Enhanced infectivity of an R5-tropic simian/human immunodeficiency virus carrying human immunodeficiency virus type 1 subtype C envelope after serial passages in pig-tailed macaques (Macaca nemestrina)

Abstract

The increasing prevalence of human immunodeficiency virus type 1 (HIV-1) subtype C infection worldwide calls for efforts to develop a relevant animal model for evaluating strategies against the transmission of the virus. A chimeric simian/human immunodeficiency virus (SHIV), SHIV(CHN19), was generated with a primary, non-syncytium-inducing HIV-1 subtype C envelope from a Chinese strain in the background of SHIV(33). Unlike R5-tropic SHIV(162), SHIV(CHN19) was not found to replicate in rhesus CD4(+) T lymphocytes. SHIV(CHN19) does, however, replicate in CD4(+) T lymphocytes of pig-tailed macaques (Macaca nemestrina). The observed replication competence of SHIV(CHN19) requires the full tat/rev genes and partial gp41 region derived from SHIV(33). To evaluate in vivo infectivity, SHIV(CHN19) was intravenously inoculated, at first, into two pig-tailed and two rhesus macaques. Although all four animals became infected, the virus replicated preferentially in pig-tailed macaques with an earlier plasma viral peak and a faster seroconversion. To determine whether in vivo adaptation would enhance the infectivity of SHIV(CHN19), passages were carried out serially in three groups of two pig-tailed macaques each, via intravenous blood-bone marrow transfusion. The passages greatly enhanced the infectivity of the virus as shown by the increasingly elevated viral loads during acute infection in animals with each passage. Moreover, the doubling time of plasma virus during acute infection became much shorter in passage 4 (P4) animals (0.2 day) in comparison to P1 animals (1 to 2 days). P2 to P4 animals all became seropositive around 2 to 3 weeks postinoculation and had a decline in CD4/CD8 T-cell ratio during the early phase of infection. In P4 animals, a profound depletion of CD4 T cells in the lamina propria of the jejunum was observed. Persistent plasma viremia has been found in most of the infected animals with sustained viral loads ranging from 10(3) to 10(5) per ml up to 6 months postinfection. Serial passages did not change the viral phenotype as confirmed by the persistence of the R5 tropism of SHIV(CHN19) isolated from P4 animals. In addition, the infectivity of SHIV(CHN19) in rhesus peripheral blood mononuclear cells was also increased after in vivo passages. Our data indicate that SHIV(CHN19) has adapted well to grow in macaque cells. This established R5-tropic SHIV(CHN19)/macaque model would be very useful for HIV-1 subtype C vaccine and pathogenesis studies.

FIG. 1

Schematic representation of subtype C SHIV construction. The viral genome is constructed in two plasmids, 5′-PVP-1 and 3′-pCHN19. Since the structure of plasmid PVP-1 was unchanged, the reconstruction was limited to the 3′-SHIV₃₃ genome. The 5′-tat/rev and partial vpu were replaced with the corresponding regions of 3′-SHIV₃₃ by an overlapping PCR without disrupting the subtype C env gene. The 3′-tat/rev of 3′-pCHN19 was replaced by the counterpart of 3′-SHIV₃₃ by using restriction enzyme AvrII, which cuts at a site in the gp41 region shared by SHIV₃₃ and HIV-1_CHN19. In order to enhance the viral replication in vivo, the mutant vpu gene of SHIV₃₃ was opened by a PCR mutagenesis method to ensure vpu expression (vpu⁺).

FIG. 2

Phylogenetic analysis of full-length env genes of HIV-1_CHN19 and HIV-1_IN108. The reference sequences were obtained from the HIV sequence database in GenBank (28). The tree was constructed by using full-length env nucleotide sequences by a neighbor-joining method as described previously (9). The branches are additive, and they represent genetic distances between viruses. The solid scale bar indicates a genetic distance of 10%. The accession number of the HIV-1_CHN19 env gene is AF268277.

FIG. 3

The replication kinetics of SHIV_CHN19 in human PBMC (A) and in CD4⁺ T lymphocytes derived from a pig-tailed macaque (B). Human PBMC or macaque CD4⁺ T lymphocytes (2 × 10⁶) were infected with a virus input of 100 TCID₅₀. The replication of SHIV and SIV was monitored by using the p27 assay (Cellular Products). The y axis represents the level of p27 production in the culture supernatants.

FIG. 4

The plasma viremia and the absolute CD4⁺ and CD8⁺ T-cell counts in four P1 animals p.i. Animals T899 and T817 (upper panel) are two P1 pig-tailed macaques, whereas AG11 and AG36 (lower panel) are two P1 rhesus macaques. The primary y axis represents viral RNA copies per milliliter of plasma. The secondary y axis indicates the absolute number of CD4⁺ or CD8⁺ T cells per microliter of whole blood.

FIG. 5

The changes in CD4/CD8 ratios over time in P1 to P4 pig-tailed macaques postinfection. Animals from the same passage are plotted in the same panel, which include two P1 (T899 and T817, upper left), two P2 (T625 and T677, upper right), two P3 (T767 and V874, lower left), and two P4 (T909 and T910, lower right) macaques.

FIG. 6

The plasma viremia and the absolute CD4⁺ and CD8⁺ T-cell counts in P2 to P4 pig-tailed macaques p.i. Animals include two P2 (T625 and T677, upper panel), two P3 (T767 and V874, middle panel), and two P4 (T909 and T910, lower panel) macaques. The primary y axis represents viral RNA copies per milliliter of plasma. The secondary y axis indicates the absolute number of CD4⁺ or CD8⁺ T cells per microliter of whole blood.

FIG. 7

Comparison of the replication kinetics of the viral isolate (SHIV_CHN19P) from P4 animal T910 to parental SHIV_CHN19 in rhesus PBMC. Rhesus PBMC (2 × 10⁶) were infected with a virus input of 100 TCID₅₀. The y axis represents the level of p27 production in the culture supernatant.

FIG. 8

Comparison of percentage of CD4⁺ cells among total CD3⁺ T lymphocytes from different compartments. Four animals were tested, including two P4 (T909 and T910) and two naïve (AE15 and AV25) pig-tailed macaques. T lymphocytes derived from three compartments, including blood, colonic lymph node, and jejunal lamina propria, were assayed. The y axis represents the percentage of CD4⁺ T lymphocytes. The number above each vertical bar indicates the average percentage of two separate stainings of the same specimen. Specimens from the two P4 animals were collected 2 weeks p.i.