Viruses of GuineaPigs: Considerations Research · DNA Herpesvirus Caviidherpesvirustype 1 GPCMVguineapig Jackson (47) salivary glandvirus ColeandKuttner(11) Caviidherpesvirus type2

MICROBIOLOGICAL REVIEWS, Sept. 1980, p. 468-490 Vol. 44, No.30146-0749/80/04-0468/23$02.00/0

Viruses of Guinea Pigs: Considerations for BiomedicalResearch

G. D. HSIUNG,* F. J. BIA, AND C. K. Y. FONGVirology Laboratory, Veterans Administration Medical Center, West Haven, Connecticut 06516; and

Departments ofLaboratory Medicine and Internal Medicine, Yale University School ofMedicine, NewHaven, Connecticut 06510

INTRODUCTION ............................................................. 469VIRUS ISOLATIONS FROM GUINEA PIGS: HISTORICAL BACKGROUND .. 469GUINEA PIG HERPESVIRUSES: INFECIION IN VTRO 470Cytopathology and Growth Rate in Cell Culture 470Ultrastructural Development in Infected Cells ......... 471Molecular and Biochemical Analysis ......... 471

Properties of guinea pig herpes-like virus deoxyribonucleic acid . 471Lack ofgenetic relatedness between guinea pig cytomegalovirus and guineapig herpes-like virus. 471

Effect of Heparin on Guinea Pig Herpesvirus Replication . 474Antigenic Distinctiveness . 474

GUINEA PIG CYTOMEGALOVIRUS: INFECTION IN VIVO . 474Natural Infection . 474Experimental Infection . 475Viremia during acute primary infection . 475Viruria during chronic persistent infection . 475Mode of transmission 475

(i) Transplacental transmission . 475(ii) Contact infection . 475

GUINEA PIG HERPES-LIKE VIRUS: INFECTION IN VIVO . 477Natural Occurrence . 477

Inbred versus random-bred strains and age variations . 477Experimental Infection.. 478

Pathogenicity and latency.. 478Transplacental transmission 478Oncogenicity: cell transformation and induction of tumors. 479

GUINEA PIG HERPESVIRUSES: ANIMAL MODELS FOR HUMAN HERPES-VIRUS INFECTION. 479

Comparison of Guinea Pig Herpes-Like Virus and Guinea Pig Cytomegalo-virus In Vivo Pathogenicity.... 479

Similarities Between Guinea Pig Herpes-Like Virus and Human Epstein-BarrVirus ...........480

Comparison of Guinea Pig Cytomegalovirus and Human Cytomegalovirus .. 480PARAMYXOVIRUSES .... .... 480

Naturally Occurring Infection 480Antibody Response After Experimental Infection . 481

RETROVIRUSES OF GUINEA PIGS . 482Nomenclature for the Guinea Pig Retroviruses . 482Observation of Virus Particles in Cells of L2C Leukemic Guinea Pigs and in*

Placental, Fetal, and Nerve Tissues of Normal Guinea Pigs . 483Induction of Guinea Pig Retrovirus in Cultured Cells . 483Biochemical Studies of Guinea Pig Retrovirus.. 484

MIXED INFECTIONS..... 484Mixed Infections In Vitro . 484Guinea pig herpes-like virus and guinea pig retrovirus . 484Guinea pig cytomegalovirus and guinea pig paramyxovirus . 486

Mixed Infection In Vivo. 486Synergistic reaction with guinea pig herpes-like virus and guinea pigretrovirus. 486

Interference between guinea pig cytomegalovirus and guinea pig herpes-like virus. 486

CONCLUDING REMARKS . 486LITERATURE CITED 488

468

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VOL. 44, 1980

INTRODUCTION

The guinea pig, Cavia porcellus, is commonlyused in both biological and immunological re-

search. The use of this animal species has beenso widespread for so many years that the ternguinea pig has become synonymous with exper-imental animal. This species has proven to beof considerable value in microbiological researchfor isolation of microbial organisms and studiesof their pathogenesis, for studies of antibodyproduction, and as a source of "complement."The guinea pig is an apparently healthy andhardy animal, subject to relatively few naturallyoccurrmng viral diseases (53).Although several viruses have been isolated

from this animal species, there have been rela-tively few reports concerned with viral diseasesof guinea pigs. In this paper emphasis will be onthose viruses of guinea pigs which are eitherendogenous or acquired, the methods by whichthey are recognized, and their pathogenicityafter experimental infection. Endogenous vi-ruses, in particular, potentially complicate re-

VIRUSES OF GUINEA PIGS 469

search in both virology and immunology; hence,investigators must be aware of their presenceand the pathogenesis of their infections in orderto interpret data correctly.Anatomic similarities between guinea pigs and

humans have been noted. The structure of theguinea pig placenta (19), for example, closelyresembles that ofhumans and permits the trans-placental transmission of many viruses, includ-ing herpesviruses. Thus, guinea pigs provide an

important animal model for studying humanviral diseases, especially congenital infectionswith cytomegalovirus (CMV) (9, 28a, 48, 49).

VIRUS ISOLATIONS FROM GUINEAPIGS: HISTORICAL BACKGROUNDSeveral deoxyribonucleic acid (DNA)- and ri-

bonucleic acid (RNA)-containing viruses havebeen isolated from guinea pigs (Table 1). Amongthe DNA viruses, herpesviruses are most com-mon. In 1920 Jackson described an intracellularparasite present in the duct cells of guinea pigsalivary gland tissues (47). However, in 1926

TABLE 1. Naturally occurring virus infections ofguinea pigsNucleicacid con- Virus group Virus type Name commonly used Referencetained

DNA Herpesvirus Caviid herpesvirus type 1 GPCMV guinea pig Jackson (47)salivary gland virus Cole and Kuttner (11)

Caviid herpesvirus type 2 GPHLV Hsiung and Kaplow (41)Caviid herpesvirus type 3 Guinea pig X-virus Bia et al. (J. Virol., in

press)Poxvirus Guinea pig poxlike virus Hampton et al. (29)

RNA Paramyxovirus Parainfluenza virus type 1 Sendai virus Van Hoosier andRobinette (74)

Parainfluenza virus type 2r Van Hoosier andRobinette (74)

Parainfluenza virus type 3a Van Hoosier andRobinette (74)

Parainfluenza virus type 5 Guinea pig parainfluenza Hsiung et al. (36)virus 5 Bia and Hsiung

(unpublished data)Mumps virus0 Hsiung et al. (36)Pneumonia virus of mice' Van Hoosier and

l Robinette (74)Retrovirus GPRV Guinea pig leukemia virus Nadel et al. (59), Opler

(63)Guinea pig C-type Hsiung (33), Nayak and

Murray (62)Guinea pig B-type Dahlberg et al. (15)Guinea pig G-type Hsiung (34)

Reovirus Reovirus type 30 Van Hoosier and, Robinette (74)Arenavirus Lymphocytic LCM Van Hoosier and

choriomeningitis viruSa Robinette (74)Enterovirus Murine encephalomyelitis GD-VII (Theiler virus) Van Hoosier and

avirus Robinette (74)a Antibody studies only.


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470 HSIUNG, BIA, AND FONG

Cole and Kuttner demonstrated that these in-tracellular inclusions were caused by a filterableagent (11, 50) or salivary gland virus which waslater named guinea pig CMV (GPCMV). Nev-ertheless, in vitro cultivation of GPCMV wasnot accomplished until 1957, by Hartley et al.(30). In fact, GPCMV was the first among theCMVs to be recognized as a filterable agent thatcaused the distinctly swollen cells in salivaryglands, from which the name of the virus groupwas derived. GPCMV has been classified by theInternational Committee on Nomenclature ofViruses as caviid herpesvirus type 1 (68).

