Department of Virology, Biomedical Primate Research Centre, PO Box 3306, 2280 GH Rijswijk, The Netherlands1
Author for correspondence: Jonathan Heeney. Fax +31 152843986. e-mail heeney{at}bprc.nl
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Abstract |
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Introduction |
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Viral factors such as infectious dose, route of infection, repeated exposure and viral virulence have been proposed to influence the rate of progression to AIDS (Asjo et al., 1986 ; Coffin, 1986
; Kestler et al., 1991
; Marthas et al., 1993
, 1995
; Nielsen et al., 1993
; Phair et al., 1992
; Tersmette et al., 1989
) To date, only the issue of viral virulence has been addressed in depth, but only with regard to attenuation of viral virulence. The Nef protein of SIV and HIV is one of the best defined viral factors and is widely considered to be a critical factor for the pathogenesis of AIDS in both humans (HIV-1/HIV-2) and rhesus macaques (SIVsm/mac) (Kestler et al., 1991
; Kirchhoff et al., 1999
; Whetter et al., 1999
). The molecular determinants of virulence appear to be encoded by several viral genes in different regions of the genome and in general appear to evolve in an interrelated fashion (Edmonson et al., 1998
; Marthas et al., 1993
). In a series of SIV studies using single Nef and multiple deletion mutants, Baba et al. (1995)
showed that AIDS could develop in neonatal animals when given high doses of the virus. Subsequently, the viral threshold hypothesis was proposed to explain in part the pathogenic potential of attenuated viruses if high enough levels of viraemia were achieved (Ruprecht et al., 1998
). More recently, a pathogenic threshold of plasma viral load has been defined which not only distinguishes between pathogenic and non-pathogenic infections, but also predicts the rate of disease progression (Ten Haaft et al., 1998
). Based on these as well as observations from other infectious disease models, we set out to determine the influence of the dose of the inoculum on the initial viral load, the threshold achieved, and thus the influence on disease progression. To address this question, different dilutions of the SIV8980 isolate were administered intravenously to ten mature rhesus monkeys. Animals were monitored for evidence of infection, plasma viral load, CD4+ T-cell decline and the rate of progression to AIDS. These results were compared to data compiled from other animals infected previously with different doses of the same virus stock.
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Methods |
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The second study consisted of retrospective data for rhesus monkeys which had been infected with the exact same stock virus diluted with the exact same procedure as before and administered by the same route but given different doses. The clinical and pathological follow-up of each animal was performed using the same standardized criteria. AIDS-defining criteria in macaques included one or more of the following; 10% weight loss, intractable diarrhoea, neurological abnormalities, oral lesions (i.e. thrush or herpes-like lesions); together with evidence of persistently low CD4 T-cell counts, and/or thrombocytopenia, anaemia, neutrophilia, low albumin with evidence of increased plasma RNA loads (>1x105 copies/ml).
Virus stock and virological analysis.
All animals were infected with the same SIV8980 stock (TCID50 1x10-4·8/ml) for comparative purposes. The TCID50 was determined by the Kärber formula as previously described (Bogers et al., 1997 ). The stock was derived from animal 8980 (P4) following four in vivo passages of SIVB670 in juvenile rhesus monkeys born and purpose-bred from the BPRCs breeding colony of Indian rhesus monkeys (Holterman et al., 1999
). The SIV8980 stock was propagated on autologous PBMCs. Within 10 days of autologous 8980 PBMC cultivation the supernatant was harvested, clarified to remove cellular debris, aliquotted and preserved at -135 °C. The in vivo macaque infectious dose 50 (MID50) was determined by intravenous administration of serial fold dilutions of the SIV8980 virus stock. The endpoint was based on the following virological criteria. The MID50 value was estimated based on the dose of the viral stock which infected approximately half of the animals to which it was administered (Bogers et al., 1997
). Virological analysis of inoculated animals included plasma antigen (p27 pg/ml), ELISA for anti-SIV antibodies, virus isolation, DNA-PCR on PBMC and quantitative RTPCR on plasma. Plasma antigen and anti-SIV ELISA (SIV antigen kindly provided by MRC/programme EVA reagent repository, Potters Bar, UK) were measured as described previously (Bogers et al., 1998
). The DNA-PCR assay was performed on genomic DNA isolated from PBMC from inoculated animals. Genomic DNA was isolated from separated and washed PBMC by proteinase K/Triton X-100-based lysis followed by ethanol precipitation. Nested PCR was performed on each sample using SIV-gag primers as follows:
