Latent Virus Infections in Primate Tissues with Special Reference to Simian Viruses G

Home , Simian foamy virus

BACTERIOLOGICAL REVIEWS, Sept., 1968, p. 185-205 Vol. 32, No. 3 Copyright"@ 1968 American Society for Microbiology Printed in U.S.A. Latent Virus Infections in Primate Tissues with Special Reference to Simian Viruses G. D. HSIUNG Yale University School of Medicine and Veterans Administration Hospital, West Haven, Connecticut 06516 INTRODUCTION...... 185 CLASSIFICATION OF SIMIAN VIRUSES BASED ON PHYSICOCHEMICAL PROPERTES. . 186 BRIEF DESCRIPTIONS OF THE MAJOR SIMIAN VIRUS GROUPS . .186 DNA Viruses ..186 Papovavirus group ..186 Adenovirus group ..187 Herpesvirus group ..187 Poxvirus group ..187 RNA Viruses ..187 Picornavirus group. 187 Reovirus group ..192 Myxovirus group ..192 Pseudomyxovirus group ..193 EPIDEMIOLOGICAL ASPECTs OF LATENT VIRUS INFECTIONS IN SIMIAN TISSUES. . 193 Detection ofLatent Infection in Cell Cultures by Prolonged Cultivationv... 194 Incidence of Virus Infections in "Normal" Monkey Kidney Cell Cultures. . 194 Comparison of Virus Types Recoveredfrom Tissues ofMonkeys With or Without Quar- antine. 195 Mixed Infections.196 Distribution of Virus Types According to Conditions ofMonkey Shipment.197 Acquisition of Parainfluenza Virus Antibody in Monkeys During Captivity.198 POSSIBLE ORIGIN OF VIRUS INFECTION IN KINEY TISuEs.199 Model Study in Viremia and Viruria in Monkeys.199 Experimentally Induced Latent Infection ofSV40.199 Persistent Infection ofSV5 Group of Viruses in Monkeys after Intranasal Inoculation.. 200 LATENT VIRUS INFECTIONS IN HUMAN KIDNEY TIssuEs.200 CONCLUDING REMARKS.201 LITERATURE CITED.201

