Simian Betaretrovirus Infection in a Colony of Cynomolgus Monkeys (Macaca fascicularis)

Abstract

Of the 419 laboratory-bred cynomolgus macaques (Macaca fascicularis) in a breeding colony at our institution, 397 (95%) exhibited antibodies or viral RNA (or both) specific for simian betaretrovirus (SRV) in plasma. Pregnant monkeys (n = 95) and their offspring were tested to evaluate maternal–infant infection with SRV. At parturition, the first group of pregnant monkeys (n = 76) was antibody-positive but RNA-negative, the second group (n = 14 monkeys) was positive for both antibody and RNA, and the last group (n = 5) was antibody-negative but RNA-positive. None of the offspring delivered from the 76 antibody-positive/RNA-negative mothers exhibited viremia at birth. Eight of the offspring (including two newborns delivered by caesarian section) from the 14 dually positive mothers exhibited SRV viremia, whereas the remaining 6 newborns from this group were not viremic. All of the offspring (including 2 newborns delivered by caesarian section) of the 5 antibody-negative/RNA-positive mothers exhibited viremia at birth. One neonatal monkey delivered by CS and two naturally delivered monkeys that were viremic at birth remained viremic at 1 to 6 mo of age and lacked SRV antibodies at weaning. Family analysis of 2 viremic mothers revealed that all 7 of their offspring exhibited SRV viremia, 6 of which were also antibody-negative. The present study demonstrates the occurrence of transplacental infection of SRV in viremic dams and infection of SRV in utero to induce immune tolerance in infant monkeys.

Although simian betaretrovirus (SRV) causes symptoms of immunodeficiency, including anemia, tumors, and persistent refractory diarrhea, in some infected macaques,^1,7,10 most infected monkeys exhibit few or no clinical signs.² Macaques free of SRV are important in many types of experiments to avoid associated immunologic and virologic effects. Establishing an SRV-free breeding colony is paramount for a steady supply of appropriate monkeys for various experiments.⁸

We previously reported that SRV-T, a novel subtype of SRV, was found in the cynomolgus colony of our institution.³ Approximately 20% of the colony monkeys tested in 2005 were viremic and shed SRV-T virus in saliva, urine, and feces.^4,5 The viruses shed by these monkeys are a potential source of horizontal SRV-T infection, as occurred in a rhesus monkey colony.^6,7 In the present study, we investigated the actual prevalence and transmission of SRV in the closed cynomolgus colony through several generations, to prevent the spread of the virus and to establish an SRV-free colony.

Materials and Methods

Animals.

The Tsukuba Primate Research Center (Tsukuba, Japan) maintains approximately 1500 cynomolgus monkeys as a breeding and rearing colony and has been maintained as a closed colony for 30 y. All adult monkeys are kept in single cages. Pregnant monkeys are produced by timed mating system in which a female monkey is placed into a male monkey's cage for 3 d; pregnancy is confirmed by ultrasonography 5 wk after mating.

Dams nurse their offspring until weaning at approximately 6 mo. Weaned infants are paired with infants of similar size. Artificial nursing is performed when the dams do not exhibit appropriate nursing behavior.

The housing and care procedures of this study were approved by the Animal Welfare and Animal Care Committee of Tsukuba Primate Research Center of the National Institute of Biomedical Innovation.

Samples.

Blood samples were collected from 419 breeders (female, 364; male, 55). All of these monkeys were born at Tsukuba Primate Research Center and are the second and third generations from the founder monkeys, which originated from the Philippines, Malaysia and Indonesia.

We selected 95 pregnant monkeys that exhibited SRV-specific antibodies by Western blotting or the virus as detected by RT-PCR (or both) as the subjects of the study. Blood samples from the mothers and the newborn infant monkeys were collected within 12 h after parturition.

Western blotting.