In 1969, as part of a longitudinal survey oflatent virus infection in laboratory animals, an-other herpesvirus of guinea pigs, caviid herpes-virus type 2, was isolated first from leukemicand then from nonleukemic strain 2 guinea pigs(41) and later from Hartley guinea pigs (42);subsequently, several laboratories reported sim-ilar findings (3, 14, 60, 65). Because the originalisolation of the guinea pig herpes-like virus(GPHLV) was from leukemia-susceptible strain2 guinea pigs (41), the natural history of thisvirus infection in guinea pigs gained considerableattention during the early 1970s (see Guinea PigHerpes-Like Virus: Infection In Vivo). More re-cently, a third herpesvirus, caviid herpesvirustype 3, was isolated from inbred strain 2 guineapigs (F. J. Bia, W. C. Summers, C. K. Y. Fong,and G. D. Hsiung, J. Virol., in press). Serologi-cally, this new herpesvirus of guinea pigs showedno antigenic cross-reaction with either GPCMVor GPHLV by either the neutralization test orthe immunoferritin electron microscopic tech-nique. In addition, biological and molecularcharacterization demonstrated that this thirdherpesvirus is distinctly different from the twowell-known herpesviruses of guinea pigs (Bia etal., in press).With regard to other DNA viruses, there has

been one report in the literature of pox-typevirus particles observed in cell cultures derivedfrom fibrovascular growth on the thigh musclesof guinea pigs in one colony (29). The source ofvirus infection in these guinea pigs was not dis-cussed.

In the RNA virus group, evidence of parain-fluenza virus infection in guinea pigs was firstrecognized by the presence of antibody titers toparainfluenza viruses (36), especially parainflu-enza virus type 5 (SV5) antibody, in guinea pigsera. Normal guinea pig sera and commerciallyavailable complement often contained parainflu-enza virus type 5 antibody, which was a problemwhen guinea pigs were used for preparing hy-perimmune type-specific antisera for the para-influenza viruses (75). (For details, see below.)

On one occasion parainfluenza virus type 1, orSendai virus, was isolated from a guinea pigcolony which was in proximity with mice (74).Recently, a parainfluenza virus isolate serologi-cally identical to SV5 was obtained from thesalivary gland of a guinea pig (see Mixed Infec-tions).

Other RNA viruses included a retroviruswhich was originally observed in the leukemiccells of strain 2 guinea pigs with L2C leukemia(59, 63). Since similar virus particles were notfound in normal guinea pigs during early inves-tigations, it was thought that these virus parti-cles were actually the guinea pig leukemia virus.Not until 1972, when attempts to cultivate theso-called guinea pig leukemia virus were suc-cessful, did we demonstrate that a similar virus,later named guinea pig retrovirus (GPRV), wasinduced in cultured guinea pig cells which weremaintained in a medium containing 5-bromo-2'-deoxyuridine (BUdR) (33). (Details will be dis-cussed below.)

Antibodies to other RNA-containing viruses,including reovirus, arenavirus, and enterovirus,have been noted (74), and antibody-positive re-actors have been found in this animal species.Occasionally, mixed infection with DNA- andRNA-containing viruses have been obtainedfrom the same culture derived from the sameanimal; and these will be discussed in MixedInfections.

GUINEA PIG HERPESVIRUSES:INFECTION IN VITRO

At least two well-characterized herpesviruses,GPCMV and GPHLV, are commonly harboredby guinea pigs; therefore, it is important forinvestigators to recognize their presence and tohave some means for differentiating them. Inthe following paragraphs, the growth character-istics of these two guinea pig herpesviruses incell cultures are compared with regard to cyto-pathology and ultrastructural development, aswell as some recent studies on molecular virol-ogy.

Cytopathology and Growth Rate in CellCulture

The growth rates of GPCMV and GPHLV inguinea pig embryo (GPE) and guinea pig kidney(GPK) cells were compared by Hsiung et al. (44)(Fig. 1). There was no evidence of cytopathiceffect or significant numbers of intranuclear in-clusions in GPK cells infected with GPCMV,although the virus persisted in the GPK cells for8 to 10 days with low infectivity titers. As deter-mined by both cytopathic effect and nuclear

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FIG. 1. Growth curves of GPCMV and GPHLV in GPE and GPK cell cultures. (Note: the number ofinclusion-bearing cells in GPCMV-infected GPK cells uas very small.) Abbreviations: TCID-,, 50Or tissueculture infective dose; CPE, cytopathic effect. Reproduced from reference 44 with permission.

inclusion counts, GPHLV was found to replicatesomewhat more rapidly than GPCMV in GPEcells. Although both viruses show narrow hostspectra, rabbit kidney cells are susceptible toGPHLV (42), but not to GPCMV (44).

Ultrastructural Development in InfectedCells

Certain distinct differences are apparent inguinea pig cells infected with either GPCMV orGPHLV when examined by electron microscope(Fig. 2). In GPCMV-infected cells, numeroustubular structures are seen within the nuclearinclusions (24, 57), but are not found in theGPHLV-infected guinea pig cells. On the otherhand, clusters of enveloped virus particles en-closed in large vacuoles are often seen inGPHLV-infected nuclei (27), but rarely found inGPCMV-infected GPE cells. Only mature en-veloped virus particles are found extracellularlywhen cells are infected with GPHLV, but bothdense bodies and virus particles are commonlyseen with GPCMV-infected GPE cells; the vastmajority of extracellular particles consist ofdense bodies (24).

Molecular and Biochemical Analysis

Properties of guinea pig herpes-like virusdeoxyribonucleic acid. In early studies Nayakreported that GPHLV had a DNA buoyant den-sity of 1.716 g/cm3 (60). More recently, Huangshowed two major populations of GPHLV DNAprepared from noncloned-virus-infected culture(unpublished data). Approximately 80% of viralDNA, with a density of 1.716 g/cm;, containedonly a portion of the viral genome (Fig. 3, sam-ples Al, A,, and B,), and 20% of the DNA, withdensity of 1.705 g/cm3, contained the whole ge-

nome sequence (Fig. 3A3 and B2). These resultssuggest defectiveness of GPHLV grown in GPEcells. In contrast, 80% of viral DNA obtainedfrom cells infected with cloned virus at low mul-tiplicity (0.1 to 0.2) was intact (Fig. 4, clone 1,fraction C, and clone 2, fraction C), whereas theother 20% was defective, with great repetition(Fig. 4, clone 1, fractions A and B, clone 2,fraction B). These repetitive sequences were de-tected even at low multiplicity of infection im-mediately after cloning.Lack of genetic relatedness between

guinea pig cytomegalovirus and guinea pig

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FIG. 2. Electron micrographs ofguinea pig herpesvirus-infected cells. (A) GPCMV-infected GPE cell; noteintranuclear nucleocapsids, tubules (T), intracytoplasmic and extracellular dense bodies (double arrous), andvirions (x13,400). (B) GPHLV-infected GPK cell; note intranuclear nucleocapsids, viral package (VP)containing enveloped virions, and intracytoplasmic and extracellular virusparticles (single arrows) (x 16,800).(Modified from reference 44.)

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FIG. 3. Restriction endonuclease analysis ofGPHLV and GPCMV DNA. Purified 32P-labeledGPHLV and GPCMV DNAs (approximately 2 x 1WMcpm per gel) were digested with restriction enzymeXbaI at 37°C for 2 h and then subjected to 1% slabgel electrophoresis. Kodak X-omat R film uas usedfor the autoradiograph. DNA samples A and B ofGPHLV uere obtained from two separate experi-ments. Al, A2, and B, had a density of 1.716; A: andB2 had a density of 1.705. (Courtesy of E. S. Huang.)

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FIG. 4. Restriction fragment patterns of GPHLVDNAs obtained from two purified cloned virus stocks.DNAs used in gels for fractions A and B of clone 1and the gel for fraction B of clone 2 each had adensity of 1.716, and DNAs used in the gels forfraction C of clone I and fraction C of clone 2 eachhad a density of 1.705; center C is a duplication offraction C of clone 1. (Courtesy of E. S. Huang.)

