In situ hybridization and immunolabelling study of the early replication of simian immunodeficiency virus (SIVmacJ5) in vivo

Abstract

The distribution of virus-infected cells in cynomolgus macaques was determined at 4, 7, 14 and 28 days following intravenous challenge with 1000 TCID 50 of the wild-type simian immunodeficiency virus SIVmacJ5 (stock J5C). At each time-point, pairs of macaques were killed humanely and the presence of SIV was determined and quantified in blood, spleen, peripheral and mesenteric lymph nodes, thymus, lung and ileum by virus co-cultivation with C8166 cells, by quantitative DNA PCR or by in situ hybridization (ISH). At day 4 post-infection (p.i.), detection of the virus was sporadic. By day 7 p.i., however, significant SIV loads were detected in the blood and lymphoid tissues by DNA PCR and virus co-cultivation. Large numbers of cells expressing SIV RNA were detected in mesenteric lymph nodes by ISH and significantly fewer (P 0n05) in the spleen. Significant numbers of ISH-positive cells were also observed in sections of ileum. By day 14 p.i., the distribution of SIV was more even in all lymphoid tissues analysed. By day 28, most of the tissues were negative by ISH, but all remained positive by virus isolation and DNA PCR. Immunolabelling of sections of mesenteric lymph node with monoclonal antibodies specific for SIV envelope and Nef largely confirmed the observations from ISH. These results indicate that, even following intravenous challenge, a major site of the initial replication of SIV is gut-associated lymphoid tissue. Vaccines that induce protection at this site may therefore be superior, even against parenteral challenge.

Social Stress Enhances Sympathetic Innervation of Primate Lymph Nodes: Mechanisms and Implications for Viral Pathogenesis

“…In vitro analyses of SIV replication used adult male rhesus macaque peripheral blood mononuclear cells (PBMCs) that were infected with SIV mac251 , stimulated with phytohemagglutinin (PHA) (Sigma, St. Louis, MO), and subsequently cultured in the presence of 0.5, 5, or 50 ng/ml biologically active recombinant human mature NGF␤ (US Biological, Swampscott, MA) or saline control. Viral replication at 4 and 6 d after infection was quantified by real-time RT-PCR (see below for description) for gag and env mRNA in total cellular RNA using previously published primer sequences (Canto-Nogues et al, 2001). Results were normalized to expression of cellular mRNA for ␤-actin (ACTB; primer sequences below).…”

Enhanced Replication of Simian Immunodeficiency Virus Adjacent to Catecholaminergic Varicosities in Primate Lymph Nodes

“…Frozen sections were fixed in 4% paraformaldehyde, digested in 1 mg/ml proteinase K (Sigma) for 5 min at 37°C, and prehybridized in hybridization buffer (50% formamide, 10% dextran sulfate, 0.05% sodium dodecyl sulfate, 0.05% polyvinylpyrrolidone, 500 g/ml boiled salmon sperm DNA, 260 g/ml Saccharomyces cerevisiae tRNA, 50 mM Tris HCl, pH 7.4, 300 mM NaCl, 2 mM EDTA) for 2 h at 37°C. Single-stranded DNA probes were synthesized by PCR and labeled with digoxigenin (Boehringer Mannheim) as previously described (4). A cocktail of all three complementary probes (or three sense probes as a negative control) were mixed in hybridization buffer, denatured at 65°C for 5 min, and hybridized to the sections at 37°C overnight.…”

Antigenic Stimulation Specifically Reactivates the Replication of Archived Simian Immunodeficiency Virus Genomes in Chronically Infected Macaques

“…Moreover, the major sequences identified at day 14 in both individuals are different, and none of them were found within the 30 clones sequenced from the viral stock, suggestive of the existence of a "bottleneck" during the first days of infection. Such bottlenecks could be attributed to early infection of particular cells such as mucosal CD4 ϩ cells that have been shown to be the principal target of SIV infection and rapidly depleted during primary infection in both macaques and human (4,48).…”