1 Department of Medical Microbiology and Immunology, University of California-Davis, Davis, CA 95616, USA
2 Center for Comparative Medicine, University of California-Davis, Davis, CA 95616, USA
4 Department of Medical Pathology, University of California-Davis, Davis, CA 95616, USA
5 California National Primate Research Center, University of California-Davis, Davis, CA 95616, USA
3 Department of Veterinary Pathobiology, College of Veterinary Medicine, Oklahoma State University, Stillwater, Oklahoma, USA
Correspondence
J. L. Huff (at Department of Medical Microbiology and Immunology)
jlmshuff{at}ucdavis.edu
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ABSTRACT |
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Published ahead of print on 23 October 2002 as DOI 10.1099/vir.0.18808-0.
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Introduction |
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B virus is an alphaherpesvirus that is closely related to herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) of humans. Zoonotic human infection with B virus can be fatal and working with B virus-infected animals or culture of the virus carries substantial occupational risk (Holmes et al., 1995; Palmer, 1987
). The few studies of B virus shedding in rhesus macaques have been based on virus detection in cell culture (Lees et al., 1991
; Weigler et al., 1993
; Weir et al., 1993
; Zwartouw & Boulter, 1984
). Although B virus isolation is generally considered the standard for virus detection, studies with other viruses indicate that virus isolation is a less sensitive method than other techniques such as PCR (Cone et al., 1994
; Wald et al., 1995
). Non-quantitative PCR techniques have been described for B virus (Black & Eberle, 1997
; Scinicariello et al., 1993
; Slomka et al., 1993
; Weigler et al., 1995
). A sensitive, quantitative PCR-based assay to determine the presence of B virus DNA would help assess both the frequency and the titre of shed viral DNA. In addition, a PCR-based test would eliminate lengthy culture detection and the inherent risks of working with the virus in the laboratory.
RhCMV is another endemic herpesvirus of rhesus macaques that is shed at mucosal surfaces. RhCMV is closely related to human CMV (HCMV) by DNA sequence, life cycle and natural history (Barry et al., 1996; Kravitz et al., 1997
; Vogel et al., 1994
). In contrast to B virus, RhCMV does not pose any known risks to humans working with macaques. However, infection of rhesus macaques with RhCMV provides an excellent model for HCMV persistence and pathogenesis (Lockridge et al., 1999
; Sequar et al., 2002
; Tarantal et al., 1998
). HCMV is the most common congenital infection in the world (Daniel et al., 1995
; Stagno et al., 1982
) and a significant cause of morbidity and mortality in immunocompromised patients (Flo et al., 1995
). Use of this method to determine the frequency and quantify the magnitude of RhCMV shedding would expand our understanding of the natural history of this infection.
B virus and RhCMV infect rhesus macaques for life (Vogel et al., 1994; Weigler, 1992
). Both viruses are spread by periodic reactivation and asymptomatic shedding from mucosal sites (Lees et al., 1991
; Lockridge et al., 1999
; Weigler, 1992
). Neither virus normally causes significant disease in immunocompetent macaques during primary or recurrent infection. These viruses have different temporal patterns of acquisition and different reservoirs of persistent viral genomes (Alford & Britt, 1993
; Whitley, 1996
). Little is known about the frequency with which B virus and RhCMV are shed in healthy adult animals and what factors contribute to shedding.
To increase the understanding of non-human primate herpesviruses, a sensitive assay for the detection of B virus DNA was developed using real-time PCR methodology and compared to a RhCMV real-time PCR assay described recently (Sequar et al., 2002). Real-time PCR provides a rapid, safe and accurate method of screening for these viruses. Results of this and future studies may have important implications for colony management and occupational safety.
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Methods |
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Real-time PCR.
