Susceptibility of Chinese rhesus monkeys to SIV infection

Abstract

The use of China-derived monkeys in AIDS research has been limited by reports of reduced susceptibility to SIV. We performed a serial passage of SIV in Chinese macaques, which resulted in a viral stock capable of inducing simian AIDS and high levels of replication in these animals. Similar to HIV in humans, SIV pathogenesis in non-human primates is not limited by geographical origin. Chinese macaques are useful in pathogenesis, vaccine, and therapeutic studies in AIDS.

Most studies of SIV pathogenesis to date have used rhesus macaques of Indian origin, which have proved quite valuable as an experimental model of HIV infection in humans. However, there is currently a severe shortage of India-derived rhesus monkeys available for research ^[1,2]. Not only is the shortage of Indian rhesus macaques hampering progress towards alleviating the AIDS pandemic, but also the concentration of studies on animals of a single origin may be misleading. AIDS is a disease affecting humans of diverse origins; the study of animals of a single origin may lead to findings that are not broadly applicable to the human circumstance.

Rhesus monkeys from China are available, but their use in AIDS research has been limited by reports that the course of SIV infection is different compared with those from India. Following infection, steady-state plasma viral loads were found to be generally lower in monkeys of Chinese origin than those found in Indian monkeys, and although survival time is not as well documented in China-origin monkeys, experiments in which both Indian and Chinese monkeys were infected with the same SIV stock revealed a longer survival in Chinese animals ^[3,4]. This is not surprising, as the relative pathogenicity of SIV infection is linked to the level of the plasma viral load ^[5–7]. This lower viral load in China-derived rhesus macaques, however, may more closely parallel that found in untreated HIV-infected humans ^[3].

We hypothesized that the serial passage of SIV in Chinese rhesus macaques would result in a viral stock capable of high levels of viral replication when used as an inoculant in Chinese rhesus macaques and would result in disease. We began with the SIVmac182 stock (which was derived from serial microglia passage from SIVmac251 in Indian-origin monkeys ^[8–12]) to infect a Chinese rhesus macaque intravenously, and performed a serial passage, using plasma from days 13–14 postinoculation to inoculate two additional Chinese macaques. From the third animal, a viral stock was made from day 9 postinoculation plasma and was used for the intravenous inoculation of nine Chinese rhesus macaques, whose origin was verified by DNA typing for 12 unlinked microsatellite (STR) markers ^[13,14]. All nine were treated identically for the first 8 weeks after infection. Six were then utilized for other studies, and the remaining three were maintained until 14–15 weeks postinfection, when they were sacrificed.

Longitudinal analysis of CD4 and CD8 T cells by FACS analysis in the peripheral blood revealed, relative to each animal's baseline levels, a mild decrease in the CD4 cells, and an increase in the CD8 cells, resulting in a 40% drop in the CD4/CD8 ratio beginning at 2 weeks postinoculation and lasting through the 8 week postinoculation measurement period (Fig. 1a). Sequential viral loads were determined using the branched DNA assay (Bayer Reference Laboratory, Emeryville, California, USA). The plasma viral load peaked at day 10 postinoculation and then dropped to a steady state (Fig. 1b). The steady-state plasma viral load, taken as the mean of determinations between days 28 and 72 postinoculation, was 6.19 log₁₀ copy equivalents of SIV RNA/ml in these Chinese rhesus monkeys infected with Chinese-passaged virus (CC). The viral load achieved in the CC group is similar to the mean steady-state viral load (5.93 log₁₀ copy equivalents) reported in a study by others of Indian monkeys infected with the pathogenic stock SIVmac251 ^[15] (which was itself derived by serial passage in Indian monkeys ^[16]).

Fig. 1: Decrease in CD4/CD8 T-cell ratio and high plasma viral load in Chinese monkeys infected with Chinese-passaged SIV. (a) Average CD4 cell levels and CD8 cell levels in the nine Chinese rhesus macaques infected with the Chinese-passaged virus. The data were normalized to the levels at the day of infection. The error was reported as a standard error of the mean. —▪— CD4 T cells; —•— CD8 T cells; —▴— CD4/CD8 cells. (b) Plasma viral loads were taken periodically from 10 days to at least 8 weeks postinoculation. The plasma viral load was measured in log₁₀ copies RNA/ml using branched DNA assays. The squares and dashed line represents the mean viral loads in 24 Indian macaques infected with the Indian-passaged SIVmac182 (II). As this group was assembled retrospectively from other studies, the dates postinoculation were not precisely matched, so for the II group the data were binned by weeks postinfection. The circles and dotted line represents the mean viral load in plasma of the six Chinese rhesus macaques infected with SIVmac182 at the given timepoint (CI). The triangles and solid line is the mean viral load of the nine Chinese animals infected with the Chinese-passaged virus for the given timepoint (CC). The error was measured as the standard error of the mean.

We then compared the viral load in the CC group with that found when we infected Indian or Chinese monkeys with the parental Indian-derived SIVmac182 stock (II and CI groups, respectively). Analysis of variance revealed significant differences between the groups (P < 0.05). In these animals, similar to the findings of others, we found that the steady-state plasma viral loads achieved in Indian rhesus monkeys (II, mean setpoint viral load 6.23 log₁₀ copies RNA/ml, n = 24) were higher than those found in Chinese rhesus monkeys (CI, mean 5.48, n = 6) when the Indian-derived SIVmac182 stock was utilized (Fig. 1b). The CI and II groups showed a statistically significant difference at the 95% confidence level upon post-hoc testing using the Tukey's HSD procedure. Importantly, there is no statistical difference between the mean steady-state viral load of the II group and the CC group.

Infection in three of the CC animals was allowed to proceed until scheduled sacrifice at 14–15 weeks postinoculation, by which time one of the three animals became symptomatic with simian AIDS (rapid weight loss, poor fluid and food intake, diarrhea, and fever). Histopathological examination of this animal revealed the presence of severe SIV encephalitis.

Our objective in these studies was to test the hypothesis that the serial passage of SIV in Chinese macaques would result in a viral stock capable of high levels of replication and pathogenesis in Chinese rhesus macaques. Previous reports have indicated that Chinese macaques more effectively controlled SIV infection when compared with Indian macaques ^[3,17]. However, upon serial passage of SIV in the Chinese animals, we found that steady-state viral loads were increased from those found using Indian monkey-derived SIV, and in one case simian AIDS developed early after infection.

One drawback to our study was that six of the nine CC animals were only followed for 8 weeks before their use in separate studies. A time course study is needed to follow additional Chinese rhesus macaques infected with Chinese-passaged virus over a longer course of disease. Nonetheless, this paper is the first report that Chinese macaques are consistently capable of high levels of steady-state viral loads after SIV infection.

Acknowledgements

The authors would like to thank Claudia Flynn, Debbie Watry and Michelle Zandonatti for technical assistance.

Sponsorship: These studies were supported by NIH grants MH62261, NS045534, and MH073490 (awarded to H.S.F.). T.H.B. was supported by NRSA post-doctoral fellowship F32 NS048830.

This is manuscript no. 17520-NP from The Scripps Research Institute.