Neuropathogenesis of Simian Immunodeficiency Virus in Neonatal Rhesus Macaques
1999; Elsevier BV; Volume: 155; Issue: 4 Linguagem: Inglês
10.1016/s0002-9440(10)65224-8
ISSN1525-2191
AutoresSusan V. Westmoreland, Kenneth C. Williams, Meredith A. Simon, Mary E. Bahn, Amy E. Rullkoetter, Michelle W. Elliott, Colin deBakker, Heather Knight, Andrew A. Lackner,
Tópico(s)Cytomegalovirus and herpesvirus research
ResumoNeonatal human immunodeficiency virus (HIV) infection usually occurs intrapartum or postpartum and results in a higher incidence of neurological dysfunction than is seen in adults. To explore the neuropathogenesis of neonatal HIV infection, we infected neonatal macaques with simian immunodeficiency virus (SIV) and followed the course of infection focusing on early time points. Infected neonates had decreased brain growth and mild histological changes in brain that resembled those seen in pediatric AIDS, including perivascular infiltrates of mononuclear cells, mineralization of vessels in the basal ganglia, and gliosis. The perivascular lesions and gliosis were associated with the presence of occasional infected cells that required in situ hybridization with radiolabeled riboprobes for detection. Using this technique, SIV-infected cells were detected in the brain parenchyma within 7 days of infection. These findings were confirmed by nested PCR for SIVgag DNA in brain and RT-PCR for viral RNA in cerebrospinal fluid. Together, these techniques revealed SIV infection of the CNS in 12 of 13 neonates infected with SIVmac239, 3 of 3 infected with SIVmac251, and 2 of 2 infected with SIVmac239/316. The prevalence of CNS infection was indistinguishable from that of older animals infected with the same dose and stock of virus, but neonates appeared to have fewer infected cells in the CNS and detecting them required more sensitive techniques. This observation was true regardless of inoculum and despite the fact that neonates had equal or greater viral loads in the periphery compared with older animals. These data suggest that maturation-dependent host factors have a major impact on the neuropathogenesis of pediatric AIDS. Neonatal human immunodeficiency virus (HIV) infection usually occurs intrapartum or postpartum and results in a higher incidence of neurological dysfunction than is seen in adults. To explore the neuropathogenesis of neonatal HIV infection, we infected neonatal macaques with simian immunodeficiency virus (SIV) and followed the course of infection focusing on early time points. Infected neonates had decreased brain growth and mild histological changes in brain that resembled those seen in pediatric AIDS, including perivascular infiltrates of mononuclear cells, mineralization of vessels in the basal ganglia, and gliosis. The perivascular lesions and gliosis were associated with the presence of occasional infected cells that required in situ hybridization with radiolabeled riboprobes for detection. Using this technique, SIV-infected cells were detected in the brain parenchyma within 7 days of infection. These findings were confirmed by nested PCR for SIVgag DNA in brain and RT-PCR for viral RNA in cerebrospinal fluid. Together, these techniques revealed SIV infection of the CNS in 12 of 13 neonates infected with SIVmac239, 3 of 3 infected with SIVmac251, and 2 of 2 infected with SIVmac239/316. The prevalence of CNS infection was indistinguishable from that of older animals infected with the same dose and stock of virus, but neonates appeared to have fewer infected cells in the CNS and detecting them required more sensitive techniques. This observation was true regardless of inoculum and despite the fact that neonates had equal or greater viral loads in the periphery compared with older animals. These data suggest that maturation-dependent host factors have a major impact on the neuropathogenesis of pediatric AIDS. Pediatric human immunodeficiency virus (HIV) infection usually occurs intrapartum or immediately postpartum and results in a higher incidence of neurological disease than is seen in adults.1Wilfert CM Wilson C Luzuriaga K Epstein L Pathogenesis of pediatric human immunodeficiency virus type 1 infection.J Infect Dis. 1994; 170: 286-292Crossref PubMed Scopus (39) Google Scholar, 2Ammann AJ Human immunodeficiency virus infection/AIDS in children: the next decade.Pediatrics. 