Primers and probe for real-time PCR were designed for the conserved glycoprotein B (gB) gene of B virus (Table 1). Sequences were based on the sequence of B virus rhesus strain E2490 and designed using the PRIMER EXPRESS software (Applied Biosystems). Characterization of the RhCMV primers and probe combination has been presented elsewhere (Sequar et al., 2002
). The Taqman probe for RhCMV gB detection was fluorescently labelled with TET at the 5' end and TAMRA at the 3' end. The Taqman probe for B virus gB detection was fluorescently labelled with FAM at the 5' end and TAMRA at the 3' end. All primers and probes were synthesized by Applied Biosystems.
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A standard curve was generated using a plasmid containing either the full-length B virus (pND-BVgB) (Loomis-Huff et al., 2001) or RhCMV gB (Sequar et al., 2002
). Serial 10-fold dilutions of each plasmid containing 106 to 100 copies per 5 µl were made in 20 µg calf thymus DNA ml-1 (Sigma) in water. Both assays reproducibly detected between 1 and 10 copies of RhCMV or B virus DNA.
Animal selection and screening.
Study protocols were approved by the Institutional Animal Use and Care Committee of University of California, Davis, USA (UC Davis), prior to implementation. UC Davis is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care. Juvenile rhesus macaques were selected for the group 1 study on the basis of B virus and RhCMV seropositivity. Outdoor-housed 3- to 4-year-old animals were bled during routine physical examination. Plasma was screened by ELISA for reactivity to B virus and RhCMV (Lockridge et al., 1999; Loomis-Huff et al., 2001
). A total of 13 seropositive animals (4 females, 9 males) with a range of antibody titres were brought indoors and same-sex pair-housed with an animal unknown to them. Changes in housing conditions and pairing with an unknown animal are known stressors for macaques (Capitanio & Lerche, 1998
). One animal, 29937, was included in two phases of the group 1 study (described below). Group 2 animals included 43 adult males (age 610 years), seropositive for both RhCMV and B virus, from which saliva samples were obtained during a screening phase of animals entering an unrelated study (described below).
Sample collection and processing.
Group 1 monkeys were sampled in three phases. The first phase occurred in December, during the breeding season. The second and third phases occurred in April and July. During the third phase, daily mucosal swab samples were obtained for 8 days, then, after a 5 day rest period, mucosal swab samples were collected daily for 10 days during dexamethasone treatment. A low to moderate intravenous dose of dexamethasone (2 mg kg-1) was administered daily for 7 days, then tapered off over the next week.
Mucosal swab samples were taken at the same time every day by one of two technicians and all samples for a single day were obtained by the same technician. Mild ketamine anaesthesia was used to facilitate sample collection. Animal weights were monitored routinely to assure the animals were not losing weight due to multiple days of anaesthesia.
For group 1 animals, mucosal swab samples were taken of the oral cavity, each eye and the genital region using sterile Dacron swabs (Fisher). Oral swabbing was done by running the swab inside the lower lip, into the buccal pouch and along the gumline. Separate swabs of each eye were taken by swabbing the upper and lower conjunctival surface. For the genital samples, the vaginal mucosa was swabbed in females; the swab was inserted in the prepuce of male animals. Each swab was placed into a tube containing 1 ml DMEM (Invitrogen) with 10 % foetal bovine serum, 200 units penicillin ml-1 (Sigma), 200 µg streptomycin ml-1 (Sigma) and 2·5 µg fungizone ml-1 (Invitrogen). Only the oral samples were of sufficient volume to saturate the swab with mucosal fluid prior to placement in media.
Swab samples were processed on the day of collection. Samples were vortexed and aliquotted for DNA extraction (400 µl) and virus isolation (volume ranged from 350 to 600 µl). Samples for DNA extraction were mixed with an equal volume of AL Lysis buffer (Qiagen) and both aliquots were stored at -80 °C.
