Human immunodeficiency virus type one (HIV-1) epidemic in China is largely driven by several high-risk factors.1 One of the risk factors is paid blood donation (PBD), which was initially identified in Henan province, where many poor farmers were infected through illegal blood collection practices without adequate screening and sterilization procedures.2,3 Many of the infections occurred when contaminated blood cells were transfused back to donors once the plasma had been removed from the pooled blood. Over the past years, such activities, which peaked between 1992 and 1996, have caused hundreds of thousands of new infections among paid blood donors in rural areas in Henan and neighboring provinces including Anhui, Hubei, Hebei, Shaanxi, and Shanxi,2,4 creating the tragic “AIDS villages”5,6 and attracting tremendous public attention.7,8 Although illegal PBD has been banned by the Chinese government, the increasing prevalence of HIV-1 subtype B' through other risk behaviors including unprotected sexual contacts still calls for efforts to fight the epidemic.
To fulfill the goal of controlling HIV-1 dissemination from high-risk groups into general populations,9,10 there is a need to understand the viral transmission and to develop an effective preventive means. Because many preventive means cannot be tested in humans, it is desirable to develop a relevant animal model. Therefore, a lot of efforts have been put on the development of the chimeric simian/human immunodeficiency virus (SHIV).11-13
Most of currently available SHIVs utilize envelope genes derived from HIV-1 subtype B strains, either from laboratory-adapted, syncytium-inducing, T-tropic viruses (HIV-1HXB2 and HIV-1NL43) or from primary, nonsyncytium-inducing, M-tropic (HIV-1SF162), syncytium-inducing T-tropic (HIV-1SF33) and dual-tropic (HIV-189.6 and HIV-1DH12) isolates.14,15 In addition, a few nonsubtype B SHIVs were generated for subtype C and E including the SHIVCHN19 that we previously generated to study the major HIV-1 epidemic caused by subtype C and CB recombinant viruses in China.16-18 Till now, however, no SHIV has been generated based on the envelope of subtype B' HIV-1, another major epidemic strain in China and Southeast Asia. Moreover, it remains unknown whether or not a SHIV generated from a freshly isolated human HIV-1 would be pathogenic in vivo during acute phase of infection. Here, we report the construction and characterization of a new SHIVB'WHU both in vitro and in vivo.
MATERIALS AND METHODS
Amplification of HIV-1 tat/rev/vpu/env Genes
The genomic DNA was extracted from peripheral blood mononuclear cells (PBMCs) of an HIV-1-infected PBD (02HNSMX2).2 The HIV-1 tat/rev/vpu/env fragment was amplified from genomic DNA by a nested polymerase chain reaction (PCR) using the EXPAND high-fidelity DNA polymerase according to the manufacturer's specifications (Boehringer-Mannheim, Indianapolis, IN). The outer primer pair is 4759F1 (5′-GTT TTT CAG AAT CTG CTA TAA GAA ATG CC-3′), BU5 (B) (5′-CTC TAG TTA CCA GAG TCA CAC AAC AGA CGG GCA CAC AC-3′) and the inner primer pair is VprA (5′-TGCC GAATTCGCATGC TA TAG A TAG AGG AGA GCA AGA AAT GGA G -3′) and EnvB (5′-TGCC CTCGAG CT TAT AGT AAA GCC CTT TCG AGG-3′), where the introduced stop codons are underlined and the restriction enzyme sites (EcoRI, SphI, and XhoI) are italicized. Products from the second-round amplifications were purified from agarose gels using QIAquick gel extraction kit (Qiagen, Valencia, CA).