1994; 93: 930-935PubMed Google Scholar, 3Belman AL HIV-1-associated CNS disease in infants and children.Res Publ Asso Res Nerv Ment Dis. 1994; 72: 289-310PubMed Google Scholar The central nervous system (CNS) disease in children is also considered to be more severe than in adults but has a different spectrum of manifestations due to the developmental state of the brain. In children, the neurological disease is characterized by progressive or static loss of previously acquired neurodevelopmental milestones, impaired brain growth with cortical atrophy, and progressive motor dysfunction.3Belman AL HIV-1-associated CNS disease in infants and children.Res Publ Asso Res Nerv Ment Dis. 1994; 72: 289-310PubMed Google Scholar, 4Epstein LG Sharer LR Oleske JM Connor EM Goudsmit J Bagdon L Robert-Guroff M Koenigsberger MR Neurologic manifestations of human immunodeficiency virus infection in children.Pediatrics. 1986; 78: 678-687PubMed Google Scholar, 5Chase C Vibbert M Pelton SI Coulter DL Cabral H Early neurodevelopmental growth in children with vertically transmitted human immunodeficiency virus infection.Arch Pediatr Adolesc Med. 1995; 149: 850-855Crossref PubMed Scopus (42) Google Scholar, 6Nozyce M Hittelman J Muenz L Durako SJ Fischer ML Willoughby A Effect of perinatally acquired human immunodeficiency virus infection on neurodevelopment in children during the first two years of life.Pediatrics. 1994; 94: 883-891PubMed Google Scholar Histopathological findings are similar to those seen in adults and include perivascular mononuclear cell infiltrates with occasional multinucleated giant cells (MNGCs) and gliosis.4Epstein LG Sharer LR Oleske JM Connor EM Goudsmit J Bagdon L Robert-Guroff M Koenigsberger MR Neurologic manifestations of human immunodeficiency virus infection in children.Pediatrics. 1986; 78: 678-687PubMed Google Scholar, 7Sharer LR Epstein LG Cho E-S Joshi VV Meyenhofer MF Rankin LF Petito CK Pathologic features of AIDS encephalopathy in children: evidence for LAV/HTLV-III infection of the brain.Hum Pathol. 1986; 17: 271-284Abstract Full Text PDF PubMed Scopus (249) Google Scholar, 8Sharer LR Dowling PC Michaels J Cook SD Menonna J Blumberg BM Epstein LG Spinal cord disease in children with HIV-1 infection: a combined biological and neuropathological study.Neuropathol Appl Neurobiol. 1990; 16: 317-331Crossref PubMed Scopus (57) Google Scholar These lesions have a predilection for specific regions of the CNS, including basal ganglia, central white matter in cerebral hemispheres, and brain stem. In contrast to the neurological disease in adults, infants have a lower incidence of CNS opportunistic infections, peripheral neuropathies, and vacuolar myelopathy, but an increased incidence of vessel-associated mineralization.3Belman AL HIV-1-associated CNS disease in infants and children.Res Publ Asso Res Nerv Ment Dis. 1994; 72: 289-310PubMed Google Scholar, 7Sharer LR Epstein LG Cho E-S Joshi VV Meyenhofer MF Rankin LF Petito CK Pathologic features of AIDS encephalopathy in children: evidence for LAV/HTLV-III infection of the brain.Hum Pathol. 1986; 17: 271-284Abstract Full Text PDF PubMed Scopus (249) Google Scholar In the CNS of pediatric AIDS patients, HIV has been detected primarily within macrophages, microglia, and MNGCs, similar to what has been described in adults.7Sharer LR Epstein LG Cho E-S Joshi VV Meyenhofer MF Rankin LF Petito CK Pathologic features of AIDS encephalopathy in children: evidence for LAV/HTLV-III infection of the brain.Hum Pathol. 1986; 17: 271-284Abstract Full Text PDF PubMed Scopus (249) Google Scholar, 9Lyman WD Kress Y Kure K Rashbaum WK Rubinstein A Soeiro R Detection of HIV in fetal central nervous system tissue.AIDS. 1990; 4: 917-920Crossref PubMed Scopus (74) Google Scholar, 10Shaw GM Harper ME Hahn BH Epstein LG Gajdusek DC Price RW Navia BA Petito CK O'Hara CJ Cho E-S Oleske JM Wong-Staal F Gallo RC HTLV-III infection in brains of children, and adults with AIDS encephalopathy.Science. 