VOL. 44, 1980

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herpes-like virus. The relatedness of GPCMVand GPHLV was examined by DNA-DNA reas-

sociation kinetic analysis and restriction endo-nuclease fragmentations of viral DNAs. Therewas no detectable homology between these twoguinea pig herpesviruses in the DNA-DNA reas-

sociation kinetic analysis. Furthermore, the frag-mentation patterns of GPCMV and GPHLV as

analyzed by the restriction endonuclease XbaIwere distinctly different (Fig. 3, GPHLV sampleB, and GPCMV). In addition, there was no

genetic homology between GPCMV and humanCMV Towne strain (E. S. Huang, unpublisheddata).

(Molecular and Biochemical Analysis was

contributed by E. S. Huang, University of NorthCarolina, Chapel Hill.)

Effect of Heparin on Guinea PigHerpesvirus Replication

The presence of as little as 0.1 U of heparinper ml of medium resulted in a 10-fold reductionin the infectivity titer of GPCMV, but not ofGPHLV (10). This property provides a usefultool for differentiating between these two her-pesviruses of guinea pigs, especially when a

mixed infection of the two viruses is present inthe same culture. Inhibition was shown to in-crease as the heparin concentration increased.However, complete inhibition was not obtainedwhen a high concentration of virus was used. Inaddition, the inhibitory process was found tooccur on the cell surface before virus attachmentand penetration. When heparin was added tocultures after virus adsorption, the effect wasnegligible. Other anticoagulants, including Al-

sever solution (sodium citrate, 3.8% in saline)and ethylenediaminetetraacetic acid, exerted no

inhibitory effect on GPCMV replication (10).

Antigenic DistinctivenessThe absence of any antigenic relationship be-

tween these two well-known herpesviruses ofguinea pigs has been reported (42, 44). This was

determined by either inhibition of cytopathiceffect or plaque reduction neutralization tests inGPE cells or by immunoferritin electron micros-copy. Furthermore, cross-neutralization testshave shown that GPHLV and GPCMV are an-

tigenically different from known human andother animal herpesviruses (30, 42, 44), althoughthere has been one report indicating a one-waycross-reaction between human herpes simplexvirus and GPHLV by the complement fixationtest (65). Rabbits are better hosts for productionof specific antiserum to each virus type, sinceguinea pigs may be latently infected with otherherpesviruses. However, guinea pigs producehigher titers of antibody to GPCMV than dorabbits.

GUINEA PIG CYTOMEGALOVIRUS:INFECTION IN VIVO

Natural InfectionThe observation of an unusual change in the

submaxillary glands of guinea pigs was made as

early as 1920, by Jackson, and was interpretedas a protozoan infection (47). Of 48 guinea pigsexamined, 26 were found to have "intracellularprotozoan parasites" in the duct cells of salivaryglands (Table 2). These observations were con-

TABLE 2. Natural GPCMV infectionEvidence of GPCMV infection

No. of Virus inclusion or isola-Date re- Age of .inea guinea tion Antibody studiesported or pigs (M pigs ____ _ ___ _ Referencestudied pigs (m)Ipgas

studied Positive PositiveTesting method Testing method"

No. %c No. %i1920 Adult 48 26 54 Histology ND" Jackson (47)1926 >6 75 63 84 Histology ND Cole and Kuttner (11) and

<1 43 3 7 Histology ND Kuttner (50)1930 Adult 19 6 32 Histology ND Andrews (2)1957 5 39 3 8 Histology NK' 58 NT (CPE) Hartlev (30)1959 1-8 NK NK 33 Histology NK 38 CF SSmith (71)1974 Adult 50 7 14 Histology ND Doisi and Georgescu (18)1975-1979 2-4 (Hartley) 204 6 2 Virus isolation 15 25 NT (CPE oriPFU) Hsiung et al. (37), Choi and

Hsiung (9), Griffith andHsiung (28a)

1976-1979 2-4 (strain 2) 133 0 0 Virus isolation 0 0 NT (CPE) Bia et al. (4)

"NT, neutralization test either by inhibition of cytopathic effect (CPE) or by plaque reduction (PFIJ) (neutralizing antibodytiters of 1:5 or greater were considered positive); CF, complement fixation test.

'ND, Not done.NK, Not known.

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firmed by Cole and Kuttner 6 years later, atwhich time they noted that 84% of full-grownguinea pigs showed swollen epithelial cells in theducts of submaxillary glands but that only 7% ofguinea pigs under 1 month old showed thesechanges (11, 50). The latter investigators furtherdemonstrated that a filterable virus, the salivarygland virus or CMV, was responsible for theatypical cells with nuclear inclusions seen insalivary gland duct cells of guinea pigs. Eversince these early observations, typical intranu-clear and intracytoplasmic inclusions within theduct cells of the salivary glands (Fig. 5A and B)have been considered reliable indications of ananimal's being infected (2, 30, 71). Under elec-tron microscopic examination, the intranuclearinclusions contained immature virus particlesand intracytoplasmic inclusions contained nu-merous mature virus particles (Fig. 5C). Morerecently, we have found that animals showedmaximum numbers of inclusions at 3 to 4 weeksafter primary infection with salivary gland-pas-saged virus (23). Thereafter, the numbers ofinclusions decreased significantly, although vi-rus infectivity titers persisted for 30 weeks. Dur-ing 1975 to 1979 a longitudinal study was under-taken in order to learn the incidence of naturallyoccurring GPCMV infection among commer-cially available animals (Table 2). GPCMV wasisolated from the salivary glands of 6 animals ina total of 204 Hartley strain guinea pigs exam-ined during the 5 years of surveillance, althoughlow titers of GPCMV-neutralizing antibody wereobserved in 25% of the animals tested. The per-centages of antibody-positive animals obtainedfrom different sources varied from shipment toshipment, with as few as 8% at one time and asmany as 50% at another time. Occasionally,guinea pigs in certain shipments showed no ev-idence of prior GPCMV infection. During thesame time period a total of 133 strain 2 guineapigs were tested; none showed antibody toGPCMV before inoculation, and they werehighly susceptible to infection (4).

Experimental InfectionViremia during acute primary infection.

Guinea pigs without preexisting antibody showvirus in their blood 2 to 14 days after intraperi-toneal inoculation with GPCMV; occasionallyviruria is detected as well. The viremic stage isbrief but reproducible (37). Infectious virus isalso recovered from the spleen, kidneys, andlungs of infected animals during the first 2 weeksof infection (Table 3). Virus recovery from thesalivary gland and pancreas commences laterthan that from other tissues during primaryinfection and is followed within 14 days post-


inoculation by detectable titers of neutralizingantibody.Viruria during chronic persistent infec-

tion. Once guinea pigs have been infected withGPCMV, chronic persistent infection is readilyestablished. In animals sacrificed from 3 to 10weeks after inoculation, high levels of infectiousvirus have been consistently recovered from thesalivary glands and pancreas despite the pres-ence of high levels of circulating neutralizingantibody. It was apparent from the experimentsof Hsiung et al. (37) that as antibody titersincreased, virus was generally not recoveredfrom tissues other than the pancreas and sali-vary glands 11 weeks post-inoculation (Table 3).However, virus has been recovered from theurine of some animals, especially inbred strain 2guinea pigs, as late as 16 weeks post-inoculation(4). Furthermore, female strain 2 guinea pigsappear to excrete GPCMV in urine more oftenthan do male guinea pigs (4).Mode oftransmission. (i) Transplacental

transmission. Transplacental transmission ofGPCMV has been demonstrated in guinea pigsduring acute primary maternal infection, i.e., 5to 24 days post-inoculation (Table 4). In studiesby Hsiung and co-workers, infectious virus wasrecovered from 27 of 44 placental tissues and 9of 37 fetal tissues, including brain, lungs, andkidneys, tested after initiation of maternal infec-tion and independent of stage of gestation (9,28a). Infectious virus was also isolated from cer-vical swabs of the infected mothers (28a), Novirus was detectable in the tissues of 43 fetusestaken from female guinea pigs which were in-fected for 40 or more days (Table 4). Animals inthe latter groups showed significant levels ofcirculating antibody, concurrent with infectiousvirus in the maternal salivary glands, typical ofchronic persistent infection. Newborn guineapigs whose mothers had been inoculated morethan 30 days before delivery of the neonates,when tested at birth, showed levels of antibodycomparable to maternal antibody levels, whichdeclined significantly 1 to 2 months after birth(28a). Transplacental transmission of GPCMVwas also reported recently by other investigators(48, 49).