Construction of SHIVB'WHU
The env sequence was subjected to the phylogenetic analysis as previously described.16 PVP-1 plasmid contains the 5′-half SIVmac239 genome from the 5′ long terminal repeat, a generous gift of Dr Paul Luciw. 3′ half vpu+-B'WHU plasmid (3′-p02HNSMX2-B'WHU) contains the 3′-half of SHIVB'WHU genome. The 3′- p02HNSMX2-B'WHU plasmid was made by replacing counterparts of SHIVSF33 (a generous gift of Dr Cecilia Cheng-Mayer) with newly obtained subtype B' tat/rev/vpu/env genes. To make a replication-competent recombinant SHIVB'WHU, linear 5′-PVP-1 and 3′-p02HNSMX2-B'WHU plasmids were co-transfected into 293T cells using Effectene Transfection Reagent (Qiagen, Valencia, CA). Forty-eight hours later, 1 × 107 CEM × 174-5.25M7 cells in DMEM media were placed into the 60 mm dishes containing the transfected 293T cells and supernatant. The culture supernatants were harvested for p27 measurement and used to infect macaque PBMCs. Some transfected 293T cells were also subjected to the cell fusion assay to test the expression and phenotype of Env as we previously described.16
Genetic Analysis of Amplified HIV-1 Genes
Full-length tat/rev/vpu/env amplicon was cloned into the expression vector pcDNA-I/Amp (Invitrogen, Carlsbad, CA) and was subsequently sequenced. The full env sequence was subjected to phylogenetic analysis.1,2 The phylogenetic tree was generated using the neighbor-joining method implemented in the ClustalX1.8 program. The tree was plotted using the TreeView program.19 The SimPlot and BootScan analysis was used to determine the possible intersubtype recombination.20
Replication of SHIVB'WHU in Chinese Macaque PBMCs
Macaque blood was obtained from healthy adult Chinese rhesus monkeys that were provided by Wuhan University Laboratory Animal Center. PBMCs were separated from the whole blood by centrifugation through Ficoll-Paque PLUS (GE Healthcare, Piscataway, NJ) density gradients. To test the infectivity of newly generated SHIVB'WHU strain, 1 × 107 stimulated macaque PBMCs were infected by cell-free virus with an equal inoculum (p27 titer: 0.5 ng/mL). The infected PBMCs were cultured in 10% FBS RPIM-1640 containing 20U IL-2. On day 5, 10 and 15 postinfection, the viral supernatants were harvested for p27 measurement. The p27 level was measured using a kit purchased from ZeptoMetrx Corporation (Buffalo, New York, NY).
Fluorescence-Activated Cell Sorter (FACS) Analysis
The number of peripheral CD4+ and CD8+ lymphocytes in SHIV-infected animals was determined by fluorescence-activated cell sorter using fluorochrome-conjugated monoclonal antibodies as described previously.16
Inoculation of Chinese Macaques With SHIVB'WHU
Four juvenile Chinese rhesus macaques were infected with SHIVB'WHU in this study. A dose of 1.1 μg p27 in a volume of 15 mL of cell-free viral supernatant was infused intravenously via the femoral vein into a Chinese macaque (P1-#0002). For serial passage, 10 mL of heparinized whole blood and 5 mL of bone marrow collected from the P1-#0002 macaque at 2 weeks postinoculation were inoculated intravenously, also via the femoral vein, into another healthy Chinese macaque (P2-#0296). Subsequent passages for P3 (P3-#1032) and P4 (P4-#1050) animals were carried out in the same manner as for P2-#0296 animal. Our experimental protocols were reviewed and approved by the Institutional Animal Care and Use Committee at the Laboratory Animal Center in Wuhan University Laboratory Animal Center, where the experimental animals were housed.
Viral Load Measurement
The level of plasma viremia and viral RNA and proviral DNA loads in tissues was measured by a quantitative real-time PCR method as described previously.16,17 A molecular beacon-based real-time PCR assay was used to quantify the copy number of SHIVB'WHU in plasma using the ABI 7700 PRISM spectrofluorometric thermal cycler (Applied Biosystems, Foster City, CA). The limit of detection for plasma viral load was approximately 400 RNA copies per mililiter after the adjustment of the dilution factors.
Tissue sections from formalin-fixed paraffin-embedded small intestines (SIs) (jejunum) were stained with a mouse anti-human CD4 monoclonal antibody (NCL-CD4-1F6; Novacastra Laboratories, Ltd, London, United Kingdom) using the DakoCytomation-Labelled Streptavidin-Biotin2 System, Horseradish Peroxidase (LSAB2 System, HRP, DAKO Corporation, Carpinteria, CA) and AEC Substrate-Chromogen solution (DAKO Corporation) as previously described.21,22
Determination of Coreceptor Usage
The coreceptor usage of the postpassage SHIVB'WHU was determined by using GHOST CD4 cells expressing each of the following proteins including CCR1, CCR2b, CCR3, CCR4, CXCR4, CCR5, CCR8, Bob, Bonzo, and V28. Viral isolates were used to infect each of the cell types in duplicate in 96-well plates at a multiplicity of infection of 1.0. After incubation at 37°C for 2 hours, the cultures were washed 3 times with phosphate-buffered saline and maintained in the culture medium. The cells were photographed by fluorescent microscopy to detect GFP expression 24-48 hours postinoculation.