INTRODUCTION scope of this review. Furthermore, certain viral In recent years, there have been increasing agents have even been recovered from germ-free numbers of reports concerning the recognition of animals (4, 79). Thus, one cannot be assured that latent virus infections in tissues of primates as the organs of any animal species are microbiolog- well as nonprimates. The original isolation of ically sterile. adenovirus from adenoid tissues (87) and the sub- While searching for the viral etiology of human sequent recovery of adenovirus in tonsils (26), Kuru disease, Gajdusek and associates (30, 83) cytomegalovirus in adenoids (86), and several isolated 47 strains of viruses from a total of nine other virus types in kidney tissues (8, 22, 63) chimpanzees which had been experimentally in- are well-documented instances of latent virus oculated with Kuru 1 to 3 years previously. Some infections in tissues of man. Various methods of these isolates were identified as adenovirus, have been used for the detection of hidden reovirus, and foamy virus. All virus isolates were viruses in human tissues and these methods have obtained from tissue explants from chimpanzee been reviewed by Melnick et al. (68, 70). organs, such as brain, spinal cord, spleen, and Latent virus infections have also been recog- kidney. It became evident that some of these ani- nized in cell cultures derived from tissues of non- mals had been latently infected by these viruses primates. These include the isolation of cyto- prior to the inoculations of Kuru. megalovirus from the salivary glands of guinea Simian tissues have been of special interest, pigs and mice (38, 94), of herpesvirus from dog since monkey kidney cell cultures are commonly (95) and horse (51) kidney cell cultures, of adeno- used in the studies of animal viruses and in the virus from avian (16) and swine (11) kidney tis- preparation of virus vaccines for human use. As a sues, and of many others which are beyond the result of the extensive use of primate cell cultures, 185 186 HSIUNG BACTERIOL. REV. a great number of simian viruses have been re- determination by membrane filtration was em- covered from a variety of monkeys, baboons, and phasized (6). It was shown that SV5, measles, and marmosets (9, 12, 14, 15, 24, 25, 29, 40, 41, 47, foamy virus did not pass through a membrane 52, 56-58, 60, 65, 66, 72, 88, 89, 98, 99, 100). filter having a 100 nm limiting pore diameter; These indigenous viruses have caused consider- thus, these were considered large viruses. SVl1 able frustration and economic loss to workers in and SV12, which passed through the 100-nm filter terms of contaminated virus stocks and rejected with some reduction in infectivity titer, were in cultures. In this paper, I have reviewed the avail- the medium range; SV16 and SV40 passed able data, although in many instances the infor- through both 100- and 50-nm filters and were con- mation is rather limited regarding latent virus in- sidered small viruses. Thus, the use of membrane fections in primate tissues with special reference filtration has facilitated the grouping of unknown to viruses isolated from the monkey kidney tissues viruses (44). of apparently healthy animals. A brief descrip- The use of electron microscopy has led to tionoftheclassification of simian viruses and some highly detailed studies of the morphological struc- of the physicochemical and biological properties ture and the size of some simian viruses, especially ofthe major virus groups is also included. In addi- SV40 (31, 34). The fine structure of other simian tion, attempts have been made to elucidate the viruses has been described. These include simian origin of virus infections in primates, the mech- adenoviruses (2, 28), enteroviruses (3, 39), myxo- anism of viral persistence in kidney tissues, and viruses (19), foamy virus (61), and Yaba poxvirus the problems in the control of virus infections in (21). Based upon physicochemical properties, primate cell cultures. such as nucleic acid type and ether sensitivity, and on the results obtained by ultrafiltration and CLASSIFICATION OF SIMIAN VIRUSES electron microscopy, the classification of simian Simian viruses are, by defintion, a group of viruses has been proposed (45) in a manner simi- viruses isolated from tissues or excreta of pri- lar to that described for other animal viruses (1, mates other than man. The isolation of virus-like 44). This classification includes the DNA and agents from monkey kidney tissue cultures was RNA viruses, as shown in Table 1. In the DNA first reported by Rustigian et al. in 1955 (89). virus group, there are four major subgroups: the S-ubsequently, as a result of the extensive use of simian papovavirus, adenovirus, herpesvirus, monkey kidney cell cultures, especially in the and poxvirus. Similarly, there are four major sub- preparation of virus vaccines, a great number of groups among the RNA viruses: the simian pi- simian viruses have been recovered as endogenous cornavirus, reovirus, myxovirus, and pseudo- tissue contaminants by Hull and associates (56- myxovirus (monkey measles and foamy agents). 58). Since such a large number of viral agents have been isolated from monkeys, Hull et al. have BRIEF DESCRIPTIONS OF THE MAJOR designated all viruses isolated from simian origin SIMIAN VIRus GROUPS as "SV" with serial numbers, irrespective of their Recognition and characterization of simian properties or taxonomic order (56). In the mean- viruses in cell cultures are ofpractical importance, time, Malherbe and Harwin of South Africa since monkey tissues often harbor a variety of isolated several viral agents from vervet monkeys viruses. Some of the physicochemical properties and designated them "SA" with serial numbers, of the major simian virus groups are summarized indicating simian viruses isolated from South in Table 1. Methods used for recognition of each African monkeys (65, 66). simian virus group have been reported by various On the basis of certain biological properties, investigators and are described briefly below. especially cytopathic effect (CPE), simian viruses Some of the simian viruses, for example, the pox- were originally divided into four groups by Hull virus, were not recognized as latent virus infections et al. (57). Other biological properties, including in primate tissues. But, for completeness in listing plaque morphology, host-cell susceptibility, and the simian virus groups, the poxvirus group is hemagglutinin production, have also been used included. For a detailed discussion on the classifi- for grouping these viruses (54, 81). cation of the major groups of animal viruses, the More recently, the physicochemical properties reader is referred to Andrewes (1) and to Melnick of certain simian viruses have been studied. Infec- and McCombs (71). tious deoxyribonucleic acid (DNA) was obtained from SV40-infected cultures soon after the virus DNA Viruses was discovered (32), and infectious ribonucleic acid (RNA) has been successfully extracted from Papovavirus groups. SV40 or the vacuolating several strains of simian enterovirus (39). During virus of monkeys was found to be a small DNA, our study characterizing the simian viruses, size ether-resistant virus (52). This virus was first VOL. 32, 1968 LATENT VIRUS INFECTIONS IN PRIMATE TISSUES 187 isolated from kidney cell cultures derived from bodies to the B virus, and the percentage rose to rhesus and cynomolgus monkeys by Sweet and 60 to 70% when the monkeys were confined in Hilleman (98) and was isolated from patas mon- "gang-cages." Other studies indicated that 100% keys in our laboratory (52). Subsequently, SV40 of the experimental monkeys showed B virus was also isolated from kidney tissues of African antibody rise when the monkeys were housed green monkeys (47). In rhesus monkey cells, together over a period of 6 weeks (67). SV40 produced intranuclear inclusions, although Herpes T or Herpesvirus tamarinus, a new mem- CPE generally was not apparent (52). In patas, ber of the herpesvirus group, was isolated from African green monkey, and baboon cell cultures, marmoset, a New World monkey obtained from SV40 induced extensive vacuolations in the cyto- South America (41, 72). Although herpes T was plasm of infected cells (Fig. 1, Bi). Intranuclear highly pathogenic to marmosets, it did not pro- inclusions of a basophilic Feulgen-positive nature duce disease when inoculated into rhesus or green were observed in infected monkey cells regardless monkeys or baboons (72). of monkey species (Fig. 1, B2). Nonsimian cells Both herpes B and T grew readily in cultures of were generally resistant to SV40 infection (52). monkeys, human, and rabbit cells and produced However, transformation has been observed in extensive cellular degeneration (Fig. 1, Dl). The human (64, 93) and hamster cells (13) infected type A eosinophilic inclusions observed in herpes with SV40. Since the discovery that SV40 pro- B-infected monkey cells resembled those produced duced tumors in hamsters (23), this virus type has by herpes simplex virus-infected human cells. been used by hundreds of investigators as a Pinpoint pocks were produced on the chorio- model for oncogenic studies. allantoic membrane ofembryonated eggs. Follow- Adenovirus group. A total of 18 simian adeno- ing inoculation with virus B or T strains, these virus types have been reported (81). Most of these pocks resembled those produced by other mem- were isolated from rhesus and cynomolgus mon- bers of the herpesvirus group. key kidney cell cultures by Hull and co-workers Cytomegalovirus was isolated from kidney cell during 1954-1956. Additional types have been cultures prepared from African green monkeys obtained from green monkeys (66), patas monkeys (12). The virus underwent a long latent period (101), and chimpanzees (83). Clusters of rounding before the appearance of CPE. The production of cells were commonly seen throughout the cell intranuclear DNA inclusions in the monkey cells sheet of infected cultures (Fig. 1, Cl). In hematox- (Fig. 1, D2) confirmed its presence. ylin-eosin stained preparations, characteristic Poxvirus group. Monkey pox was described by basophilic inclusions were seen in the nuclei of von Magnus et al. during an epidemic among infected cells (Fig. 1, C2). Morphologically, captive cynomolgus monkeys (106) where it simian adenoviruses were found to resemble appeared to exist as a silent infection. Another human adenovirus. They were 80 nm in diameter poxvirus, the Yaba virus, was obtained from sub- and icosahedral in shape (2, 28). All simian ad- cutaneous tumors of rhesus monkeys (76). The enoviruses share a complement-fixing antigen replication ofYaba virus in tissue culture was first with human adenovirus strains, but no serolog- described by Yohn et al. (109), who demonstrated ical cross between human and simian adeno- cytopathology in an infected green monkey cell virus types was noted when viruses were tested line, BSC-1. by neutralization methods (56, 57). As with human adenoviruses, most simian adenovirus-infected RNA Viruses culture fluids showed hemagglutination with rhesus monkey or rat erythrocytes, or with both. Picornavirus group. Originally, the name On the basis of these hemagglutinating proper- ECMO (enteric cytopathic monkey orphan) or ties, simian adenoviruses were divided into four enteroviruses of monkeys was used to describe subgroups by Rapoza (81). Recently, Hull and this group of viruses. Most of the virus strains associates (55) reported that several simian were isolated from the stools of monkeys; the adenovirus types were capable of producing others were isolated from kidney tissues (39, 40, tumors in infant hamsters. Among the different 56). The CPE produced in monkey kidney cells types of simian adenoviruses tested, SV20 (Fig. 2, A) was similar to that produced by the and SA7 appeared to be most oncogenic. enteroviruses isolated from man. However, no Herpesvirus group. In monkeys of the Old antigenic relationship has been demonstrated be- World species, herpes B or Herpesvirus simiae tween the monkey and human strains tested (56). produced a naturally occurring mild infection. Plaque morphology and host cell spectrum of sus- However, most human cases of B virus infection ceptibility have provided additional bases for sub- have been fatal. Hull and Nash (59) showed that dividing these viruses into two groups (54). 10% of newly caught rhesus monkeys had anti- Studies of the physicochemical properties of 188 HSIUNG BACTERIOL. REV.