Group 2 monkeys were part of another study assessing the psychosocial aspects of disease progression. A baseline saliva sample was obtained and then monkeys were subjected to seven daily chair restraint sessions. Animals were not anaesthetized during chair restraint. At the end of each chair session, a saliva sample was obtained by allowing the animal to chew a length of braided cotton rope that had been saturated with a sugar solution. After 1 min of chewing, the distal 1 cm of saliva-saturated rope was cut off and placed into the barrel of a 6 ml syringe. Saliva was expressed into a sterile tube by compressing the rope sample with the plunger of the syringe (approximately 400 µl). An aliquot of each saliva sample was mixed 1 : 1 with AL Lysis buffer and stored at -80 °C for batch processing.
DNA extraction.
DNA was extracted from mucosal swab and saliva samples using the QIAmp Blood kit in a 96-well plate format (Qiagen). Each sample was processed according to the manufacturer's instructions. The final elution volume was 200 µl. Samples were stored at -20 °C until PCR analysis was performed.
Virus isolation for B virus.
Virus isolation was performed on subconfluent monolayers of Vero cells grown in 24-well tissue culture plates. Samples (100500 µl) were inoculated onto cells and incubated at 37 °C for 818 h. The inoculum was then removed and fresh media added (1 ml per well). Media was changed every other day for 1 week. Cultures were checked twice daily for cytopathic effect. No culture for RhCMV was attempted.
ELISA.
ELISA assays were performed according to protocols published previously (Lockridge et al., 1999; Loomis-Huff et al., 2001
). Briefly, a Triton X-100 extract of HVP-2-infected Vero cells was used as antigen for B virus antibody detection (Loomis-Huff et al., 2001
; Ohsawa et al., 1999
). Reactivity to uninfected Vero cell extract was subtracted from the absorbance value (measured at 450 nm) for HVP-2 reactivity. RhCMV ELISAs were performed using a Triton X-100 extract of RhCMV-infected primary skin fibroblasts. Little to no reactivity to uninfected primary skin fibroblast extract was detected. Plasma samples were screened initially at 1 : 100 and 1 : 500. Plasma samples of selected animals were serially diluted to determine the antibody titre. Titres were calculated by taking the inverse of the last dilution for which the absorbance value was greater than 0·1 absorbance units above negative control sample absorbance values (measured at 450 nm).
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Results |
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A plasmid containing the HSV-2 gB gene was undetectable even at a very high concentration by the B virus real-time PCR assay. Consistent with the lack of detection of HSV-2 with this assay, sequence alignments of the B virus primer and probe with the published sequence of HSV-2 (accession no. Z86099) indicated that there is one mismatched base pair in the forward and two each in the reverse primer and probe sequences. Though SA8 DNA from African green monkeys was not tested with this assay, one primer and two probe base pair mismatches suggest that there would be inefficient amplification with this primer/probe set as well.
B virus DNA detection in mucosal samples
After demonstrating the sensitivity and specificity of the quantitative real-time B virus PCR assay, experiments to evaluate clinical mucosal swab samples for the presence of B virus were conducted. In captive rhesus macaques B virus infection usually occurs at 34 years of age (Weigler, 1992; Whitley, 1996
). Studies of HSV-2 infection in humans have demonstrated that reactivation frequency is greater in the period immediately following infection (Benedetti et al., 1994
, 1999
; Koelle & Wald, 2000
). This age group was selected to identify recently infected seropositive animals that would be likely to reactivate and shed virus under stress.