(ii) Contact infection. Several experimentswere undertaken by Choi and Hsiung to deter-mine the degree to which GPCMV is spread byanimal-to-animal contact (9). Among uninocu-lated guinea pigs that were housed with inocu-lated animals of the same sex, 4 of 13 contacteesexhibited rises in antibody titer 2 to 3 monthsafter contact, and infectious virus was recoveredfrom the salivary gland of 1. When uninoculatedguinea pigs were housed in pairs with inoculated


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TABLE 3. Distribution ofGPCMV in the blood, urine, and various tissues during acute and chronicinfection after intraperitoneal inoculation of Hartley guinea pigs with tissue culture-passaged virus stock'

No. of animals showing GPCMV infection/no. examined in:Wk post-inocula-

tion Blood Urine Kidney Lung Spleen Pancreas gland1-2 18/23 2/16 10/14 10/11 9/14 6/11 16/233-10 1/12 0/2 1/7 1/5 2/7 3/3 12/1211-37 0/16 0/11 0/15 0/13 0/15 4/9 11/16

aModified from reference 37.

TABLE 4. Transplacental transmission ofGPCMV in experimentally infected guinea pigs'Mothers Fetuses

No. of No.

Virusinoculation ~~aftrsi showing AntibodyVirus inoculation afteruvi- No. GPCMV titer No. GPCMV GPCMVrus inoc studied in sali- range' at studied in pla- in fetalulation vary sacrifice centa" tissues'

gland

During last 20 days of gestation 5-10 7 4 5-40 23 16/23 5/17During first 10 days of gestation 15-24 6 6 10-20 23 11/21 4/20On the day of conception or 5 to 10 days

before conception 40-70 6 6 40-160 16 0 050 to 60 days before conception 90-150 8 7 80-640 27 0 0

a Modified from reference 9.b Reciprocal dilutions of serum.c Number that showed virus/number studied; fetal tissues include brain, lungs, and kidneys.

animals of the opposite sex, rises in antibodywere observed in seven of seven contactees, andinfectious virus was recovered from the salivaryglands of five of the seven. Since five of theseven females became pregnant, sexual contactin the latter experiment can be assumed. Thissuggests that sexual contact is a more efficientmeans of spreading GPCMV than is environ-mental contact.

GUINEA PIG HERPES-LIKE VIRUS:INFECTION IN VIVO

Natural OccurrenceInbred versus random-bred strains and

age variations. Although GPHLV was initiallyisolated from a spontaneously degenerated kid-ney cell culture derived from a leukemic strain2 guinea pig (41), subsequent study revealed thatnonleukemic strain 2 guinea pigs also harbor thesame virus in their blood and various organs(42). It should be noted that isolations ofGPHLV from blood (leukocytes) or tissues ofinfected animals were accomplished only by cul-tivation of tissue cells or cocultivation with sus-ceptible cell cultures (6, 42, 72). Infectious viruscould not be isolated from cell-free tissue ex-tracts obtained from infected animals or from

infected tissues exposed to freezing and thawingbefore virus isolation attempts (42).Guinea pigs, particularly older animals from

inbred strains, show widespread infection withGPHLV. Hsiung found that inbred strain 2 andstrain 13 guinea pigs, 6 to 12 months old, ob-tained from several different sources, con-sistently showed a high percentage of GPHLVinfection, whereas relatively few random-bredHartley strain guinea pigs exhibited naturalGPHLV infection (Table 5) (34). However, later

TABLE 5. Natural GPHLV infection in differentguinea pig strains'

Age at GPHLVGuinea pig strain tismted studied solation

(mo) No. %

Strain 2 (inbred) <6 41 9 226-12 35 28 80

Strain 13 (inbred) <6 6 0 06-12 6 6 100

Hartley (random <6 474 21 4bred) 6-12 17 3 17

a Reproduced from reference 34 with permission.bVirus isolations were made from leukocytes or

spleen tissues by cocultivation.

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studies showed that hybrids derived from mat-ing strain 2 with Hartley guinea pigs showed aninfection rate comparable to that observed inthe inbred strain 2 animals (Hsiung, unpublisheddata). Detailed study revealed that in strain 2and hybrid guinea pigs tested at monthly inter-vals, GPHLV infection was evident in theseguinea pigs by age 5 to 6 months or older (38).Natural GPHLV infection was present in almostall inbred strain 2 and strain 13 guinea pigs byage 10 to 12 months; virus was distributed widelyin the blood and various tissues, but spleensalways showed the highest virus content. In onetnstance, virus was recovered from the fetal lungtissues of a naturally infected strain 2 guinea pig,suggesting that transplacental transmission ofthis virus had occurred during natural infection(51).

Experimental InfectionPathogenicity and latency. Hsiung and co-

workers (6, 42, 51, 72) found that in experimen-tally infected Hartley guinea pigs, GPHLV couldbe recovered from a wide variety of tissues,including leukocytes, bone marrow, spleen, liver,lungs, kidneys, salivary glands, brain, etc. How-ever, the highest titers of virus were alwaysrecovered from the spleen, regardless of route ofinoculation. Figure 6B shows the distribution ofGPHLV in the blood, spleen, and other tissuesof guinea pigs during acute and chronic infectionafter intraperitoneal inoculation (72). Virus ti-ters increased rapidly during the first 2 weeks ofinfection and reached the highest titer at week3. Thereafter, significant virus titers persistedthroughout the animal's life. No disease attrib-utable to the virus was found, nor were anyvirus-induced intracellular inclusions noted inthe infected tissues. As indicated above, recoveryof GPHLV from infected guinea pigs requirescultivation or cocultivation of infected tissuecells (6, 42, 72). Neutralizing antibody, however,was detectable at about 2 weeks after inoculationand persisted at low levels throughout the testperiod of 12 to 18 months. Once GPHLV infec-tion has been established, the virus can be re-covered from the blood of the infected guineapig throughout its lifetime.Lam and Hsiung showed that experimental

infection of rabbits with GPHLV revealedprompt antibody response regardless of route ofinoculation (52). Virus persistence in rabbit tis-sues was difficult to demonstrate, although in-fectious virus could be recovered readily beforethe development of antibody. Rats and miceinoculated with GPHLV produced minimal, ifany, antibody response, and no virus could be

0LUw

LLz

z

zw0)wr0AJ

GPCMVA100.

504-

0 1.