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_s . .w | _ 6 ..... F''fi'2L'"'';'.;;s.,: .:.*.: '.:....'s li_ a..;.s'w....:' FIG. 1. Simiani DNA virutses. Top row: Monikey kidniey cells ini flu,id culltulres, X 100. Bottomi1 row: Heiema- toxyliln-eosinl stainled preparationls ojmonkev kidney cells, X 970. (Al amid 2) Rhesuts momnkel kidiniey cell culture colitrol. (B! amid 2) SV40-iimfrcted patas monkey kicdniey cells. (Cl anid 2) SVI-injected r/heslus monmkev kitldieyv cells. (Dl) Herpes B-imifected r-hesuts monkeyv* cells.°,....;(D2):S::...... Cvtomegalovirus-infected...... w,og.Sw'- rhesus monikeY cells. Vote nitranumclear imiclutsionIs iln B2, C2, amidt D2. From..;.,:referentce.,..Sg9:<45...'!.b::'ty..: some 15 strains of simian enteroviruses.. revealed-.-aX}-:

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FIG. 2. Simian RNA viruses. Top row: CPE in monkey cellfluid cultures, X 100. Bottom row: Hematoxylin- eosin stained preparations of infected monkey kidney cells, X 400 (except E). (A) SV16-infected rhesus monkey kidney cells. (Bi and 2) SV12-infected rhesus monkey kidney cells showing intracytoplasmic inclusions. (CI anid 2) Monkey measles virus-infected green monkey kidney cells showing intranuclear inclusions in the nuclei of a synicytical cell. (Dl and 2) Foamy agent-infected rhesus monkey kidney cells; note absence of intranuclear in- clusionis. (E) SV5-infected rhesus monkey cells showing adsorption of guinea pig erythrocytes, X 100. From reference 45.