SRV-specific Abs were assessed by Western blotting using SRV-T.⁵ Purified virus for this analysis was obtained from the culture supernatant of cloned SRV-T–infected A549 cells by ultracentrifugation through a sucrose gradient; purified viruses were disrupted by 1% SDS for use as antigen in Western blotting. The criterion for a positive reaction was detection of 2 or more virion-specific bands (that is, Gag and Env proteins).

RT-PCR.

RNA was extracted from serum of the monkeys (QIAamp Viral RNA Mini Kit, Qiagen, Tokyo, Japan, or MagNA Pure Compact Nucleic Acid Isolation Kit I, Roche, Mannheim, Germany). Reverse transcription was performed (ThermoScript RT-PCR System, Invitrogen, Tokyo, Japan) by using gene-specific reverse primers. PCR analysis was performed (Premix ExTaq Hot-Start Version, Takara, Shiga, Japan) by using published sets of external primers (SRVenv1E and SRVenv2E) and nested primers (SRVenv3N and SRVenv4N).⁹

Results

SRV infection status of the 419 laboratory-bred breeders.

Of the 419 (female, 364; male, 55) cynomolgus macaques evaluated, 22 were negative for both SRV-specific antibodies and RNA. Of the remaining 397 breeders, 340 were positive for SRV-specific antibodies but were not viremic, 29 were positive for both viral RNA and antibodies, and the remaining 28 monkeys had viremia without antibodies.

SRV infection status of 95 pairs of mothers and offspring at birth.

RT-PCR and Western blotting of samples from 95 pairs of mothers and offspring at the time of birth revealed that the dams could be grouped into 1 of 3 categories based on the presence of SRV-specific antibodies and viremia.² Among the 95 dams, 76 developed SRV-specific Abs without viremia, 14 had both antibodies and viremia, and the remaining 5 were viremic without SRV-specific antibodies.

None of the offspring of the 76 dams that were antibody-positive but RNA-negative were viremic at birth. Eight infants (including 2 delivered by caesarian section) of the 14 dually positive dams were viremic at birth; the remaining 6 infants of dams in this group were viral RNA-negative. All 5 progeny (including 2 infants delivered by caesarian section) of viremic but antibody-negative dams were viremic at birth.

Plasma SRV-specific antibodies and RNA in viremic newborns during the first 6 mo.

We then tested the SRV-specific antibody and RNA status of 3 representative viremic newborns at 1, 2, and 6 mo after birth (Table 1). All 3 of the dams exhibited SRV viremia at delivery, and 2 of them also were positive for SRV-specific antibodies. All 3 infants exhibited SRV-specific RNA at all time points, but none was antibody-positive at weaning.

Table 1.

SRV-specific antibodies and RNA in the plasma of viremic newborns during their first 6 mo

						Status of infant at
				Status of dam at parturition		0 d	1 mo	2 mo	Weaning (approximately 6 mo)
Infant ID	Method of delivery	Dam ID	Method of nursing	Antibodies	RNA	RNA	RNA	RNA	Antibodies	RNA
1310611144	Caesarean	1210003019	Artificial	+	+	+	+	+	–	+
1410508022	Natural	1319710076	Artificial	–	+	+	+	+	–	+
1420506016	Natural	1319711082	Maternal	+	+	+	+	+	−	+

Testing of infants for SRV-specific antibodies was delayed until weaning because transplacentally transferred maternal antibodies can persist at 2 mo of age.

Family analysis of two representative SRV-viremic dams.

The SRV status of all 7 offspring born to 2 representative viremic mothers was verified in 2007. Dam 1319711082 and her 4 offspring (infant 1410311011, born 2003; infant 1420506016, born 2005; infant 1420608031, born 2006; and infant 1420709050, born 2007) all demonstrated SRV RNA in tests performed during 2007. In addition, this dam and her oldest infant (1410311011) were antibody-positive, unlike the 3 youngest siblings. Dam 1319710076 and her 3 offspring (infant 1410408017, born 2004; infant 1410508022, born 2005; and infant 1420701001, born 2007) were all RNA-positive but antibody-negative according to tests performed in 2007.