Bloot

50s

0

GPHLV

(6) (8) 10)

1-3 4-9 10-20

WEEKS AFTER IP INOCULATION

BLOOD OR SPLEEN U KIDNEY U SALIVARY GLAND

TOTAL NO. TESTED

FIG. 6. Distribution of GPCMV and GPHLV indifferent organs of infected guinea pigs 1 to 20 weeksafter intraperitoneal (IP) inoculation. (Modified fromreference 72.)

recovered from the inoculated animals. Studieson experimentally infected hamsters have indi-cated that these animals are also nonpermissivehosts for GPHLV infection (Michalski andHsiung, unpublished data).Transplacental transmission. Since

GPHLV was most commonly isolated frominbred guinea pigs, the question arose as towhether or not the virus was capable of passingthrough the placenta and being transmitted tothe offspring of experimentally infected animals.Lam and Hsiung (51) inoculated female Hartleyguinea pigs with GPHLV at various times duringgestation, and their fetuses were examined forvirus infection. Representative examples ofGPHLV transplacental transmission after intra-peritoneal inoculations are shown in Table 6. Allvirus isolations were made by cocultivation ofinfected tissues with GPK cells (51). The isola-tion of virus from the fetuses seemed to berelated to the titer of virus the pregnant guineapigs received and the duration of infection in the

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TABLE 6. Distribution of GPHLV in maternal and fetal tissues after experimental infection ofHartleyguinea pigsa

Dose in- Virus isolation by cocultivationboculated

Guinea pig (log 50% Days Maternal tissue Fetal tissue EstimatedGuineapig tissue post-inoc- fetal ageno. culture ulation (days)

infective WBC Sp K SG Th P1 Lu Sp Th Corddoses)

LA 23 5.3 88 + + + - + + - + + NDc 50LA 27 5.3 141 + + + - - ND Whole embryo + 15LA 94 5.3 119 + + + + + + + - - + 50

LA 53 6.3 43 + + + - + + + + - ND 60-70LA 56 6.3 51 + + + - - + + - - ND 60-70

a Modified from reference 51.b Tissues: WBC, leukocytes; Sp, spleen, K, kidney; SG, salivary gland; Th, thymus; P1, placenta; Lu, lung;

Cord, umbilical cord. Symbols: +, virus isolated; -, virus not isolated.'ND, Not done.

animals. It was noted that when animals re-ceived 5.3 log 50% tissue culture infective dosesof virus suspension, their fetuses showed virusonly when the mothers were infected for 88 daysor longer. When animals received 6.3 log 50%tissue culture infective doses of virus suspension,infectious virus was isolated from their fetuses43 to 51 days post-inoculation. Thus, animalsreceiving a smaller dose of virus required alonger interval for the virus to get established inthe fetuses, whereas in animals receiving a largerdose of virus, infection occurred in the fetusesafter a shorter period of time. This contrastswith GPCMV transplacental transmission, inwhich infectious virus was not recovered in thefetuses when maternal infection was longer than40 days (Table 4). In the latter case, antibodyappeared to play a role in the control of fetalinfection (28a).Oncogenicity: cell transformation and in-

duction of tumors. Fong and Hsiung foundthat GPHLV strains isolated from both leukemicand nonleukemic guinea pigs were capable oftransforming hamster embryo cells in culture(25). The virus strains isolated from leukemicstrain 2 guinea pigs showed a somewhat highertransforming capacity than that of the virusisolates obtained from nonleukemic Hartleystrain guinea pigs. Infectious GPHLV was re-covered from the transformed hamster cells bycocultivation of several cell lines tested; how-ever, the number of infected cells was small.Michalski et al. found that inoculation of ham-sters with one of these transformed cell lineswhich had undergone repeated passages in cul-tures led to the development of tumors charac-terized as angioid sarcomas and fibrosarcomas(56). However, inoculation of GPHLV directly

into hamsters did not induce tumor production.More recently, malignant transformation of ratembryo cells by GPHLV has been reported (65).

GUINEA PIG HERPESVIRUSES:ANIMAL MODELS FOR HUMANHERPESVIRUS INFECTION

Comparison of Guinea Pig Herpes-LikeVirus and Guinea Pig Cytomegalovirus In

Vivo PathogenicityThe pathogenesis and distribution ofGPHLV

are significantly different from those ofGPCMVinfection (Fig. 6). After experimental infection ofguinea pigs with GPCMV, viremia is very brief,but the virus is consistently isolated from thesalivary gland 2 weeks post-inoculation andthereafter (4, 12, 37). GPCMV is rarely found inthe blood 4 weeks after primary acute infection.However in GPHLV-infected guinea pigs, virusis recovered in the blood immediately after in-oculation and persists thereafter in the leuko-cytes and all other tissues that have been tested,including spleen, kidneys, and salivary glands,although recovery of GPHLV from tissues re-quires cocultivation techniques (6, 42, 72).Whereas intranuclear inclusions and virus par-ticles have been observed in the duct cells ofsalivary gland tissue from guinea pigs that havereceived GPCMV-infected salivary gland tissuesuspension (Fig. 5A and B), no evidence ofGPHLV inclusions has been found in any tissuesof GPHLV-infected animals. Guinea pigs inoc-ulated with GPCMV promptly produce hightiters of specific neutralizing antibody to thehomologous virus; those inoculated withGPHLV develop lifelong latent infection of leu-kocytes accompanied by minimal levels of neu-

VOL. 44, 1980 VIRUSES OF GUINEA PIGS 479


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tralizing antibody to the virus. Although we havebeen able to isolate GPCMV from urine ofGPCMV-infected guinea pigs, to date there havebeen no isolations of GPHLV from the samesource (4).

Similarities Between Guinea Pig Herpes-Like Virus and Human Epstein-Barr VirusOne of the most interesting features of

GPHLV is its interaction with host cells in vivoand in vitro. Leukocytes taken from infectedguinea pigs show no intracellular viral antigenby immunofluorescence. Intranuclear inclusionsand virus particles are not found by either lightor electron microscopy (40). Nevertheless, theviral genome is present in these leukocytes, sincevirus-induced cytopathic effect and viral inclu-sions have been demonstrated after cocultiva-tion with susceptible cells and herpesvirus par-ticles were found after cultivation of the leuko-cytes. Guinea pig leukocytes carrying GPHLVresemble human leukocytes carrying Epstein-Barr herpesvirus, both showing the absence ofviral antigens in vivo and their prompt appear-ance after in vitro cultivation. Table 7 comparessome of the similarities between these two her-pesviruses (35). In each instance there was anassociation with B lymphocytes and the neo-

plastic disease, but, as yet, neither virus has beenshown definitively to be the causative agent inthe development of the neoplastic disease in itsrespective host. Although GPHLV can be iso-lated from leukocytes by cocultivation withGPK or GPE cell monolayers, lymphoblastoidcell lines carrying GPHLV genomes have notbeen established (40). It is possible that GPHLVis a more lytic virus than Epstein-Barr viruswhen cultured in vitro.

Comparison of Guinea PigCytomegalovirus and Human

CytomegalovirusIn many respects the pathogenesis ofGPCMV

infection in guinea pigs closely simulates that ofCMV in humans. Since CMVs of humans andanimals are relatively species specific, guineapigs infected with GPCMV provide an excellentanimal model for human CMV infection. Table8 lists the similarities between GPCMV andhuman CMV infections, in their respective hosts.In ultrastructural studies on development ofGPCMV both in vivo (23) and in vitro (24),surprising similarities have been noted betweenGPCMV in guinea pig cells and human CMV ininfected human cells.A most important finding has been the dem-

onstration of transplacental transmission ofGPCMV in guinea pigs (9, 28a, 48, 49). In con-

TABLE 7. Similarities between GPHLV and humanEpstein-Barr herpesvirus (EBV.a

VirusCharacteristic of infection

GPHLV EBV

Natural host Guinea pig HumanNature of infection Latent LatentPrimary site Leukocyte LeukocyteTransforming capacity

in vitroHeterologous species + +Homologous species - +

Oncogenic potentialin vivo

Disease associated L2C leukemia Burkitt'slymphoma

Cell type associated B-lymphocytes B-lymphocyteswith disease (orproliferation)

"Reproduced from reference 35, with permission.

TABLE 8. Similarities between GPCMV and humanCMV"

VirusManifestation of infection

GPCMV Human

Infection in vitroNuclear inclusions .............. + +Mature virus particles and dense

bodies ....................... + +Infection in vivoAcute infection: viremia ......... + +Chronic infection: viruria ........ + +Congenital infection and

transplacental transmission .. + +Histopathology: intranuclear and

intracytoplasmic inclusions insalivary glands ............... + +

a Summary taken from references 4, 9, 23, 24, 28a,48, and 49.

trast to the mouse system, the guinea pig pos-sesses a placenta with a single trophoblast layer,similar in structure to the human placenta (19),which may allow the passage of virus frommother to fetuses. It has been found that trans-placental transmission of GPCMV occurs inguinea pigs regardless of the time of gestation atinfection, but it depends upon the duration ofmaternal infection (9, 28a). More recently,GPCMV congenital infection with brain damagehas been demonstrated in newborn guinea pigsfrom which infectious virus has not been isolated(28a). Thus, GPCMV-infected guinea pig fetuseshave provided an excellent model for studies ofhuman congenital CMV infection.