lung tissues of patas monkeys during a severe ogy of measles virus in monkeys will be discussed outbreak of respiratory disease in these animals later. (20). The data on parainfluenza 3 and SV5 anti- Foamy virus, usually observed in aged monkey body distributions in monkeys will be discussed kidney cell cultures (89), has four serological in the later sections. types (97), with additional types, for example, the Pseudomnyxovirus group. This name has been chimpanzee foamy virus (83). In fluid cultures, adopted here for the group of viruses which re- CPE occurred in the form of large vacuoles (Fig. semble the myxoviruses morphologically, but can 2, D1) which were indistinguishable from the be easily distinguished by certain biological prop- syncytial cells produced by monkey measles virus. erties. Since agglutination by the pseudomyxo- However, the complete absence of eosinophilic viruses was not affected by RDE treatment of inclusions in the nuclei of foamy virus-infected erythrocytes, this method has been applied for cells (Fig. 2, D2) immediately distinguished them differentiation of the myxovirus and pseudomyxo- from cells infected with measles virus. Recently, virus groups. Measles and foamy viruses are in- Stiles showed that most of the rhesus monkeys cluded in the latter. were infected with foamy virus type 1, whereas Monkey measles virus, originally known as green monkeys were mostly infected with foamy minia (monkey-intranuclear-inclusion-agent) virus virus types 2 and 3 (96). (88), produced multinucleated syncytial cells in infected cultures (Fig. 2, Cl). In hematoxylin- EPIDEMIOLOGICAL ASPECTS OF LATENT VIRUS eosin stained preparations, eosinophilic inclusions INFECTIONS IN SIMIAN TissuEs located in the nuclei as well as in the cytoplasm Various investigators have reported that mon- could be readily seen (Fig. 2, C2). The epidemiol- key cells often harbor a variety of viruses, but the 194 HSIUNG BACTERIOL. REV. results obtained have varied from study to study. TABLE 2. Detectioni ojf late/it viruises il priH ary It has become evident that many factors may monkey kidnhey cell clultiures (total, influence the recognition of endogenous agents. 120 lots) These variables may include: (i) the frequency and No. slhowing X irus Percentage showing x-irus nature of contact in with other animals nature, No. of weeks infections infections (ii) the frequency and nature of contact with after cells planted man and other animals at capture and during SV40 I oam SV4O Foamy virus shipment, (iii) the stress factors imposed during capture and shipment, (iv) the period and conditions of quarantine, and (v) the methods used 2-3 3 4 2.5 3.3 3-4 15 16 12.5 13.4 for detection of latent viruses. Any of these fac- 4-8 36 36 30.0 30.0 tors could have influenced the number of virus isolations and the types of viruses obtained. Ex- amples are presented here for comparison with TABLE 3. Relatio/lship betwteenl i/li-plt multiplicity the data available in the literature. a/id cappeaCrcIIICe of virlus i/ilfectiO/i cis determileld by hiemnadsorptioni Detection of Latent Infections in Cell Cultures by Prolonged Percentage of cultures showving Cultivation hemadsorption at indicated no. of Virus in-put multiplicity days after cells planteda Primary monkey cultures have been used by per cell many laboratories for various purposes, but for 3 days 14 days 27 days 38 days most investigators latent virus infections have not been a major concern. The question arose as 10 100 100 ND ND to why these latent infections were not recognized 0.01 0 92 100 ND in the routine use of these cultures. In an attempt 0.0001 0 64 78 100 to answer these questions, an experiment subject- 0.000001 0 0 0 16 ing cells to prolonged cultivation was designed to Control, no virus 0 0 0 0 investigate the presence of latent viruses; the results were then compared with those from cul- ,1 ND = not done. tures which were kept under observation for shorter periods of time (Table 2). Of the 120 lots These infected cell suspensions were seeded of cultures examined, about 3'c showed virus into tubes. Each tube contained 3 X 105 cells. infections when examined 2 to 3 weeks after the At selected time intervals, 20 to 40 tubes cells were planted, the usual duration of most from each group were tested for the presence of virological studies. However, when the same lots the myxovirus infection by the hemadsorption of cultures were examined after prolonged culti- technique (105). As shown in Table 3, with an vation, i.e., 4 to 8 weeks after the cells were input multiplicity of 10, 100o of the cultures planted, it was noted that the percentage of iso- showed infection on the third day after planting. lations increased 10-fold. Thus, the longer the When a multiplicity of 0.01 was used, 92cr of the cultures were kept, the higher the percentage of cell cultures showed infection 14 days after plant- virus isolations obtained (46). A similar situation ing, but 100%, showed infection on day 27. When was noted in the detection of latent virus infec- the input multiplicity was 0.000001, infection tions of the human respiratory tract (26), of could not be recognized until day 38, and even chimpanzee tissues (83), and of those experi- then only 16C- of the cultures showed infection. mentally induced in monkeys (5, 17, 74). Thus, quantitative factors appeared to have an The reason for this increase in the number of important bearing on the recovery of certain types latent viruses recovered after prolonged cultiva- of latent viruses in cell cultures. tion was thought possibly to be due to the presence of a minute amount of infectious virus in the ori- Incidence of Virus Infections in "Normal" ginal tissue cell, which became evident only after Monkey Kidney Cell Cultures virus multiplication had occurred and a significant To study the incidence of latent virus infections amount of virus had been produced. This interpre- in monkey cell cultures, we conducted three lon- tation was supported by the following experiment. gitudinal survey studies during the years 1963- Freshly prepared monkey kidney cell suspensions 1968. Cultures were prepared from monkey kid- containing 3 X 105 cells per ml were divided into ney tissues obtained from apparently healthy portions of 100 ml. Each portion was infected with animals. Details of these studies have been a serial 10-fold dilution of a myxovirus with multi- described previously (45, 47). In general, a set of plicities per cell ranging from 10 to 0.0000001. 20 to 40-tube cultures, each containing an 11 X VOL. 32, 1968 LATENT VIRUS INFECTIONS IN PRIMATE TISSUES 195