1985; 227: 177-182Crossref PubMed Scopus (685) Google Scholar, 11Wiley CA Belman AL Dickson DW Rubenstein A Nelson JA Human immunodeficiency virus within the brains of children with AIDS.Clin Neuropathol. 1990; 9: 1-6PubMed Google Scholar, 12Kure K Lyman WD Weidenheim KM Dickson DW Cellular localization of an HIV-1 antigen in subacute AIDS encephalitis using an improved double-labeling immunohistochemical method.Am J Pathol. 1990; 136: 1085-1092PubMed Google Scholar Unique to pediatric AIDS is the possibility of restricted infection of astrocytes.13Tornatore C Chandra R Berger JR Major EO HIV-1 infection of subcortical astrocytes in the pediatric central nervous system.Neurology. 1994; 44: 481-487Crossref PubMed Google Scholar, 14Saito Y Sharer LR Epstein LG Michaels J Mintz M Louder M Golding K Cvetkovich TA Blumberg BM Overexpression of nef as a marker for restricted HIV-1 infection of astrocytes in postmortem pediatric central nervous tissues.Neurology. 1994; 44: 474-481Crossref PubMed Google Scholar Early events in neonatal HIV-1 infection, including the timing of neuroinvasion, the distribution of virus in the CNS, and host and viral factors that contribute to neurological disease, are poorly understood due to the difficulty of obtaining appropriate samples. Nevertheless, current data suggest that the increased severity of CNS disease in young children compared to adults is related to the time of infection (in utero, intrapartum, or postpartum) and the immaturity of the host immune system.1Wilfert CM Wilson C Luzuriaga K Epstein L Pathogenesis of pediatric human immunodeficiency virus type 1 infection.J Infect Dis. 1994; 170: 286-292Crossref PubMed Scopus (39) Google Scholar To further examine the neuropathogenesis of pediatric AIDS with a focus on early events, we have used the neonatal rhesus macaque infected with simian immunodeficiency virus (SIV) as a model of pediatric AIDS.15Marthas ML Van Rompay KKA Otsyula M Miller CJ Canfield DR Pedersen NC McChesney MB Viral factors determine progression to AIDS in simian immunodeficiency virus-infected newborn rhesus macaques.J Virol. 1995; 69: 4198-4205PubMed Google Scholar, 16Otsyula MG Miller CJ Marthas ML Van R KK A Collins JR Pedersen NC McChesney MB Virus-induced immunosuppression is linked to rapidly fatal disease in infant rhesus macaques infected with simian immunodeficiency virus.Pediatr Res. 1996; 39: 630-635Crossref PubMed Scopus (21) Google Scholar, 17Van Rompay KK Berardi CJ Dillard-Telm S Tarara RP Canfield DR Valverde CR Montefiori DC Cole KS Montelaro RC Miller CJ Marthas ML Passive immunization of newborn rhesus macaques prevents oral simian immunodeficiency virus infection.J Infect Dis. 1998; 177: 1247-1259Crossref PubMed Scopus (157) Google Scholar In this study, we examined the neuropathogenesis of SIV infection in neonatal macaques, focusing on the first 2 months of infection. This is the time period when SIV infection of the CNS has been shown to occur in older macaques.18Chakrabarti L Hurtrel M Maire M Vazeux R Dormont D Montagnier L Hurtrel B Early viral replication in the brain of SIV-infected rhesus monkeys.Am J Pathol. 1991; 139: 1273-1280PubMed Google Scholar, 19Smith MO Heyes MP Lackner AA Early intrathecal events in rhesus macaques (Macaca mulatta) infected with pathogenic or nonpathogenic molecular clones of simian immunodeficiency virus.Lab Invest. 1995; 72: 547-558PubMed Google Scholar, 20Lackner AA Vogel P Ramos RA Kluge JD Marthas M Early events in tissues during infection with pathogenic (SIVmac239) and nonpathogenic (SIVmac1A11) molecular clones of simian immunodeficiency virus.Am J Pathol. 