PARAMYXOVIRUSESNaturally Occurring Infection

Guinea pig sera chosen at random have dis-

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played an irregular but broad spectrum of pre-viously acquired antibodies to several parainflu-enza viruses (Tables 9 and 10). For example,Hsiung et al. found that random samples oflyophilized complement, obtained from variouscommercial sources of pooled guinea pig sera,showed significant levels of antibody to parain-fluenza type 5 (DA) virus and mumps virus inall lots tested and low levels of antibody toparainfluenza types 1 and 3 in certain lots (36).Among the various individual guinea pig seratested, the results varied from shipment to ship-ment. In general, most of the guinea pigs showedparainfluenza type 5 antibody titers when theyreached age 4 to 5 months. Interestingly, someof them also had hemagglutination inhibitionantibody to mumps virus and to a lesser degreeto parainfluenza type 2 and parainfluenza type3, but only occasionally to parainfluenza type 1.There was no difference in antibody titers insera obtained from different strains of guineapigs.We were unable to find reports in the litera-

ture of isolation of parainfluenza viruses fromguinea pigs, except for one report of parainflu-enza type 1 (Sendai) virus isolated from guineapigs when they were housed with virus-contam-inated mice (74). More recently, a parainfluenzavirus strain serologically identical to SV5 hasbeen isolated from the salivary gland of a Har-tley guinea pig simultaneously infected withGPCMV (F. J. Bia, unpublished data). Sincemost guinea pigs have demonstrated significantpreexisting levels of antibody to parainfluenzatype 5 virus, it is possible that the chances ofisolating parainfluenza type 5 virus are limited.In a study on comparison of cell susceptibilitiesto parainfluenza virus infection, it was notedthat GPE cells were more susceptible to para-influenza type 5 than all the other parainfluenzavirus types tested (M. L. Landry, unpublisheddata). SV5, a strain of parainfluenza type 5 virus,was originally isolated from monkey kidney cellcultures (46). It was not clear whether the mon-keys actually contracted parainfluenza type 5

VIRUSES OF GUINEA PIGS- 481

virus from humans or other animal species, in-cluding guinea pigs, after they were kept incaptivity (32).Antibody Response After Experimental

InfectionIn studies by Hsiung et al., guinea pigs show-

ing no detectable preexisting antibody to para-influenza type 5 virus often showed a high inci-dence of heterotypic antibody rise when inocu-lated with one of the other parainfluenza virustypes (36). It was possible that a number oftheseanimals had been infected previously with par-ainfluenza type 5, but did not possess antibodiesat a level measurable by the techniques used. Inthe early 1960s it was found that antisera pre-pared in guinea pigs against parainfluenza virustype 1, 2, or 3 occasionally contained a commonantibody, i.e., to parainfluenza type 5 virus. Thiscommon antibody in antisera prepared in thisanimal species has caused considerable confu-sion in identification of the parainfluenza virusesisolated from clinical specimens (75). In addi-tion, guinea pigs and hamsters possessing anti-body to parainfluenza type 5 virus were found tobe resistant to experimental infection with thisvirus (7).

In studies of the antigenic properties of theDA strain of parainfluenza type 5 in guinea pigs,hamsters, and rabbits, it was noted that bothhomologous and heterologous antibody re-sponses were demonstrated after immunizationwith the virus (36). The extent of heterologousantibody rise was found to be dependent uponthe degree of susceptibility of a specific animalspecies and upon the presence or absence ofpreexisting antibody to members of the parain-fluenza-mumps virus group (36). Parainfluenzatype 5 (DA) virus and mumps virus could con-ceivably be considered antigenic variants of thesame virus on the basis of data obtained fromsera taken from immunized guinea pigs (Fig. 7).In contrast, no antigenic relationship could bedemonstrated between parainfluenza type 5 andmumps viruses when hamsters were used for

TABLE 9. Hemagglutination inhibition and neutralizing antibody titers to parainfluenza viruses in pooledsera (complement) of normal, healthy guinea pigs"

Hemagglutination inhibition antibody titerh to:

Source of pooled Total no. of Newcastlesera pools tested Parainflu- Parainflu- Parainflu- Parainfluenza 5 Mumps disease vi-

enza 1 enza 2 enza 3rus

Lab A 2 20 - - 80 (20) 40 -Lab B 4 - - 40 40 (10) 40 -Lab C 2 20 - 20 320 (40) 80 -

a Modified from reference 36.b Reciprocal serum dilution. -, Titer less than 1:10. Numbers in parentheses are neutralizing antibody titers.


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TABLE 10. Hemagglutination inhibition antibody titers to parainfluenza viruses in single sera of normal,healthy guinea pigs'% with hemagglutination inhibition antibody titer greater than 1:10

Source of individual Total no. of sin- Newcastleanimals gle sera tested Parainflu- Parainflu- Parainflu- Parainflu- Mumps disease vi-

enza 1 enza 2 enza 3 enza 5rus

Lab A 240 0 21 16 84 71 0Lab B 140 5 30 10 70 48 0

a Modified from reference 36.

ANTIBODY RESPONSES

VIRUS | HAMSTERS

WEEKSAFTERI ST INO

*-.-- DA ANTSOWOY

TO DA OR MUMPS VIRUS INFECTION

o----o --o MUMPS ANTIBODY

FIG. 7. Antibody responses to parainfluenza type 5 (DA) or mumps virus in hamsters, rabbits, and guineapigs as measured by hemagglutination inhibition (HI) test. Abbreviations: ino, inoc, inoculation. (Reproducedfrom reference 36, with permission)

immunization (36). Similarly, a one-way anti-genic crossing between parainfluenza type 5(SV5) virus and parainfluenza type 2 (CA) viruswas demonstrated in antisera prepared in guineapigs (8), whereas no antigenic relationship couldbe demonstrated between parainfluenza type 5(SA) virus and parainfluenza type 2 (CA) viruswhen antisera prepared in hamsters were used(70). Since DA, SV5, and SA are serologicallyidentical, the name parainfluenza virus type 5has been used (32). Thus, investigators shouldbe aware of the possibility that prior infectionwith parainfluenza viruses, especially type 5,may have occurred in any guinea pig used forantiserum production.

RETROVIRUSES OF GUINEA PIGSNomenclature for the Guinea Pig

RetrovirusesSince the original observation of virus-like

particles in tissues and cells of L2C leukemic

guinea pigs that were absent in nonleukemicguinea pigs, the name "guinea pig leukemia vi-rus" has been used in many of the early reportsdescribing the virus particles observed (22, 59,63). In 1972, Hsiung first demonstrated that asimilar virus particle could be induced in cellculture, as had previously been shown with mu-rine C-type virus; therefore, the name "guineapig C-type virus" was used (33).

Morphogenically, the GPRV particles, espe-cially those induced by BUdR, show some sim-ilarity to the B-type viruses of the murine spe-cies. Thus, the guinea pig virus has been classi-fied with the murine B-type virus rather thanthe C type (15, 69). However, no interspecies-specific antigen has been demonstrated with theGPRV. Furthermore, the reverse transcriptaseof the guinea pig retrovirus is magnesium de-pendent (55). Since GPRV is serologically dis-tinct from the murine B-type virus as well aspossessing several unique morphological char-

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acteristics of its own, Hsiung has suggested thatGPRV be placed in a separate category and bedesignated G type, representing guinea pig virusof the Retroviridae family (34). For the conven-ience of the present discussion, we shall use theterm GPRV.

