum samples within four hours, compared to the PRNT which requires at least five days. The assay is less labor-intensive than PRNT and does not require cell culture after antigen preparation. The assay is sensitive and capable of detecting SVV antibodies in seropositive serum at dilutions as high as 1:16,000, while a serum dilution of 1:500 or less failed to detect SVV antibodies in uninfected monkeys.
A previous study employing an SVV ELISA focused on detection of IgM antibodies during the early stages of simian varicella in experimentally infected patas monkeys.18 SVV antibodies were detected using antihuman IgM and IgG alkaline phosphate conjugated antibodies. In the present study, SVV indirect ELISA determined viral IgG antibody titers within serum of infected rhesus monkeys, the most commonly used species used for experimental SVV studies. This study utilized HRP-conjugated anti-monkey IgG secondary antibody, providing a more sensitive approach compared to using anti-human alkaline phosphate conjugated secondary antibody.19 Both studies determined that SVV IgG antibodies could be detected by day 14 pi shortly after viremia and the appearance of clinical signs (fever, skin rash) in the infected monkeys. In addition, both studies demonstrated that SVV IgG titers remained elevated for at least 84 days pi.
The SVV ELISA can be especially useful for epidemiological studies of SVV seroprevalence in facilities housing nonhuman primates. After initial SVV exposure, the virus establishes latent infection within the neural ganglia of infected monkeys.20,21 Subsequently, stress or immunosuppression in naturally or experimentally SVV infected monkeys may induce viral reactivation resulting in acute or subclinical infection and shedding of infectious virus to susceptible monkeys.3,4,11 During the 1970’s and 1980’s, major simian varicella epizootics caused significant morbidity and mortality in primate facilities.5 Major outbreaks are now less common due to awareness of the disease and improvements in NHP housing conditions. However, simian varicella may still compromise research, especially in studies involving immunosuppressed monkeys.3,22,23 The SVV ELISA can facilitate the screening of large numbers of animals to determine the incidence of SVV infection in primate facilities and prevent the potential loss of valuable animals and research data.
Acknowledgments
We thank the Tulane National Primate Research Center Veterinary Medicine staff for excellent animal care.
Funding
This study was supported by Public Health Service Grant R01 AI123029 of the National Institutes of Health (W.L.G., V.T-D., G..W, A.D.) and in part with federal funds from the National Center for Research Resources and the Office of Research Infrastructure Programs (ORIP) of the NIH through grant P51 OD011104 to the Tulane National Primate Research Center (V.T-D, A.D.).
Footnotes
Ethics Statement
The ethical policies of the journal, as noted in the author guidelines, were followed. All animal housing and protocols involving animals were in accordance with the recommendations of the U.S. Department of Agriculture Animal Welfare Act regulations, the Guide for the Care and Use of Laboratory Animals, and regulations of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). All experiments were approved by the Tulane University Institutional Animal Care and Use Committee (IACUC), prior to the start of the study.
Conflicts of Interest
All of the authors declare that there are no conflicts of interest in the present study.