Sequence Analysis of SHIVB'WHUenv Gene
To generate a subtype B' SHIV, we amplified the tat/rev/vpu/env fragments of HIV-1 subtype B'WHU from the proviral genome of a patient's PBMCs (02HNSMX2) by a nested PCR. Sequencing results indicated that the open-reading frames of tat/rev/vpu/env genes were complete without any frame shift. Because viral envelope determines viral genotype and phenotype,23 a phylogenetic analysis was conducted to determine the viral genotype as previously described.16 As depicted in the neighbor-joining tree (Fig. 1), the SHIVB'WHUenv clustered tightly with subtype B' strains identified either in Henan (02HNsmx2, 02HNsc11, and 02HNsq4), Yunnan (B'YN.RL42) of China or in other Southeast Asian countries such as Thailand (subtype B'.TH.93TH067) and Myanmar (subtype B'.MM.99.mSTD101). In contrast, env sequences from western countries, including those from the United States, are divergent from subtype B' virus (Fig. 1).2,24 Furthermore, the SimPlot and BootScan analysis showed that the env of SHIVB'WHU is a pure B' without cross over or recombination with viruses of other subtypes, including subtype C/B' viruses currently circulating in China.1,16 When compared with env genes of other SHIV strains including SHIV89.6, SHIVSF33, SHIVSF162, SHIVKU2, SHIV1157ipd3N4, SHIVCHN19, SHIVMJ4, and SHIVE-CAR (Fig. 1),18,25 SHIVB'WHUenv is the only unique one representing the subtype B' viral cluster. In addition, there were significant differences between SHIVB'WHU and other SHIVs especially in variable regions (V1, V2, V3, V4, and V5) based on the aligned amino acid sequences (See Figure, Supplemental Digital Content 1, http://links.lww.com/QAI/A28). The amino acid similarity between SHIVB'WHU and each of these SHIV strains as mentioned above is 82%, 83%, 85%, 83%, 77%, 77%, 73%, and 75%, respectively.
Serial Passages of SHIVB'WHU in Chinese Macaques
We first determined that SHIVB'WHU used CCR5 as its coreceptor and was able to replicate in rhesus PBMCs (data not shown). To enhance the infectivity of SHIVB'WHU, in vivo serial passage of SHIVB'WHU was performed in 4 naive Chinese rhesus macaques.16,26 Cell-free SHIVB'WHU supernatant was inoculated intravenously into a healthy Chinese rhesus monkey (P1-#0002). Fourteen days after the inoculation, 10 mL of heparinized blood and 5 mL of bone marrow from this animal was inoculated intravenously into a second naive animal (P2-#0296). Such in vivo passage was carried out sequentially into the third and the fourth naive rhesus monkeys (P3-#1032 and P4-#1050), respectively. Unexpectedly, there was no detectable viremia in P1-#0002 during the 14 days of infection (Fig. 2). P2 animal (P2-#0296), however, developed productive infection as indicated by a burst of viremia (106 copies of viral RNA per mL of plasma) 14 days after the blood-bone marrow transfusion. Further in vivo passages of SHIVB'WHU have improved the viral infectivity because the burst of viral replication was detected earlier on day 12 with viral loads ranging 106 to 107 copies of viral RNA per milliliter of plasma (Fig. 2). During these 14 days of acute infection, there was no significant change in peripheral CD4+ and CD8+ T-cell counts in all 4 infected Chinese rhesus macaques.