22 mm cover slip, was prepared from each lot of 1966 freshly trypsinized monkey kidney cell suspension. 30 r *--. No Examined These cultures were kept incubated at 37 C for -o No Infected observation as long as the cells were in good con- 20 dition, usually 4 to 8 weeks. All cultures were 1 examined twice weekly, and the culture medium w was replaced as needed. Both CPE in fluid cul- uJ tures and cytopathology in hematoxylin-eosin O0 stained preparations were used for recognition of C,I- the possible presence of viral agents. The hemadsorption method using guinea pig erythrocytes C..) GREEN MONKEY CELLS was utilized for the detection of myxoviruses. O 20 Identification of each isolate was made by the neutralization test or histopathology of the in- 10 p- __> <_v4\ fected cells, or by both methods. Much to our surprise, an unusually high percentage of cultures that were considered "normal" showed virus infection. FEB MAR APR MAY JUNE JULY AUG SEPT OCT NOV DEC The first study, in which monkey kidney cell MONTHS cultures which had been prepared in the New FIG. 3. Incidence of virus infections in 344 lots of Haven Laboratory were used, started in October "normal" rhesus and green monikey kidney cell cultures 1963 and ended in July 1965. A total of 191 lots (February to December 1966, New York). of cultures were examined; of these, 126 lots were from rhesus monkeys and 65 lots were taken from green monkey cells. For each month, the hours to a few days before shipment to the labora- percentage of virus infection with one or two tory. It is possible that the grouping together of kinds of virus was greater than 50% (47). All the monkeys at the airport may have helped to monkeys used in this study were housed in the spread endemic virus infections in the animals same quarters and were purchased from primate used in the second study. importers. They were used without any special "conditioning." Comparison of Virus Types Recovered The second study was conducted in New York from Tissues ofMonkeys With or City from February 1966 to June 1967. The kidney Without Quarantine cell cultures were prepared from monkeys which The early studies of Hull and co-workers, had been shipped directly from the field to the undertaken in Indianapolis in 1954-1955, showed laboratory where they underwent thorough "con- that reovirus and adenovirus were the agents most ditioning." Green monkeys and rhesus monkeys frequently encountered (56, 58). However, there were housed in separate quarters (same animal was a conspicuous absence of adenovirus and reo- caretaker for both rhesus and green monkeys), virus infection during our 5-year survey, as deter- were kept in separate cages, and were quarantined mined by lack of virus isolation and by serological for 30 to 90 days before sacrifice for kidney studies (47). The methods used for virus isolations tissues. A total of 539 lots of cultures were exam- in our study differed from those used by Hull et al. ined; of these, 287 were from rhesus monkeys (56). This may account in part for the difference obtained from India, and 252 were taken from in the virus types isolated by the two laboratories. African green monkeys from Ethiopia. A segment Other epidemiological factors encountered by the of this study is presented in Fig. 3. Again, the two laboratories may also be responsible for the average rate of virus isolation for each month was differences in prevalence of virus types. This is about 50% regardless of the monkey species used illustrated in Table 4 by the results obtained in or the month during which the tissues were ob- two of our studies conducted in 1964 and in 1966 tained. (45). Although the same techniques were used for From September 1967 to January 1968, a recognition of viruses in both studies, it is third study was conducted in the West Haven apparent that SV5, one ofthe most prevalent virus Laboratory. It was noted that the percentage of types isolated in the 1964 study, was not present virus isolations in each month was less than 5%. in any of the 344 culture lots examined in 1966. In The monkeys used for the third study were trans- the second study, over 5,000 culture tubes were ported directly to the laboratory upon arrival at tested exclusively for hemadsorption with guinea the airport. All monkeys used in the second study, pig erythrocytes, a technique similar to that used however, had been kept at the airport for a few in 1964. Negative results were obtained consist- 196 HSIUNG BACTERIOL. REV. TABLE 4. Comparison of virus type obtained from Mixed Infections tissues of monkeys with or without quarantinea Mixed infections with two or three virus types Monkeyswithout Monkeys with 30- have occasionally occurred in both rhesus and quarantine, New 90 days quarantine, greenmonkey cells (Table 4). It was not possible to Determination Haven, 1964 New York, 1966 recognize the presence of two or more virus types Rhesus Green Rhesus Green in the fluid cultures unless cell culture preparations were fixed and stained. Figure 4 shows a Total no. of culture hematoxylin-eosin stained preparation of a mixed lots examined.... 64 34 187 157 infection. Two distinct types of intranuclear in- Virus types iso- clusions were observed in a multinucleated giant lated (%) cell. Subsequently, it was established that the SV5 ...... 25 30 0 0 eosinophilic inclusions were due to measles virus, Measles ...... 12 3 0 0 and the basophilic inclusions were due to SV40 Foamy virus ...... 29 30 37 14 (47). SV40 ...... 17 14 13 36 Mixed infections.. 25 30 6 3 When SV40 and measles virus intranuclear inclusions occurred in separate cells, or when a a From reference 45. single cell showed inclusions of the two viruses at different sites, the type of mixed virus infection was not difficult to recognize. In hematoxylin- ently. (It might be pointed out here that SV5 was eosin stained preparations, SV40 intranuclear used in the laboratory for other experiments while inclusions were basophilic and purplish-blue in the second survey study was being conducted. color, whereas measles intranuclear inclusions There has been, however, no evidence of SV5 were eosinophilic and appeared pinkish. However, cross-contamination of survey cultures in the it was difficult to recognize a doubly infected laboratory.) Neither SV5 nor measles virus was nucleus containing inclusions of both virus types. isolated in cultures from the kidney tissues of Recognition of those nuclei doubly infected with monkeys conditioned for 30 to 90 days. SV40 and measles virus was achieved by utilizing Additional variations were noted in regard to the modified method of acridine orange staining foamy agents and SV40, which occurred with with phosphate buffer at pH 8.0 instead of at a equal frequency in both monkey species in 1964. low pH (48). The doubly infected nuclei appeared There was, however, a distinctly different percent- reddish-brown, and SV40 inclusions were brilliant age of virus recovery in 1966 (Table 4). green. Nuclei doubly infected with SV40 and Apparently, heavy cross-infections had occurred measles virus have been further identified by in 1964 among the animals when both species of electron microscopy (49). Mixed infections of monkeys were housed together. In the 1966 SV40 and foamy agents, SV5 and foamy agents, study, the green monkeys and rhesus monkeys and SV5 and measles virus were also observed were housed in separate quarters, although the (47). In addition, experimentally induced double same individual took care of both monkey species. infections of reovirus and SV40 were obtained. It was clear that cross-contaminiation between the Single cells doubly infected with SV12, a reovirus two monkey species was minimized, since foamy type 1, and SV40 are shown in Fig. 5 (50). Bright agents appeared more frequently in the rhesus pinkish eosinophilic inclusions of reovirus in the cultures and SV40 occurred more frequently in cytoplasm and purplish-blue basophilic inclusions the green monkey cultures. Other investigators of SV40 in the nucleus could be easily recognized. indicated that green monkeys, if not exposed to Other investigators have reported mixed infec- rhesus monkeys, were usually free from SV40 tions of SV40 and an adenovirus, and SV40 and infections (5, 9, 74). It was possible that in our herpes simplex virus (77, 80). Since adeno-SV40 1966 study the same animal caretaker could have hybrids were obtained in monkey cultures doubly brought SV40 from the rhesus monkey quarters to infected with adenovirus type 7 and SV40 (82, the green monkey quarters, even though all 84), other hybrids could conceivably occur in possible precautions were taken. However, a multiple infected cultures. recent report indicated that SV40 "T" and "V" Separation of mixed infections has been accom- antigens could be detected in kidney cells derived plished by membrane filtration. For example, from African green monkeys obtained directly measles virus was in the large size range, whereas from the field (78). It is not clear at the present SV40 was in the small virus group (Table 1). time whether the African green monkeys are Thus, a mixture of SV40 and measles virus can actually free from SV40 infection. be conveniently separated by filtration with mem- VOL. 32, 1968 LATENT VIRUS INFECTIONS IN PRIMATE TISSUES 197