References
- 1.Gray WL. Simian varicella: A model for human varicella-zoster virus infections. Rev Med Virol 2004;14:363–381. [DOI] [PubMed] [Google Scholar]
- 2.Gray WL. Pathogenesis of simian varicella virus. J Med Virol. 2003;70 Suppl 1:S4–8. [DOI] [PubMed] [Google Scholar]
- 3.Kolappaswamy K, Mahalingam R, Traina-Dorge V, et al. Disseminated simian varicella virus infection in an irradiated rhesus macaque (Macaca mulatta). J Virol 2007;81:411–415. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Mahalingam R, Traina-Dorge V, Wellish M, et al. Latent simian varicella virus reactivates in monkeys treated with tacrolimus with or without exposure to irradiation. J Neurovirol 2010;16:342–354. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Gray WL. Simian varicella in Old World monkeys. Comp Med 2008;58:22–30. [PMC free article] [PubMed] [Google Scholar]
- 6.Pahar B, Gray WL, Phelps K, et al. Increased cellular immune responses and CD4+ T-cell proliferation correlate with reduced plasma viral load in SIV challenged recombinant simian varicella virus - simian immunodeficiency virus (rSVV-sIV) vaccinated rhesus macaques. Virol J 2012;9:160. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Gray WL. Recombinant varicella-zoster virus vaccines as platforms for expression of foreign antigens. Adv Virol 2013; 10.11551/2013/219439 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Traina-Dorge V, Pahar B, Marx P, et al. Recombinant varicella vaccines induce neutralizing antibodies and cellular immune responses to SIV and reduce viral loads in immunized rhesus macaques. Vaccine 2010;28:6483–6490. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Ward TM, Traina-Dorge V, Davis KA, Gray WL. Recombinant simian varicella viruses expressing respiratory syncytial virus antigens are immunogenic. J Gen Virol 2008;89:741–750. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Gray WL, Gusick NJ, Fletcher TM, Solke KF. Simian varicella virus antibody response in experimental infection of African green monkeys. J Med Primatol 1995;24:246–251. [DOI] [PubMed] [Google Scholar]
- 11.Traina-Dorge V, Doyle-Meyers LA, Sanford R, et al. Simian varicella virus is present in macrophages, dendritic cells, and T cells in lynph nodes of rhesus macaques after experimental reactivation. J Virol 2015;89:9817– 9824. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Gray WL, Williams RJ, Chang R, Soike KF. Experimental simian varicella virus infection of St. Kitts vervet monkeys. J Med Primatol 1998;27:177–183. [DOI] [PubMed] [Google Scholar]
- 13.Classen DC, Morningstar JM, Shanley JD. Detection of antibody to murine cytomegalovirus by enzyme-linked immunosorbant and indirect immunofluorescence assays. J Clin Microbiol 1987;25:600–604. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Traina-Dorge V, Palmer BE, Coleman C, et al. Reactivation of simian varicella virus in rhesus macaques after CD4 T cell depletion. J Virol. 2019; 10.11281/JVI.01375-18. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Fletcher TM 3rd, Gray WL Simian varicella virus: characterization of virion and infected cell polypeptides and the antigenic cross-reactivity with varicella-zoster virus. J Gen Virol 1992;73:1209–1215. [DOI] [PubMed] [Google Scholar]
- 16.Kolappaswamy K, Mahalingam R, Traina-Dorge V, et al. Dissemiated simian varicella virus infection in an irradiated rhesus macaque (Macca mulatta). J Virol 2006;81:411–415. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Felsenfeld AD, Schmidt NJ. Varicella-zoster virus immunizes patas monkeys against simian varicella-like disease. J Gen Virol 1979;42:171–178. [DOI] [PubMed] [Google Scholar]
- 18.ILtis JP, Achilli G, Madden DL, Sever JL. Serologic study by enzyme-linked immunosorbant assay of the IgM antibody response in the patas monkey following experimental simian varicella infection. Diag Immunol 1984;2:137–142. [PubMed] [Google Scholar]
- 19.Beyzavi K, Hampton S, Kwasowski P, Fickling S, Marks V, Clift R. Comparison of horseradish peroxidase and alkaline phosphatase-labeled antibodies in enzyme immunoassays. Ann Clin Biochem. 987;24:145–152. [DOI] [PubMed] [Google Scholar]
- 20.Mahalingam R, Clarke P, Wellish M, et al. Prevalence and distribution of latent simian varicella virus DNA in monkey ganglia. Virology 1992;188:193–197. [DOI] [PubMed] [Google Scholar]
- 21.Ou Y, Davis KA, Traina-Dorge V, Gray WL. Simian varicella virus expresses a latency-associated transcript that is antisense to open reading frame 61 (ICP0) mRNA in neural ganglia of latently infected monkeys. J Virol 2007;81:8149–8156. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22.Gulani J, Koch A, Chappell M, et al. Cercopithecine herpesvirus 9 (simian varicella virus) infection after total-body irradiation in a rhesus macaque (Macaca mulatta). Comp Med 2016;66:150–153. [PMC free article] [PubMed] [Google Scholar]
- 23.Choi SH, Yoon CH, Lee HJ, et al. Long-term safety of systemic immunosuppression in pig-to-nonhuman primate corneal xenotransplantation. Xenotransplant 2018; 10.1111/xen.12442. [DOI] [PMC free article] [PubMed] [Google Scholar]