Observation of Virus Particles in Cells ofL2C Leukemic Guinea Pigs and Placental,

Fetal, and Nerve Tissues of NormalGuinea Pigs

Electron microscopic studies by Fong andHsiung (26, 27) revealed two morphologicallydistinct types of virus particles in various tissuesof guinea pigs with L2C leukemia, namely, intra-cisternal A-type particles 90 to 100 nm in diam-eter with electron-lucent centers and extracel-lular, "mature" virus-like particles approxi-mately 90 to 110 un in diameter with electron-dense cores. The former were usually formed bybudding into the endoplasmic reticulum. Themature virus-like particles were always seen inthe intercellular space, and a great number werefound in the plasmas of leukemic guinea pigs(26, 27). In addition, a small number of intracy-toplasmic A-type virus particles 80 nm diameterwere also found in leukemic cells.

After an extensive search for virus particles innormal guinea pig tissues, intracisternal A-typeparticles were observed by Hsiung et al. in pla-cental cells and in gonads of both male and


female fetuses (39). Similar particles had beenseen previously in the gonad cells of fetal guineapigs (1, 5) as well as in germinal centers ofnormal guinea pigs (54). Virus particles weremost prevalent in fetal tissues at approximately30 to 40 days of gestation, decreasing in fre-quency in older fetuses and rarely found afterbirth. The intracytoplasmic A-type virus parti-cles have been found in the spiral ganglion andtrigeminal ganglion of normal guinea pigs (13,73). These intracytoplasmic A-type virus parti-cles observed in the ganglia of guinea pigs resem-ble the intracytoplasmic A-type particles ob-served in BUdR-induced virus particles in cul-tured guinea pig cells. Table 11 summarizes thedistributions of the different types of GPRVunder different conditions.

Induction of Guinea Pig Retrovirus inCultured Cells

Since GPRV particles are seldom observed innormal adult guinea pig tissues, attempts havebeen made to induce the virus in cultured guineapig cells. Hsiung et al. found that after' exposureto BUdR (40 ,g/ml of culture medium), bothintracytoplasmic and extracellular GPRV parti-cles were present in all primary cells derivedfrom normal guinea pigs regardless of tissueorigin or guinea pig strain (39) (Table 12). Thesame types of virus particles have also beenobserved in passaged normal and transformed

TABLE 11. Schematic drawing illustrating the distribution of GPRV in tissues and cultured cells fromleukemic and normal guinea pigsa

DISTRIBUTION OF GUINEA PIG ONCORNAVIRUS IN VIVO AND IN VITRO

rI- NTTRACELLULAR -IINTRACISTERNAL A INTRACYTOPLASMIC A

@0 0

BUDDING AT r- EXTRACELLULAR-1CELL MEMBRANE ENVELOPED A 'MATURE PARTICLES'

©11 0LEUKEMC GUIEA PIG'.

TISSUE

PLASMA. SER

NORMAL GUINEA PIG'FETAL TISSUE

TRIGEMINALGANGLION

PLASMA. SERUM oGdAMNIOTIC FLUID

CULTURED GUINEA PIGCELLS:WITH BrdU

WITHOUT BrdU

++

RARE -

- RARE

+ +

4- RARE

+4-+

+

a Symbols: +++, large numbers of single particles or groups, frequently found; ++, single particles andoccasional clusters of particles; +, single particles, infrequently seen; -, not seen; *, absence of cells. BrdU,BUdR (Reproduced from reference 27, with permission).


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TABLE 12. Induction ofGPRV in cultured guinea pig cellsaObservation ofGPRV in cell

Culture cultureType of culture designa- Guinea pig strain Tissue origin

tion With- Without BUdR

BUdR BdPrimary cell A 2 Adult spleen, kidney - ++

B 13 Adult spleen, kidney - ++C Hartley Adult spleen, kidney - ++AD023 2-Hartley hybrid 30-day-gestation fetus - +++

Passaged cell 106 2 7,12-Dimethylbenz[a]-transformed GPE + ++cells

74 2 3-Methylcholanthrene-transformed GPE + ++cells

LgpS 2-Hartley hybrid Leukemic spleen cell line - ++a Reproduced from reference 34, with permission.b -, No virus particles; +, a few virus particles; ++, numerous virus particles; +++, large aggregates of virus

particles.

cell lines (21) derived from various guinea pigtissues after exposure to BUdR induction. Sim-ilar findings have been reported subsequently bymany laboratories (15, 62, 67). Morphologicallyand biochemically, these extracellular virus par-ticles induced in cell cultures derived from nor-mal guinea pigs are similar to the virus-likeparticles in plasmas obtained from leukemicguinea pigs. However, repeated experiments toinduce disease in guinea pigs with GPRV eitherderived from cultured cells or obtained from theplasmas of leukemic guinea pigs have been un-successful. As yet, there is no biological methodfor assaying the infectivity of GPRV, despitenumerous studies by various investigators (26,55, 62, 64).

Biochemical Studies of Guinea PigRetrovirus

More recently, investigators have focused onthe in vivo and in vitro expression of GPRV andthe biochemical and antigenic characterizationof its virions. The proviral DNA sequences ofthe BUdR-induced GPRV have been present ina relatively constant amount in all guinea pigcell DNAs examined so far, indicating that theGPRV is an endogenous virus system (55, 61).GPRV messenger RNA is synthesized in BUdR-treated normal guinea pig cultured cells and inthe spleen and lymphoblasts of leukemic guineapigs, but not in untreated normal spleen, liver,or GPE cells (16, 17). The messenger RNA syn-

thesized appears to represent the entire GPRVgenome.

Virus particles from the BUdR-treated guineapig embryo cells have RNA that is at least 90%homologous with the virus particles obtainedfrom plasma of leukemic guinea pigs and resem-ble the latter insofar as possessing a similar

density (1.16 to 1.18 g/cm3) in sucrose gradientsand a reverse transcriptase requirement for Mg2"rather than Mn2" (69). The guinea pig retrovirusdoes not possess a group-specific antigen and isnot antigenically related to murine, hamster, rat,feline leukemia, RD-114, woolly monkey, or Ma-son-Pfizer monkey retroviruses (58, 64).

MIXED INFECTIONSMixed Infections In Vitro

Guinea pig herpes-like virus and guineapig retrovirus. Cultured GPK or guinea pigspleen cells, especially those derived from oldinbred strain 2 guinea pigs, often show sponta-neous herpesvirus cytopathic effects 5 to 7 daysafter cells are cultured. If BUdR is added tomaintain such cultured cells, both GPHLV andGPRV can be observed simultaneously by elec-tron microscopy (Fig. 8) (38). In cultures withmixed infection, progeny virus particles of bothGPHLV and GPRV can be identified on thebasis of their distinctive morphologies and dif-ferences in size (27, 28). Application of immu-noferritin electron microscopy techniques to thedoubly infected cells, using antiserum specific toeach virus, reveals a small number of virus par-ticles which exhibit retrovirus morphology butreact with antiserum to GPHLV as shown byferritin tagging to the surface of the retrovirusparticles (27, 28). Since GPRV or GPHLV par-ticles derived from singly infected cells did notreact with heterologous antiserum, Fong andHsiung first reported that a pseudotype ofGPRV (GPHLV) was produced in culturedguinea pig cells doubly infected with both GPRVand GPHLV (27). Although pseudotype virusparticles had been reported in mixed experimen-tal infections of cultures with herpes simplex

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vi,,-'''., ;, ,Y-',+ ' :. ...