FIG. 4. Mixed infection with measles (M) and SV40 (V) viruses, X 970. Note the distinct difference of intranuclear inclusions produced by measles virus (nuclei surrounding M) and inclusions produced by SV40 in the two nuclei (V), 27 days after seeding, hematoxylin-eosin stained preparatioln. From reference 47. branes of 100 and 50 nm limiting pore diameters. monkeys that had been quarantined at the local SV40 passed through filters of both 100 and 50 quarters for less than a month, whereas SV40 and nm, but the measles virus was retained by both. foamy virus were recoverable in kidneys of mon- However, this method was not satisfactory when keys even after 60 to 72 days in quarantine. In the mixture consisted of SV5 and measles virus, one instance, measles virus was isolated from one both of which are large viruses. Consequently, the kidney of a monkey but not from the other kidney plaque purification method or limiting dilution removed only 9 days later (47). Apparently, method had to be used to separate viruses of simi- measles virus does not persist long in the kidney lar sizes. tissues of monkeys. Meyer et al. (73) showed that measles virus was transmitted to monkeys that Distribution of Virus Types According had been in contact with infected human beings, to Conditions of Monkey Shipment and it is now recognized that measles infection in During the 5-year survey, it was noted that in monkeys occurs only when the animals are ex- most instances monkeys from the same shipment posed to man. These data suggest that certain tended to acquire the same virus types. A few virus infections in monkeys may be viruses oftheir examples are presented in Table 5. Of a total of own, but others may be viruses ofmanacquired by over 50 shipments of monkeys examined, only one monkeys soon after contact with man. Some virus group of 14 rhesus monkeys, shipped on Septem- infections may persist for relatively short periods, ber 17, 1966, showed no virus infection during the as in the case of measles, but others, such as SV40 study period. In general, SV5 and measles virus or foamy virus, may persist for longer periods of could be isolated only from the kidney tissues of time. 198 HSIUNG BACTERIOL. REV.

FIG. 5. Doubly infected patas monkey kidney cells showing reovirus cytoplasmic inclusions (Reo) and SV40 intranuclear inclusions (SV40) in the same cell. Hematoxylin-eosini stainedpreparation, X 970. From reference 50.

Acquisition of Parainfluenza Virus quarantine period. This interpretation was sup- Antibody in Monkeys During Captivity ported by the results obtained from the serological In the early studies of Schultz and Habel, it testing of 351 paired sera collected from the mon- was noted that SA virus antibody was present in keys of the 1966 study during the period of sera obtained from normal monkeys (90). We quarantine (Table 6). The initial sera, SI, of all reported that monkeys were free from DA virus monkeys were taken at the time of arrival at the antibodies while in their natural habitat but laboratory, and the second sera, SII, were col- developed antibodies after capture (53). These lected 30 to 90 days later, at which time the kid- findings were confirmed by recent studies (62, 92, neys were used for tissue cultures. The tests 99) which showed that SV5 antibody was absent revealed that 45 % ofthe 190 rhesus monkeys and in free-living monkeys and was acquired after 56% of the 161 green monkeys showed antibody contact with man. [SV5, SA, and DA viruses are conversion to SV5. The percentage of monkeys serologically identical; it was suggested at the that showed a rise from a negative antibody titer VIlIth International Congress of Microbiology in (. 1:5) to a positive antibody titer (1:20 or Montreal, Canada (1962) that SV5-SA-DA group greater) appeared to be related to the number of of viruses be designated as parainfluenza 5 virus.] days the monkeys were quarantined. A similar In the preceding sections, it was noted that SV5 result was obtained when the same paired sera was one of the most prevalent virus types found in were tested against parainfluenza 3 virus (Table 6). the cultures prepared from kidney tissues of mon- Of the 168 rhesus monkeys studied, 26% showed keys without quarantine, but SV5 was found to be parainfluenza 3 virus antibody conversion, and completely absent in cultures prepared from tis- 41 % ofthe 141 green monkeys showed conversions sues of monkeys which were quarantined in the to parainfluenza 3. The prevalence of parain- local quarters for 30 days or longer. It was con- fluenza 3 antibodies in captive monkeys has been sideredpossiblethat theconditioned animals could noted by other investigators (10, 17, 20, 92). have acquired SV5 infection during transit or soon However, there was no evidence that para- after arrival and then became immune during the influenza types 1 or 2 virus infections had oc- VOL. 32, 1968 LATENT VIRUS INFECTIONS IN PRIMATE TISSUES 199

TABLE 5. Distribution of virus types in rhsuse TABLE 6. Proportion of monkey sera showing monkeys according to shipmenzt and duration antibody conversion to parainfluenza virusesa in captivity Rhesus Green

Parainfluenza virus Percentage Percentage dcn type tested No. of showing No. of showing Labora- Date of monkeys antibody monkeys antibody tory W0~ tested conve r- tested conver- arrival studied sionb sion

36. 1 96 0 97 0 2 96 0 97 0 10/7/63 N.H. 9 6 0 3 168 26 141 41 16 7 0 5 (SV5) 190 45 161 56 23 9 0 30 8 SV5 a Modified from reference 45. 37 6 0 b SI taken on the day of arrival showed a neu- tralizing antibody titer of 1:5 or less and SII 3/3/64 N.H. 25 1 5 0 taken on the day of sacrifice showed a neutraliz- 8 8 0 ing antibody titer of 1:20 or greater. 22 6 Foamy agents + SV5 isolation of virus from human urine (33, 35-37, 36 6 Foamy Agents 85, 102-104, 107), but the origins of the viruria + SV5 are obscure. In an attempt to demonstrate that 11/19/64 N.H. 22 5 8 Measles viruria follows viremia, monkeys were experi- 12 9 SV5 + foamy mentally inoculated intravenously with poliovirus 20 5 SV5 + foamy type 1. Virus was recovered from the urine 30 min later (50). The titers of poliovirus recovered in the 2/10/66 N.Y. 22 38 6 Foamy agents serum fell rapidly during the first 7 hr of sampling. 45 6 Foamy agents The titers of poliovirus in the urine, although 52 6 Foamy agents lower than those in the serum, also fell rapidly 60 4 Foamy agents and in unison with the titers of the blood samples. Similar results have been obtained by Schultz 9/17/66 N.Y. 14 36 6 0 43 3 0 and Neva with poliovirus injected intravenously 50 5 0 into mice and rats (91). Although poliovirus is not one of the viruses involved in the latent infections 10/28/66 N.Y. 16 36 2 SV40 discussed in the present paper, these model studies 45 6 SV40 indicated that, once viremia was established with a 65 4 SV40 sustained level of virus in the blood, viruria fol- 72 4 SV40 lowed immediately. However, the degree of virus multiplication in the kidney cells and the persistence of the virus in this organ probably depend curred in these monkeys, or in those tested by upon the properties of the different virus types. Shah et al. (92) or by Kalter et al. (62). Because of the complete absence of parainfluenza 2 anti- Experimentally Induced Latent Infection body in monkeys and the high prevalence of SV5 by SV40 antibody in this animal species, the antigenic et al. showed that intranasal inocu- relationship between these two viruses as reported Meyer (74) previously (18) requires reconsideration. lation of SV40 into monkeys resulted in the multiplication of this virus in the nasopharynx and POSSIBLE ORIGIN OF VIRUS INFECTION digestive tract (Fig. 6). Viremia occurred in the IN KIDNEY TIssuEs inoculated monkeys and also in an uninoculated cage mate. Moreover, SV40 was recovered from Model Study on Viremia and Viruria in cell cultures derived from kidney tissues of these Monkeys monkeys 4 to 5 months after initial infection. As noted above and from the reports of other Ashkenazi and Melnick showed that SV40 could investigators, a variety of viruses have been iso- be recovered in the urine of all monkeys inoculated from the kidney tissues of apparently lated intracerebrally, intracutaneously, or directly healthy animals. It is not known, however, how into the kidneys (5). In addition, they found that often and to what extent the kidney is infected. SV40 persisted in the kidney tissues 6 to 8 weeks Several reports have been concerned with the after virus administration, regardless of the route 200 HSIUNG BACrERIOL. REV.