Quantification of Proviral DNA and Viral RNA in Tissues
Because SHIVB'WHU is a R5-tropic virus, we were curious to understand viral infection and pathology in the guts during the acute phase of infections, which has not been previously studied with any SHIVs before in vivo adaptation. Therefore, all animals were sacrificed 2 weeks postviral inoculation. Proviral DNA and viral RNA loads in tissues were determined by a quantitative real-time PCR assay as previously described.17,27 In this analysis, small intestine (SI), axillary lymph node (ALN) and mesenteric lymph node were examined in all animals (Fig. 3). Interestingly, despite the absence of detectable viral RNA in peripheral blood of P1-#0002, there was significant level of viral RNA load and proviral DNA load in ALN of this animal (Fig. 3A). In P2-#0296 monkey, accompanying the burst of viremia on day 14 post transfusion, proviral DNA and viral RNA were mainly detected in SIs in addition to low levels in ALN. In P3-#1032 and P4-#1050 monkeys, with the earlier burst of viremia on day 12 postinoculation, the viral RNA load and provial DNA load were significantly higher in SIs. Moreover, both proviral DNA and viral RNA load were significantly higher in mesenteric lymph nodes (Fig. 3B), which belong to mucosal tissue compartments. It is possible that the enhanced viral infectivity is related to the increasing infection of CD4+ T lymphocytes in mucosal tissue compartments. As for the failure of detectable viral load in other tissues, we believed that it was probably due to the limit of our viral load assay because viral genes were readily detected in all tissue compartments tested by nested PCR.
Immunohistochemistry Analysis of CD4+ T Cells in SIs
To further determine the pathogenicity of CCR5-tropic SHIVB'WHU during in vivo passages, the cytopathic effect of viral infection was investigated in the SIs. We were unable to analyze the CD4+ T cells using the flow cytometry technique due to the lack of equipment at our study site. Immunohistochemistry analysis, however, was performed on formalin-fixed paraffin-embedded SIs to determine the loss of CD4+ T cells using a mouse anti-human CD4 monoclonal antibody.22 CD4+ T cells were stained red as shown in Fig. 4. For each monkey, we analyzed multiple tissue sections and multiple fields of each tissue section. Compared with uninfected rhesus monkeys, the number of CD4+ T cells in SHIVB'WHU-infected monkeys was significantly reduced especially among the intraepithelial lymphocytes (IEL) of SIs. CD4+ T cells were detected among the lamina propria lymphocytes but with reduced numbers, and in mucosa-associated lymphocyte aggregates or tissues (Fig. 4).
Unchanged CCR5-Tropism of SHIVB'WHU After Serial Passage
To determine the viral coreceptor usage, viruses were reisolated from P2-P4 passaged monkeys. These viral isolates were used to infect GHOST CD4 cells expressing each of the coreceptors, including CCR1, CCR2b, CCR3, CCR4, CXCR4, CCR5, CCR8, Bob, Bonzo, and V28. These isolates infected GHOST CD4 Hu-CCR5 cells but not other cells (See Figure, Supplemental Digital Content 2, http://links.lww.com/QAI/A29) with a pattern similar to that of the parental SHIVB'WHU. Furthermore, there were only low frequent sporadic amino acid substitutions identified in their env fragments. These results suggest that, despite in vivo serial passage, SHIVB'WHU has maintained CCR5 specificity with no evidence of altered coreceptor utilization.
Over the past years, Asia has become the second most heavily affected continent by HIV/AIDS in the world. Because Asia has the world's largest human population, effective measures must be implemented to control the increasing prevalence of HIV-1 infection. To this end, the development of a relevant animal model to facilitate the testing of various preventive measures (eg vaccine, microbicide, antivirals) remains necessary. HIV-1 subtype B' is a major HIV-1 genotype not only in China but also in other Southeast Asia countries.2,28 Because there is a significant amount of sequence variation in env genes of subtype B' and western B viruses (See Figure, Supplemental Digital Content 1, http://links.lww.com/QAI/A28), currently available B SHIVs are not necessarily suitable for studies of subtype B' viral transmission and pathogenesis. For example, recent studies indicated that some subtype B' HIV-1 strains are less susceptible to a few potent neutralizing antibodies derived from patients infected with western B viruses.29 The newly constructed SHIVB'WHU is therefore particularly useful for studying issues related to subtype B' env-mediated infection in a nonhuman primate model.