1994; 145: 428-439PubMed Google Scholar For these studies animals were infected with equal doses of the pathogenic molecular clone SIVmac239 (n = 13), the macrophage-tropic derivative of SIVmac239 known as SIVmac239/316 (n = 2), or uncloned SIVmac251 (n = 3). Although the prevalence of CNS infection was indistinguishable from that of older animals infected with the same dose and stock of virus, neonates appeared to have fewer infected cells in the CNS and detecting them required more sensitive techniques. This was true regardless of inoculum and despite high viral loads in peripheral blood and peripheral lymphoid organs. Thus, although neuroinvasion by SIV occurred rapidly in neonatal macaques, viral replication and neuropathology were limited. This suggests that maturation-dependent host factors have a major impact on the neuropathogenesis of pediatric AIDS. A total of 18 rhesus macaque (Macaca muletta) neonates were obtained by cesarean section at 155 ± 5 days of gestation and inoculated intravenously within 24 hours of birth with 20 ng p27/kg (approximately 103 50% tissue culture infectious doses/kg) of one of three isolates of SIV: SIVmac239 and SIVmac239/316, which are molecular clones, and uncloned SIVmac251 (Table 1). These are the same stocks and doses of virus (per kilogram) that have been used previously in our juvenile macaque studies.19Smith MO Heyes MP Lackner AA Early intrathecal events in rhesus macaques (Macaca mulatta) infected with pathogenic or nonpathogenic molecular clones of simian immunodeficiency virus.Lab Invest. 1995; 72: 547-558PubMed Google Scholar, 20Lackner AA Vogel P Ramos RA Kluge JD Marthas M Early events in tissues during infection with pathogenic (SIVmac239) and nonpathogenic (SIVmac1A11) molecular clones of simian immunodeficiency virus.Am J Pathol. 1994; 145: 428-439PubMed Google Scholar, 21Wykrzykowska JJ Rosenzweig M Veazey RS Simon MA Halvorsen K Desrosiers RC Johnson RP Lackner AA Early regeneration of thymic progenitors in rhesus macaques infected with simian immunodeficiency virus.J Exp Med. 1998; 187: 1767-1778Crossref PubMed Scopus (85) Google Scholar, 22Veazey RS DeMaria M Chalifoux LV Shvetz DE Pauley DR Knight HL Rosenzweig M Johnson RP Desrosiers RC Lackner AA The gastrointestinal tract as a major site of CD4+ T cell depletion and viral replication in SIV infection.Science. 1998; 280: 427-431Crossref PubMed Scopus (1219) Google Scholar, 23Lane JH Sasseville VG Smith MO Vogel P Pauley DR Heyes MP Lackner AA Neuroinvasion by simian immunodeficiency virus coincides with increased numbers of perivascular macrophages/microglia and intrathecal immune activation.J Neurovirol. 1996; 2: 423-432Crossref PubMed Scopus (136) Google Scholar The origin, gene sequence, and biological behavior of these viruses have been described extensively.24Kestler H Kodama T Ringler D Marthas M Pedersen N Lackner A Regier D Sehgal P Daniel M King N Desrosiers R Induction of AIDS in rhesus monkeys by molecularly cloned simian immunodeficiency virus.Science. 1990; 248: 1109-1112Crossref PubMed Scopus (517) Google Scholar, 25Mori K Ringler DJ Kodama T Desrosiers RC Complex determinants of macrophage tropism in env of simian immunodeficiency virus.J Virol. 1992; 66: 2067-2075PubMed Google Scholar, 26Kodama T Mori K Kawahara T Ringler DJ Desrosiers RC Analysis of simian immunodeficiency virus sequence variation in tissues of rhesus macaques with AIDS.J Virol. 1993; 67: 6522-6534PubMed Google Scholar, 27Desrosiers RC The simian immunodeficiency viruses.Annu Rev Immunol. 1990; 8: 557-578Crossref PubMed Scopus (342) Google Scholar, 28Lackner AA Pathology of simian immunodeficiency virus induced disease.in: Desrosiers RC Letvin N Current Topics in Microbiology and Immunology: Simian Immunodeficiency Virus. Springer Verlag, Berlin1994: 35-64Google Scholar Briefly, SIVmac239 is the prototypical pathogenic molecular clone; it replicates poorly in monocyte/macrophages in vitro. SIVmac239/316 is a macrophage-competent derivative of SIVmac239 that differs by eight amino acids in envelope. SIVmac251 is a highly pathogenic uncloned isolate that replicates well in both lymphocytes and monocyte/macrophages.Table 1Animals, Viral Inoculum, and Major Pathological Findings in Neonates Infected with SIVVirusAnimal no.Days piMajor pathological findings*Excludes the CNS.SIVmac23968–973NSL69–973Hepatitis, periportal minimal102–977Thymic dysinvolution, mild; lymphoid hyperplasia, moderate103–977Thymic dysinvolution, moderate; lymphoid hyperplasia, moderate136–9714Lymphoid