Seropositive 3- to 4-year-old monkeys (four females and nine males, designated group 1) were stressed by relocation to new housing and pairing with a new animal in three phases over 1 year. Each phase involved new animals, with one exception, at a different time of the year: December (breeding season, n=4), April (n=4) and July (n=6). Animal 29937 was included in both the December and July phases. B virus DNA was detected only in mucosal swab samples obtained from animals moved during the breeding season. Each of the four breeding season animals yielded a unique pattern of B virus-positive swabs (Table 3). Although swabs were collected up to 28 days after relocating the animals, all B virus-positive swabs occurred within the first 10 days of sampling. B virus DNA was detected from multiple sites on multiple days for two animals (29937 and 30200). The highest copy number of B virus DNA was in the genital tract samples. Intermittent detection of B virus was seen also in both ocular and oral samples. The other two animals had only a single site that was B virus DNA positive and was confined to a single day. The overall frequency of positive swabs during the first 10 days of sampling during the breeding season was 14·6 % (21/144) as compared to 0 % (0/376) in April and July. For the two animals with multiple positive swab samples (29937 and 30200), 26·4 % (19/72) of swabs were positive for B virus DNA during the first 10 days.
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RhCMV detection in mucosal samples
To demonstrate key differences in the natural history of two endemic macaque herpesviruses, the shedding profile of RhCMV DNA was assayed with the same mucosal samples and compared with that of B virus. RhCMV was detected more frequently than B virus DNA, though there was wide variability between animals within a group and between group 1 and group 2 animals. Results of RhCMV DNA detection for the four animals from which B virus DNA was detected are shown in Table 4. The variability seen in these four animals is representative of that seen from other animals in group 1. RhCMV DNA detection occurred more frequently and at higher copy number in the younger group 1 animals compared to group 2 animals (Table 5
).
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Dexamethasone treatment
Reactivation of latent HSV and shedding of infectious virus in response to glucocorticoid administration has been well documented in clinical settings (Mosimann et al., 1994; Shane et al., 1994
) and in animal models (Cook et al., 1991
; Kaufman et al., 1999
). Real-time PCR was used to determine whether dexamethasone treatment resulted in increased detection of B virus DNA. Six of the monkeys of group 1 sampled in July were kept indoors for a second round of sample collection while being treated with dexamethasone. After a 5 day rest period, swab samples were obtained for 10 days during daily dexamethasone treatment (2 mg kg-1).
B virus DNA was not detected in any swab samples taken while the monkeys were being treated with dexamethasone. The frequency of RhCMV DNA detection was also unchanged with dexamethasone treatment (Table 6) and the number of positive days remained unchanged. An increase in the amount of virus shed did occur for one animal (29938), with 2·8x106 genome copy numbers ml-1 mucosal fluid being detected (Table 6
).
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Discussion |
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The relationship between B virus copy number and infectious virus titre is not known. Studies of HSV shedding demonstrated values 100-fold higher for copy number by traditional PCR than virus titres determined by virus culture (Koelle & Wald, 2000; Wald et al., 1997
). No virus was detected by virus isolation in this study. However, the limit of detection of the assay is not known. It is possible that low titres of infectious virus were present in the samples. In addition, inactivation of small amounts of infectious virus may have occurred during the single freezethaw cycle samples were subjected to before being inoculated onto Vero cell monolayers. Freezing and thawing samples for B virus culture has been shown to decrease virus detection by 12 logs (Krech & Lewis, 1954
; Weir et al., 1993
). The real-time PCR assay described here is probably not more sensitive for B virus than traditional PCR but it is more specific due to the use of an internal probe that appears to differentiate between B virus and other primate alphaherpesviruses, including HSV. While further study is needed, there is a potential for application of this assay to diagnose B virus infection in an exposed human that is also infected with HSV.