The infectivity of SHIVB'WHU was enhanced in Chinese macaques after in vivo serial passage. Before in vivo passage, the phenotype and replication kinetics of SHIVB'WHU was determined by in vitro assays. SHIVB'WHU was found to be CCR5-tropic similar to its primary parental HIV-1 isolate (See Figure A, Supplemental Digital Content 3, http://links.lww.com/QAI/A30). This phenotype is crucial because it represents the dominant type of sexually transmitted HIV-1 strains.23,30 Furthermore, SHIVB'WHU was demonstrated to be replication-competent in PBMCs of 2 randomly selected Chinese macaques (See Figure B, Supplemental Digital Content 3, http://links.lww.com/QAI/A30), which warranted the subsequent in vivo experiments. Although no detectable viremia was found in peripheral blood of P1-#0002 monkey at the acute phase of infection, the subsequent increasing viremia detected in P2-P4 animals suggested that SHIVB'WHU has adapted to grow in vivo and therefore became infectious in Chinese macaques (Fig. 2). In general, the observed pattern of SHIVB'WHU replication in macaques is similar to previous findings of SHIV infection in a new monkey host.16 Consistently, the improved growth kinetics and the broader tissue tropism of SHIVB'WHU in P2-P4 animals suggested the enhanced viral infectivity in vivo (Figs. 2, 3). The enhanced infectivity, however, did not result in altered CCR5-tropism and therefore, unlike pathogenic X4-tropic and X4R5-tropic SHIV strains which induce severe CD4+ T-cell loss in the periphery,23,31 no significant CD4+ lymphocytopenia was found in peripheral blood of SHIVB'WHU-infected macaques. In addition, although we see enhanced viral replications in P3 and P4 macaques, we cannot exclude the possibility that the target cells of P1 and P2 are less susceptible to SHIVB'WHU infection.
The rapid in vivo serial passage improved the infectivity of SHIVB'WHU probably by improving viral fitness and extending the involvement of tissue CD4+ T lymphocytes in vivo. Previous investigators demonstrated that serial passage of SHIV in macaques resulted in virus with high pathogenecity.18,32,33 The pathogenicity was often indicated by high viral load, CD4+ T lymphocytopenia, and even simian AIDS. In this study, we found that a freshly made (before adaptation in macaques) CCR5-tropic SHIV was able to deplete CD4+ IELs and to less extent the CD4+ lamina propria lymphocytes in all 4 macaques tested. This finding suggested that CD4+ IELs were probably more susceptible to the acute infection of SHIVB'WHU even without the overtime adaptation of the virus in vivo. However, the number of experimental animals is small in our study. Because similar studies were not conducted for other CCR5-tropic SHIVs, it remains to be determined whether our finding is a general phenomenon for viruses of the same phenotype. Moreover, a more quantitative FACS analysis is needed to determine the quantitative loss of CD4+ IEL in SHIVB'WHU-infected animals in future study. Previous studies indicated that some changes were also detected in the IELs population during SIV infection,34 it might be critical to determine the role of IELs in seeding the early transmission and replication of CCR5-tropic HIV-1. Because the viral tropism was not changed in P3 and P4 monkeys, it is therefore possible that the improved infectivity of SHIVB'WHU was not simply due to its phenotypic changes. It is likely that the early involvement of CD4+ T lymphocytes in broad tissue compartments also contributed to the rapid burst of viremia especially in P3 and P4 animals (Fig. 3). To confirm the improved infectivity of the virus, 3 additional Chinese macaques were recently inoculated with SHIVB'WHU derived from the P4 animal. All 3 animals have become infected as indicated by peak viremia and subsequent seroconversion. These animals will be used to determine whether or not SHIVB'WHU would lead to persistent infection and subsequent simian AIDS in Chinese macaques. In addition, to build a useful SHIVB'WHU/Chinese macaque model, it would also be necessary to determine the mucosal transmissibility of SHIVB'WHU with enhanced infectivity in future studies. Lastly, because different macaque species may respond to SIV or SHIV infection differently, future studies should determine the pathogenicity and disease course of SHIVB'WHU infection in Indian or pig-tailed macaques.
We thank Drs D. D. Ho and K. Y. Yuen for scientific advice and C. Cheng-Mayer for SHIV33 molecular clone and discussions.
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