hyperplasia, moderate; thymic dysinvolution, mildHepatitis, typhlocolitis, myocarditis (Clostridium pilliforme)137–9714Lymphoid hyperplasia, mild; thymic dysinvolution, markedHepatitis, typhlocolitis, myocarditis (Clostridium pilliforme)190–9721Lymphoid hyperplasia, moderateTyphlocolitis (Clostridium pilliforme)191–9721Lymphoid hyperplasia, moderateTyphlocolitis (Clostridium pilliforme)296–9750Lymphoid hyperplasia, moderate297–9750Lymphoid hyperplasia, moderate415–97†Animals euthanized when moribund with AIDS.79Lymphoid depletion, severeAdenoviral enteritis484–97†Animals euthanized when moribund with AIDS.141Lymphoid depletion, severeAdenoviral enteritis, severe412–97†Animals euthanized when moribund with AIDS.209Lymphoid depletion, severeDisseminated adenoviral infectionPneumocystis carinii pneumoniaSIVmac251163–9821Lymphoid hyperplasia, moderate; thymic dysinvolution, mild162–98†Animals euthanized when moribund with AIDS.35Lymphoid depletion, moderateDisseminated adenoviral infection160–9850Lymphoid depletion, moderateAdenoviral enteritisSIVmac239/316109–9850Lymphoid hyperplasia, mildAdenoviral enteritis110–9850Lymphoid hyperplasia, mildAdenoviral enterocolitisNSL, no significant lesions.* Excludes the CNS.† Animals euthanized when moribund with AIDS. Open table in a new tab NSL, no significant lesions. Thirteen animals were inoculated with SIVmac239 and two animals were euthanized at 3, 7, 14, 21, and 50 days postinfection (dpi). Three additional animals were similarly infected and allowed to progress until they became moribund with AIDS, at which time they were euthanized at 79, 141, and 209 dpi. Two animals were inoculated with SIVmac239/316 and euthanized at 50 dpi. Three animals were inoculated with SIVmac251 and euthanized at 21, 35, and 50 dpi. The SIVmac251-infected infant euthanized at 35 dpi was moribund with AIDS. All animals were housed in accordance with standards of the American Association for Accreditation of Laboratory Animal Care. The investigators adhered to the Guide for the Care and Use of Laboratory Animals prepared by the Committee on Care and Use of Laboratory Animals of the Institute of Laboratory Resources, National Research Council. All dams were negative for antibodies to HIV-2, SIV, Type D retrovirus, and Simian T-cell leukemia virus type 1 before cesarean section. Peripheral blood was collected from all animals before inoculation, at days 3, 7, 14, 21, 35, and 50, monthly thereafter, and terminally. CSF was collected at similar intervals, but only from animals inoculated with SIVmac251, SIVmac239/316, and two of the three animals inoculated with SIVmac239 that survived more than 50 days. Peripheral blood was used for quantitation of cell-associated viral loads and determination of plasma SIV RNA levels. Quantitative viral cultures were performed on each blood sample as described previously.29Gibbs JS Lackner AA Lang SM Simon MA Sehgal PK Daniel MD Desrosiers RC Progression to AIDS in the absence of a gene for vpr or vpx.J Virol. 1995; 69: 2378-2383Crossref PubMed Google Scholar Briefly, serial threefold dilutions were performed in duplicate beginning with 106 PBMC. PBMC dilutions were cocultured with 105 CEMX174 cells in a volume of 1 ml. Cultures were split 1:2 twice weekly until day 21, when the cultures were assayed for virus production by enzyme-linked immunosorbent assay for SIV p27 (Coulter Corp., Hialeah, FL). Results are expressed as the number of SIV-infected cells/106Nozyce M Hittelman J Muenz L Durako SJ Fischer ML Willoughby A Effect of perinatally acquired human immunodeficiency virus infection on neurodevelopment in children during the first two years of life.Pediatrics. 1994; 94: 883-891PubMed Google Scholar PBMC. Virion-associated SIV RNA in plasma and CSF was quantified by using a real-time reverse transcription-polymerase chain reaction (RT-PCR) assay on an Applied Biosystems (Foster City, CA) Prism 7700 sequence detection system as described previously.30Desrosiers RC Lifson JD Gibbs JS Czajak SC Howe AY Arthur LO Johnson RP Identification of highly attenuated mutants of simian immunodeficiency virus.J Virol. 