Multiple factors can trigger reactivation from latency to virus shedding of infectious HSV (Sainz et al., 2001). Examples of the contribution of social stress to virus pathogenesis have been described for both humans (Glaser et al., 1999
; Glaser & Kiecolt-Glaser, 1997
; Padgett et al., 1998
) and non-human primates (Baroncelli et al., 1997
; Capitanio et al., 1998b
). In this study stress was created by moving, pair-housing with a new animal and submitting each animal to daily sample taking. However, results from this study suggest that factors associated with the breeding season may be more important at inducing B virus reactivation and shedding than stress introduced by the study design. The breeding season has been described as a time of stressful social interaction for rhesus macaques (Weigler et al., 1993
; Wilson & Boelkins, 1970
). In this study, baseline B virus shedding in two animals (29937 and 30141) may have been a reflection of the stress induced by the breeding season. In addition, animal 29937 was sampled in both December and July and B virus was detected only during the breeding season (December). B virus-positive mucosal swab samples have also been detected in 1/28 monkeys (3·6 %) in a cross-sectional survey performed during the breeding season (unpublished observations). The timing of B virus detection in this study supports the idea that B virus shedding is relatively rare and is possibly associated more with the breeding season than thought previously. While the study size for group 1 was small (n=14) and only a few animals had detectable B virus, the intriguing association between B virus reactivation and the breeding season warrants further investigation.
As with studies of HSV asymptomatic shedding (Koelle et al., 1992; Wald et al., 1995
), no differences were apparent in the humoral immune response between animals that were B virus DNA-positive versus those with no B virus detected in this study (data not shown). A clear role for the cellular immune response in controlling HSV reactivation and shedding has been established (Koelle et al., 1998
; Posavad et al., 1997
, 1998
). HSV is also more frequently shed in human immunodeficiency virus (HIV)-infected individuals with low CD4+ T cell counts (Augenbraun et al., 1995
; Schacker et al., 1998
). For this reason, dexamethasone immunosuppression was attempted but did not result in the detection of B virus by PCR. The concentration of dexamethasone used in this study has been reported to alter lymphocyte function in rhesus macaques (Capitanio et al., 1998a
; Pachner et al., 2001
). While other investigators have detected reactivation and shedding of B virus following immunosuppressive treatment, dosing for 3 months was required and only a few animals (3/14) in the highest dose range reactivated (Chellman et al., 1992
). Thus, while chronic immunosuppression can probably exacerbate shedding of B virus in some animals, the significance of transient immunosuppression relative to the stress of the breeding season was not defined by this study.
The pattern of RhCMV detection is very similar to what is known for HCMV. Numerous groups have demonstrated clearly that CD34+ haematopoietic stem cells are reservoirs of truly latent HCMV genomes (Hahn et al., 1998; Soderberg-Naucler & Nelson, 1999
). However, recent evidence has demonstrated that mucosal shedding of HCMV occurs commonly in many healthy individuals (do Canto et al., 2000
; Lucht et al., 1998
; Shen et al., 1996
) and very frequently in immunocompromised patients (Diamond et al., 2000
; Fidouh-Houhou et al., 2001
; Lucht et al., 1998
; Mostad et al., 2000
). In this study, RhCMV shedding was detected consistently in many animals, indicating low-level, persistent infection. RhCMV was detected most frequently in the oral cavity, which is consistent with what has been reported for CMV in humans. In addition, CMV was not detected in eye swab samples from healthy CMV-infected humans (Lee-Wing et al., 1999
), whereas genital shedding of CMV in the semen and cervical fluid has been noted (Gradilone et al., 1996
; Yang et al., 1995
), especially during HIV-infection (Diamond et al., 2000
; Mostad et al., 2000
).
The different shedding signatures of RhCMV and B virus in immunocompetent, healthy macaques imply fundamental distinctions in the natural history of these two herpesviruses. The infrequent detection of B virus DNA is consistent with other alphaherpesviruses that establish a true latent infection (Roizman & Sears, 1996). In the case of HSV, latent viral genomes are activated infrequently, resulting in a change from an extremely limited pattern of gene expression to one of active virus production (Roizman & Sears, 1996
). In contrast, the continuous detection of RhCMV suggests that this virus establishes a state of persistent virus production. While individual RhCMV-infected cells may be characterized by a non-productive pattern of gene expression (i.e., latency), sufficient numbers of such cells reactivate to generate chronic detection of RhCMV DNA in mucosal fluids.
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ACKNOWLEDGEMENTS |
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Received 2 September 2002;
accepted 4 October 2002.