Discussion

In 2005, we reported that about 20% of the cynomolgus monkeys in the colony at our institution exhibited SRV-T viremia and that virus was present in saliva, urine, and feces from the viremic monkeys.^3-5 Because the virus secreted from these monkeys was a potential source of horizontal SRV-T infection, we performed the current large-scale survey of SRV infections in our laboratory-bred monkeys and assessed the transmission of SRV through the generations represented in the colony.

The present study validated our concerns about vertical and horizontal SRV infections in the colony, because more than 90% of the laboratory-born breeders were positive for SRV-specific antibodies or virus (or both). The rate of viremia in the present study (14%) was smaller than that (20%) in the earlier survey,⁵ which involved 49 retired breeders. The rate of viremia in a colony may vary depending on the age distribution of animals and their countries of origin. In particular, we hypothesize that the 28 monkeys that exhibited SRV viremia without specific antibodies are immunotolerant to SRV because of being infected in utero, as is reported to occur in rhesus and pigtailed macaques.^7,12

To evaluate transplacental maternal–infant transmission of SRV, we tested 95 pairs of mothers and newborns, including 4 infants delivered by caesarean section, by using SRV-specific RT-PCR. The results showed that all monkeys exhibiting SRV-specific antibodies without viremia produced newborns without viremia. However, the transplacental SRV infections observed in infants included 4 newborns delivered by cesarean section from viremic mothers. In pigtailed monkeys, SRV2 was detected in the tissues and amniotic fluid of fetuses and in the blood of newborns delivered from viremic mothers.¹² In other cynomolgus monkeys, SRV was transmitted through transfusion of blood from a viremic donor but not from a nonviremic donor.¹³ These findings indicate that SRV viremia of the mother is essential to establishing transplacental infection of the fetus. However, the production of 6 SRV-negative newborns from 14 viremic dams with SRV-specific antibodies may indicate that these antibodies reduced the viral loads in the viremic mothers sufficiently to prevent transplacental infection with SRV. Further investigation to quantify SRV in blood and the occurrence of transplacental infections will resolve this question.

An important issue is whether SRV viremic newborns can convert to a nonviremic state after developing virus-specific antibodies. Three infants born from viremic mothers exhibited viremia, which was maintained at 1, 2, and 6 mo of age, with no antibodies at 6 mo of age. In addition, 7 offspring born from the representative 2 SRV-viremic mothers were all viremic, at ages of 6 mo to 4 y. Pigtailed monkey newborns infected transplacentally with SRV2 maintained a viremic state for 1 y without producing antibodies and harbored proviral DNA in many tissues.^11,12 A newborn rhesus monkey produced from a viremic mother was SRV1-positive within 24 h after birth and was antibody-negative for as long as 6 mo after birth.⁷ These findings suggest that cynomolgus infants infected in utero with SRV and born from viremic mothers are immunologically tolerant to the virus and that they then become the source of SRV infection in the colony.

The cynomolgus monkey breeding colony at our institution has been maintained as SPF with regard to B virus, SVV, SIV, STLV1, and measles virus but not SRV. The cage system used during the first 25 y was a two-story type—monkeys were able to touch feces and urine of animals in adjacent cages. In addition, cages were washed with high-pressure water, perhaps helping to spread virus-contaminated waste and increasing the likelihood of horizontal infections. After redesigning the cage system to a single-story type that prevents monkeys from touching fecal and urine waste from another macaque, we anticipate that we will be able to establish an SRV-free colony by introducing SRV nonviremic monkeys into the breeding colony. Furthermore, elimination of viremic dams, which can become a source of transplacental infection, from the breeding colony is critical to establishing an SRV-free breeding colony. The establishment of an SRV-free cynomolgus breeding colony is paramount for supplying monkeys that are appropriate for many fields of investigation, including vaccine testing, gene therapeutics, organ transplantation, and infectious disease studies.