1998; 72: 1431-1437Crossref PubMed Google Scholar, 31Suryanarayana K Wiltrout TA Vasquez GM Hirsch VM Lifson JD Plasma SIV RNA viral load determination by real-time quantification of product generation in reverse transcriptase-polymerase chain reaction.AIDS Res Hum Retroviruses. 1998; 14: 183-189Crossref PubMed Scopus (196) Google Scholar Results shown are averages of duplicate determinations. Analyses of viral RNA levels were performed by Drs. Jeffrey Lifson and Michael Piatak at Scientific Applications International Corporation (Frederick, MD). Animals were sacrificed at intervals described above and as shown in Table 1. At necropsy animals were exsanguinated and body and organ weights were recorded. A complete set of tissues, including frontal cortex, basal nuclei, thalamus, and brain stem, were collected and fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned at 6 μm, and stained with hematoxylin and eosin by routine techniques. In situ hybridization was performed on serial sections. Adjacent blocks of fresh tissue were snap-frozen for immunohistochemistry in optimum cutting temperature compound (O.C.T., Miles Inc., Elkhart, IN) by immersion in 2-methylbutane cooled in dry ice. Viral localization was examined in peripheral lymphoid tissues and at least three different regions of the brain, including cerebral cortex, basal ganglia, and brain stem, by immunohistochemistry for viral antigens and two different in situ hybridization techniques to detect RNA. The in situ hybridization techniques used either a digoxigenin-labeled, random primed DNA probe or a 35S-labeled riboprobe. The DNA probe was a combination of two plasmids: a subclone of p239SpE3′ in pBS−, which contains tat, rev, env, nef, and small part of the 3′LTR, and p239SpSp5′, which contains gag, pol, vif, vpx, vpr, and the 5′LTR in pBS+. This combination provides essentially the entire SIVmac genome. The probe was labeled with digoxigenin-11dUTP by random priming (Boehringer Mannheim, Indianapolis, IN) as previously described.29Gibbs JS Lackner AA Lang SM Simon MA Sehgal PK Daniel MD Desrosiers RC Progression to AIDS in the absence of a gene for vpr or vpx.J Virol. 1995; 69: 2378-2383Crossref PubMed Google Scholar Controls consisted of hybridizing sections with plasmid pUC19, which had been labeled with digoxigenin at the same time as the probe and matched tissues from uninfected, age-matched macaques. Labeled cells were detected using a digoxigenin-specific antibody in a standard avidin-biotin-horseradish peroxidase complex (ABC) technique as previously described.29Gibbs JS Lackner AA Lang SM Simon MA Sehgal PK Daniel MD Desrosiers RC Progression to AIDS in the absence of a gene for vpr or vpx.J Virol. 1995; 69: 2378-2383Crossref PubMed Google Scholar The second in situ hybridization technique used radiolabeled RNA probes synthesized from five DNA templates, covering 90% of the SIV genome, subcloned into pGEM4.32Fox CH Cottler-Fox M In situ hybridization for detection of HIV RNA.in: Coligan JE Kruisbeek AM Margulies DH Shevach EM Strober W Current Protocols in Immunology. John Wiley & Sons, New York1994: 12.8.1-12.8.21Google Scholar Controls consisted of hybridizing sections with sense probes and matched tissues from uninfected, age-matched macaques. The radiolabled in situ hybridization was performed by Dr. Cecil Fox at Molecular Histology, Inc. (Montgomery Village, MD). To localize viral antigen, snap-frozen tissues were used in immunohistochemical procedures as previously described.21Wykrzykowska JJ Rosenzweig M Veazey RS Simon MA Halvorsen K Desrosiers RC Johnson RP Lackner AA Early regeneration of thymic progenitors in rhesus macaques infected with simian immunodeficiency virus.J Exp Med. 1998; 187: 1767-1778Crossref PubMed Scopus (85) Google Scholar Briefly, frozen tissue sections were fixed in 2% paraformaldehyde for 10 minutes at 4°C and immunostained using an ABC technique with diaminobenzidine (DAB) as the chromogen. The primary antibody used was Senv71.1 (provided by C. Colignon, C. Thiriart, SmithKline Beecham, Rikensart, Belgium), which recognizes SIV gp120. Negative controls included serial sections processed