VIRUS- IN MONKEY NO.41 INOCULATED INTRANASALLY WITH IO0TTID5OFSV40 BLOOD ++++++++ OO 0 0 0 0 0 THROAT 0 0 0 + 0 + o + + 0o O 0 0 0 STOOL +++0000000 0 O 0 0 0 ANTIBODY BY: CF --- NEUT- 64 0 2 3 4 5 6 7 8 20-22 WEEKS POST INOCULATION VIRUS IN: UNINOCULATED MONKEY_~~~-NO.36 (CAGE CONTACT OF MONKEY NO.41) BLOOD o 0 0 0++++o0 o0 o 0 0 0 THROAT 000000o+00 0 o 0 0 0 STOOL 0000000000 0 o 0 0 0 ANTIBODY BY: CF --4096 64 I NEUT- ===7-- -V z 0 1 2 3 4 5 6 7 8 20-22 WEEKS POST EXPOSURE FIG. 6. Experimental and contact infection of green monkeys witl S V40. Reproduced by permission from reference 74. of inoculation. The recovery of SV40 from the adenovirus types 1, 4, and 7 (36, 37), cytomegalo- kidney tissues, however, succeeded only when the virus (85), mumps virus (104), WB myxovirus tissue cells were cultivated for a prolonged period (33), measles virus (35), rubella virus (107), and of time but SV40 was not recovered from test coxsackie B virus (103). Viruria in man has been cultures inoculated with the minced kidney tissue discussed by Utz (102), and the mechanism of suspensions (5). viruria has been studied by Flanagan and Schultz (27). However, there are only a few reports con- Persistent Infection of the SVS Group cerning the isolation of virus from human kidney of Viruses in Monkeys after tissues (7, 8, 22, 63). Intranasal Inoculation Human embryonic kidney (HEK) cell cultures Intranasal inoculation of the DA virus strain are available commercially and are being used by into monkeys with no preexisting DA antibodies many laboratories for the propagation of a variety resulted in the multiplication of this virus in the of viruses. However, the possibility of latent virus nasopharynx (17). Virus was recovered daily infection in the HEK cell cultures has not been from throat swabs for 3 to 7 days after virus given much attention. Human kidney cell cultures inoculation. The infected monkeys were kept for have been used in our laboratory since 1959. The long-term observation. No clinical manifestations methods used for the preparation of the human of illness were noted during the period of study. kidney cell cultures have been described pre- In several instances, one kidney from the same viously (43). Not until recently was a survey of monkey was removed on two separate occasions latent virus infections in human kidney tissues and virus isolations were attempted. In one case, initiated. Recognition and techniques for isola- virus was isolated from the kidney tissue 16 weeks tion were similar to those described earlier for the after inoculation. Again, the persistent infection by monkey cell cultures. During a 3-year period, the virus of the kidney tissues was demonstrated November 1964 to December 1967, 124 lots of only by prolonged cultivation of the cells in cul- human kidney cell cultures were examined, and tures prepared from the kidney. Direct inocula- seven viruses (6%) were isolated. These included tion of the minced kidney tissue suspensions into two measles viruses, two foamy viruses, one reo- test cultures did not result in virus isolation (17). virus, one adenovirus, and one myxovirus. One measles virus isolation was made from a lot of LATENT VIRUS INFECTIONS IN human kidney cell cultures from a 3-year-old HUMAN KIDNEY TIsSUEs child who had been exposed to measles and had There have been several reports of virus isola- been given 0.6 ml of y-globulinat thetime of expo- tion from human urine. These have included sure. The child died 2 weeks later. Attempts at VOL. 32, 1968 LATENT VIRUS INFECTIONS IN PRIMATE TISSUES 201 virus isolation from the tissue suspension and anti- kidney tissues of so-called "normal" healthy body studies provided no evidence that thechildhad monkeys is unusually high. The virus types re- been infected with measles virus. The presence of covered varied from year to year and from labora- the measles virus infection in the kidney of this tory to laboratory. These variations may reflect child would not have been recognized ifthe kidney the source from which the monkeys were ob- cell cultures had not been kept for 33 days after tained, the nature and degree of contact of mon- planting. The second isolation of measles virus keys with man and other animals, and the con- was made from a lot of HEK cell cultures pur- ditions under which the monkeys were quaran- chased commercially. There was no history avail- tined. In general practice, latent virus infections in able for this case. In fact, the HEK cells had been primary cell cultures are often not recognized. used for the propagation of a slow-growing Virus stocks derived from such latently infected adenovirus. The presence of the measles virus was cultures or virus vaccines produced from these noted only when stained preparations were exam- infected cells would undoubtedly be contaminated ined to ascertain the presence of adenovirus in- with indigenous viral agents. It is, therefore, urged clusions. To our surprise, measles virus intranu- that precautions be taken whenever primary cell clear and intracytoplasmic eosinophilic inclusions cultures are used, in order to avoid misleading occurred in both inoculated and uninoculated experimental data which might result from the control HEK cultures. Thus, the adenovirus presence of latent viruses. stock derived from the commercially made HEK The sources of latent virus infections in mon- cultures was inadvertently contaminated with a keys are obscure. Some of these agents, for measles virus. example SV40, are undoubtedly viruses of pri- The isolation of a strain of myxovirus from one mates, but others may be infectious agents lot of HEK cells prepared from the kidney tissue acquired after contact with man. This concept is of a premature baby deserves some attention. The supported by the data on measles virus infection infant died 12 hr after birth and had a respiratory in monkeys and by the antibody conversions to disease syndrome. There was no evidence of hem- parainfluenza 3 and SV5 in paired sera obtained adsorption when this lot of cultures was tested at from monkeys during quarantine. Thus, SV5, 21, 24, and 30 days after planting, but 70% of the although originally isolated from monkeys, may culture tubes showed hemadsorption when tested well be a human myxovirus, since it is prevalent on day 50. This isolate was serologically identical only in monkeys having human contacts. It has to SV5. Since monkey cell cultures were used in been suggested that monkeys be vaccinated with the laboratory for various purposes, one cannot inactivated SV5 upon arrival (99) or be held in eliminate the possibility of SV5 laboratory con- quarantine for 3 to 6 months (47) prior to their tamination. However, of over 100 lots of HEK use in order to reduce SV5 or other latent viruses cell cultures tested, only one showed distinct hem- in their kidney tissues. adsorption, and even then the virus did not appear until after prolonged cultivation. ACKNOWLEDGMENTS Benyesh-Melnick et al. (8), have reported the I am very grateful to R. H. Green for his continu- isolation of nine viruses from kidney cell cultures ous interest and encouragement during this study. This from 84 children coming to autopsy during a 2- research would not have been possible without the year period, October 1961 to September 1963. cooperation of my students and colleagues at both Their isolates included three cytomegaloviruses, Yale and New York Universities. I also want to thank 2 Kari Hastings, H. T. Hatch, and V. Mercadante for three adenoviruses, (types 1, and 7), and one their help in the preparation of this manuscript. each of measles, varicella, and coxsackie Bi The original work was supported by Public Health viruses. Thus, latent virus infections in human Service research grants AI-05313, AI-07198 and kidney tissues, although not as frequent as in AI-08648 from the National Institute of Allergy monkeys, do occur fairly often. and Infectious Diseases. CONCLUDING REMARKS LITERATURE C1TED This survey will have served its purpose if it 1. Andrewes, C. H. 1964. Viruses of vertebrates. stimulates more investigators to delve into the The Williams & Wilkins Co., Baltimore. pathogenesis, persistence, and epidemiology of 2. Archetti, I., and D. Steve-Bocciarelli. 1963. virus infections in Data on the Structure of some simian adenoviruses. latent primates. Virology 20:399-404. mechanism of latent virus infections are limited, 3. Archetti, I., D. Steve-Locciarelli, and G. Toschi. and much information remains to be gathered 1961. Structure of an enteric simian virus. before we can understand the origin and persist- Virology 13:149-152. ence of virus infections in primate tissues. 4. Ashe, W. K., H. W. Scherp, and R. J. Fitz- The incidence of latent virus infections in the gerald. 1965. Previously unrecognized virus 202 HSIUNG BACTERIOL. REV.