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Outer reaction PCR mixes contained 1 µg genomic DNA, 20 mM TrisHCl (pH 8·3), 50 mM KCl, 0·01% gelatine, 2·5 mM MgCl2, 200 µM each dNTP, 250 nM each primer and 2 U Taq DNA polymerase (AmpliTaq; PE Biosystems) in a total volume of 50 µl. Cycling conditions for outer primers consisted of an initial denaturation step (95 °C, 3 min), followed by five cycles (95 °C, 30 s; 50 °C, 30 s; 70 °C, 30 s), thirty cycles (95 °C, 30 s; 55 °C, 30 s, 72 °C, 30 s), and finally by one cycle (72 °C, 7 min; 4 °C, 7 min; 20 °C, 1 s). From the outer reaction mix, 5 µl of product was transferred to an inner reaction mix containing 20 mM TrisHCl (pH 8·3), 50 mM KCl, 0·01% gelatine, 2·5 mM MgCl2, 200 µM each dNTP, 250 nM each primer and 2 U Taq DNA polymerase in a total volume of 50 µl. Cycling conditions for inner primers were identical to the first PCR except that twenty cycles (95 °C, 30 s; 55 °C, 30 s; 72 °C, 30 s) were carried out. PCR products were analysed by agarose gel electrophoresis.
Quantification of plasma viral load.
A quantitative competitive RNA-PCR was used to estimate the virus load in plasma (Ten Haaft et al., 1998 ). In brief, RNA was extracted from 200 µl of serum or EDTA plasma using guanidine isothiocyanate-mediated lysis, followed by propan-2-ol precipitation of the RNA. A known amount of synthetic internal standard RNA was added prior to RNA purification and was co-purified to monitor the efficiency of the purification. The RNA was reverse transcribed and amplified in a single reaction protocol using rTth DNA polymerase (PE Biosystems) and biotinylated primers. The internal standard RNA was co-amplified to monitor the amplification efficiency. The amplified fragments were detected by a capture probe that was covalently bound to Nucleolink microwells (NUNC). The amplification products were detected by a streptavidinhorseradish peroxidase-mediated colorimetric reaction. The amplified internal standard was hybridized to a different capture probe in separate microwells. The number of RNA copies in the sample was calculated from the absorbance of the sample wells compared to that of the corresponding internal standard well.
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Results |
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Discussion |
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A number of host factors which may influence the rate of progression to AIDS have been documented, including age, concurrent disease and host immunogenetics. We have previously found a correlation between survival and the MHC type of the SIV-infected animals (Baskin et al., 1997 ; Bontrop et al., 1996
). Similarly, several viral virulence factors have been reported to influence the rate of disease progression in man as well as in monkeys. Since the virus used in these studies was a highly virulent, late stage variant (Holterman et al., 1999
), it may be less likely to be controlled by host immune responses, in contrast to what we have observed with other strains (Baskin et al., 1997
; Bontrop et al., 1996
). Indeed, in this study no association between serologically defined Mamu-A, -B and -DR specificities, and susceptibility/resistance to SIV8980 was found.
Certain SIVmac nef mutations may either cause an attenuated disease course and prolonged survival or cause acute haemorrhagic enteritis, but a viral virulence factor which is actually capable of accelerating the progression to AIDS has not yet been identified. In a previous study (Holterman et al., 1999 ) the in vivo passage of SIVB670 led to an acute progression to AIDS following four in vivo passages in monkeys. Comparison of the KaplanMeier plots of the survival of animals infected with pre-passage stock versus the post-passage stock clearly revealed that the virus had acquired a significant increase in virulence capable of causing an accelerated disease course (Holterman et al., 1999
). Our data indicate that the dose of the inoculum is important for establishing SIV infection. However, once systemic primary plasma viraemia is established there is no influence of dose on the rate of disease progression. Once systemic infection has occurred, it is probably the replication rate together with other virulence properties of the dominant viral variant in the viral inoculum which influence the post-primary viraemia set-point of viral load which is established (Kimata et al., 1999
; Ten Haaft et al., 1998
). It is not the dose of the inoculum. Together, the interaction of viral virulence factors with host responses appear to dictate the rate of progression to AIDS after systemic infection has been established.
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Acknowledgments |
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L. Holterman and H. Niphuis contributed equally to this work.
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References |
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Received 1 November 1999;
accepted 16 March 2000.