P

W4

5 t~~~~~~~~~~~~A

/ 4

A- e %

~~~~!"~~~~~~~~~~-~A

! *~~~~~~A

B f < S .. I fVo.~~~~~~~~~~~~~~~~~~~~~~~~~'-

40~~~~~~~~~~~~~vt

a A~~

FIG. 8. Electron micrographs showing mixed infections with GPHLVand GPRV. (A) At low magnification(x19,680), nucleocapsids ofGPHLV in the nucleus (N) and extracellular virus particles of both GPHLV andGPRV at the cell surface. (B) At higher magnification (x100,800), GPHLV (H) and GPRV (R). (B is modifiedfrom reference 35.)

485


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and vesicular stomatitis viruses (45), and alsowith vesicular stomatitis and murine leukemiaviruses (76), pseudotype virus particles occurringin natural host cells experimentally infected withtwo endogenous viruses had not been seen beforethese studies. The ability of the pseudotypevirus GPRV (GPHLV) to infect guinea pig cellsor cells of other animal species has not beendetermined.Guinea pig cytomegalovirus and guinea

pig paramyxovirus. In an attempt to recoverinfectious virus from the salivary glands of aguinea pig which was previously inoculated withGPCMV, we observed an unusual cytopathiceffect in a GPE cell culture inoculated with thesalivary gland suspension. Upon addition of a0.5% guinea pig erythrocyte suspension into theinfected monolayer, hemadsorption ofthe eryth-rocytes was observed. The hemadsorbing viruswas subsequently isolated and identified serolog-ically as SV5, a strain of parainfluenza type 5virus. This mixed infection, i.e., GPCMV, a her-pesvirus, and the paramyxovirus of guinea pigs,is illustrated in Fig. 9. Using immunoferritinelectron microscopy, the herpesvirus particlewas identified as GPCMV and the paramyxovi-rus was tagged with SV5 antiserum (Bia, unpub-lished data). Both viruses were identified also incell culture by the neutralization test, using in-hibition of cytopathic effect for GPCMV andinhibition of hemadsorption for the paramyxo-virus isolate.

Mixed Infection In VivoSynergistic reaction with guinea pig

herpes-like virus and guinea pig retrovi-rus. Since the presence of both GPRV andGPHLV has been demonstrated in guinea pigswith leukemia, it has been postulated that bothviruses may play a role in the oncogenesis of thisdisease. GPRV is apparently present in allguinea pigs, but it is expressed only under certainconditions. Expression of the latent GPHLV isgenerally age and strain dependent. Experimen-tal investigation of the role played by these twovirus types in the development of neoplasticdisease in guinea pigs has shown that inoculationof GPHLV or mixtures of GPRV and GPHLVhas led to the development of self-limited lym-phoproliferative changes characterized by hy-perplasia in the spleen and lymph nodes (Table13) (34, 43). However, when guinea pigs areinoculated with GPRV alone, the incidence ofhyperplasia is significantly lower. It is not knownwhether a comparable synergistic reaction couldoccur naturally when the two virus infectionstake place at specific times, such as during preg-nancy or in the neonatal period.

Interference between guinea pig cyto-megalovirus and guinea pig herpes-like vi-rus. During investigations of chronic persistentGPCMV infection in strain 2 guinea pigs, itbecame clear that GPHLV could not be dem-onstrated in those strain 2 guinea pigs whichwere inoculated with GPCMV early in life, be-fore emergence of active GPHLV infection (Ta-ble 14) (4). Although GPHLV was repeatedlyisolated from the parallel age-controlled strain2 guinea pigs, GPHLV was not isolated from43 animals experimentally inoculated withGPCMV at an early age (4). The reason(s) forthese findings is not clear, although viral inter-ference resulting from CMV infection has beenreported with human or murine CMV infectionsas discussed (4).

CONCLUDING REMARKSGuinea pigs are common laboratory animals

and have been used extensively for biomedicalresearch over many years. They are compara-tively clean animals, with a relatively low inci-dence ofendogenous virus infection as comparedwith other animals, especially simian species (31,46). Since guinea pigs have several distinct ana-tomical similarities to humans (19), they haveserved as excellent models for studies of patho-genesis of many viral infections of human im-portance, particularly congenital disease associ-ated with transplacental transmission of herpes-viruses (9, 48, 49, 51). However, investigatorsshould be aware of the presence of the few well-characterized viruses (Fig. 10) associated withguinea pigs and should understand the impactof the presence of these viruses in order tointerpret data properly.The study of endogenous guinea pig viruses,

on the other hand, especially those of the her-pesvirus group, has proven to be of considerablesignificance in understanding the pathogenesisof comparable latent viruses in humans. Al-though endogenous viruses are not ordinarilyassociated with overt disease, they can, underspecific conditions, be activated to produce char-acteristic disease syndromes. Thus, usingGPCMV infection in guinea pigs as an animalmodel, the serious medical complications asso-ciated with human congenital CMV infection orthe CMV syndrome after renal transplantationcan be better studied.Mixed virus infections have been reported in

humans as well as in plants, bacteria, and otheranimal species. It is not even known whethersuch mixed infections would result in the devel-opment of pseudotypes in vivo. They could con-ceivably result in a beneficial effect or lead toirreparable damage to the host. Experimental

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p=S~~~:~~~~tCMV X

,.,>ot . X~~-A8S,a ; t

.>~~~~~~c.-a7,wx t

;W 'rt t' + #, '4.;

ht.

FIG. 9. Electron micrographs showing mixed infections with GPCMV and a paramyxovirus. (A) At lowmagnification (x16,320), both GPCMV dense bodies (D) and virions, as well as many filamentous forms ofparamyxovirus (P), are seen on the cell surface. (B) At higher magnification (x100,800), a budding ofparamyxovirus (P) and a GPCMV virion (CMV) are shown.

487

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TABLE 13. Experimental infection ofguinea pigswith GPHLV and GPRVn

Lymphoid hyperplasia

No. positive/no. % PositiveMaterial inoculated examined

Spleen Lymph Spleen Lymphnode node

GPRV alone 2/11 2/11 18 18GPHLV alone 15/21 15/20 71 75GPHLV and GPRV 14/20 21/22 67 95Ultraviolet- 0/15 0/15 0 0

irradiated GPHLVa Reproduced from reference 34, with permission.

TABLE 14. Effect of experimental GPCMV infectionon natural GPHLV infection in strain 2 guinea

pigsNo. of GPHLV isolated/ % of animals with

Age of no. tested GPHLV isolatedguinea Uninocu- Uninoc- GPCMV-pigs (MO) lated con- GPCMV-ultd iou

trols inoculated controls lated

0-2 5/79 0/16 6 02-4 8/33 0/42 24 04-6 23/40 1/29 58 3

a Reproduced from Bia et al. (4), by permission ofthe University of Chicago Press. Copyright 1979, Uni-versity of Chicago.

GPCMV

(1920".1926. 19571

PARAWYXOVIRUS(1959. 1979) HARTLEY STRAII

GPHLV(1969)

GPRV ( RETROVIRUS)(1967.1972)

IN 2

GPX(1979)

DATE FIRST REPORTED

FIG. 10. Schematic diagram illustrating the fewwell-known viruses which are commonly isolatedfrom guinea pigs. GPX is a newly characterized her-pesvirus ofguinea pigs (Bia et al., in press).development ofpseudotype viruses and researchinto their pathogenesis should provide insightinto their potential for acting in either fashion.Pseudotype viruses might also provide informa-tion regarding their potential role in instances ofhuman disease that are associated with eithermixed viral infection or viruses that, by them-selves, cannot be identified as sole causativeagents.

There are many considerations that can andshould be borne in mind when one is assessingthe significance of endogenous viruses of guineapigs in biomedical research, and this reviewwould serve its purpose if it stimulates furtherthoughts on the pathogenesis of natural viralinfections and viral latency.

ACKNOWLEDGMENTSPortions of the work described were carried out

under U. S. Public Health Service (USPHS) researchgrant HD10609 from the National Institute of ChildHealth and Human Development, USPHS researchtraining grant AI 07018 from the National Institute ofAllergy and Infectious Diseases, and Veterans Admin-istration Research Fund.We are grateful to Kari Hastings, Mary Wright,

Ruth Donahue, and Barbara Nunes for their help inthe preparation of this manuscript. The data for mo-lecular and biochemical analysis of GPCMV andGPHLV DNAs were kindly supplied by E. S. Huang,University of North Carolina, Chapel Hill.

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