from submaxillary glands of gnotobiotic and 20. Churchill, A. E. 1963. The isolation of para- conventional rats. J. Bacteriol. 90:1719-1729. influenza 3 virus from fatal cases of pneu- 5. Ashkenazi, A., and J. L. Melnick. 1962. Induced monia in Erythrocebus patas monkeys. Brit. latent infection of monkeys with vacuolating J. Exptl. Pathol. 44:529-537. SV40 papova virus. Virus in kidneys and urine. 21. De Harven, E., and D. S. Yohn. 1966. The fine Proc. Soc. Exptl. Biol. Med. 111:367-372. structure of the Yaba monkey tumor pox- 6. Atoynatan, T., and G. D. Hsiung. 1964. Ultra- virus. Cancer Res. 26:995-1008. filtration of simian viruses. Proc. Soc. Exptl. 22. Deinhardt, F., and A. W. Holmes. 1963. Isola- Biol. Med. 116:852-856. tion of a latent virus from the kidney of a new- 7. Benyesh-Melnick, M., and H. S. Rosenberg. born human. J. Lab. Clin. Med. 62:872. 1964. The isolation of adenovirus type 7 from 23. Eddy, B. E., G. S. Borman, G. E. Grubbs, and a fatal case of pneumonia and disseminated R. D. Young. 1962. Identification of the onco- disease. J. Pediat. 64:83-87. genic substance in rhesus monkey kidney cell 8. Benyesh-Melnick, M., H. S. Rosenberg, and B. cultures as simian virus 40. Virology 17:65-75. Watson. 1964. Viruses in cell cultures of kid- 24. Emery, J. B., and C. J. York. 1960. Occurrence ney of children with congenital heart mal- of hemadsorption virus in normal monkey formations and other diseases. Proc. Soc. tissue culture. Virology 11:313-315. Exptl. Biol. Med. 117:452-459. 25. Endo, M., Y. Aoyama, T. Kaminema, T. 9. Bhatt, P. N., M. K. Goverdhan, M. F. Shaffer, Hayashida, and T. Kingo. 1959. A virus con- C. D. Brandt, and J. P. Fox. 1966. Viral in- sidered as spumous virus isolated from a fections of monkeys in their natural habitat culture of renal cells of a Japanese monkey. in Southern India. I. Some properties of cyto- Japan. J. Exptl. Med. 29:355-357. pathic agents isolated from bonnet and 26. 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