identically, using equivalent concentrations of irrelevant primary antibodies of the same isotype and matched tissues from uninfected macaques. To confirm the results of in situ hybridization for viral RNA and to detect viral DNA we performed nested PCR for SIVgag. Tissue sections were collected at necropsy from selected brain regions, including the frontal cortex, basal ganglia, and brain stem, in 50- to 100-mg pieces, frozen in microcentrifuge tubes on dry ice, and stored at −70°C for future use. DNA was isolated from frozen tissues using the Qiagen (Valencia, CA) DNA isolation kit as per manufacturer's recommendations with an additional overnight incubation with proteinase K at 55°C. Two hundred nanograms of genomic DNA were amplified using nested primers (10 pmol/50 μl reaction) for SIVgag (outer 5′: 5′-CTA CGA CCC AAC GGC AAG-3′; outer 3′: 5′-TTG CTT CCT CAG TGT GTT TC-3′; inner 5′: 5′-GAA AGC CTG TTG GAG AAC AAA GAA GGA-3′; inner 3′: 5′-AGT GTG TTT CAC TTT CTC TTC TGC GTG-3′) with 2 mmol/L MgCl2 and denatured at 92°C for 2 minutes. The amplification profile consisted of 30 seconds at 92°C, 30 seconds at 61°C, and 30 seconds at 72°C for 40 cycles followed by an extension time of 10 minutes at 72°C. PCR products were visualized on an ethidium bromide-impregnated agarose gel. To examine the immunophenotype of infected cells we combined nonradiolabeled in situ hybridization for viral RNA with immunohistochemistry for monocyte/macrophages. This entailed performing nonradiolabeled in situ hybridization for SIV as described above using nickel cobalt-enhanced DAB (black), followed by immunohistochemistry for monocyte/macrophages (HAM56) using DAB (brown) as previously described.22Veazey RS DeMaria M Chalifoux LV Shvetz DE Pauley DR Knight HL Rosenzweig M Johnson RP Desrosiers RC Lackner AA The gastrointestinal tract as a major site of CD4+ T cell depletion and viral replication in SIV infection.Science. 1998; 280: 427-431Crossref PubMed Scopus (1219) Google Scholar These double labels were performed on peripheral lymphoid tissues of rhesus neonates. The scarcity of infected cells in the brain precluded effective use of this technique in the CNS. All 18 animals inoculated with SIV were viremic within 3 days of inoculation and remained persistently viremic throughout the course of infection. PBMC viral loads and plasma SIV RNA levels rose quickly, peaked at 7 to 14 dpi, and remained high for the life of the animal (Figure 1). This pattern of viral infection is similar to that of adult rapid progressors.20Lackner AA Vogel P Ramos RA Kluge JD Marthas M Early events in tissues during infection with pathogenic (SIVmac239) and nonpathogenic (SIVmac1A11) molecular clones of simian immunodeficiency virus.Am J Pathol. 1994; 145: 428-439PubMed Google Scholar Notably, viral loads in neonates were as high as or higher than those seen in juveniles or adults inoculated with the same stock and dose of virus at the same time points, but the difference did not reach statistical significance (data not shown). Robust viral replication was also demonstrated by immunohistochemistry and in situ hybridization in spleen, lymph nodes, and thymus as early as 7 dpi in animals infected with SIVmac239, SIVmac251, and SIVmac239/316 (Figure 2). Overall, disease progression was rapid as evidenced by the early deaths of animals that were allowed to progress to terminal disease: 35 dpi with SIVmac251 and 79, 141, and 209 dpi with SIVmac239. Similar rapid disease progression in SIV-infected neonates has been described previously.15Marthas ML Van Rompay KKA Otsyula M Miller CJ Canfield DR Pedersen NC McChesney MB Viral factors determine progression to AIDS in simian immunodeficiency virus-infected newborn rhesus macaques.J Virol. 1995; 69: 4198-4205PubMed Google ScholarFigure 2Detection of SIV by nonradiolabeled in situ hybridization in the spleen of a rhesus neonate 7 dpi. Note the large number of infected cells. Original magnification, ×80View Large Image Figure ViewerDownload Hi-res image Download (PPT) In addition to high viral loads and rapid disease course, there was a high incidence of opportunistic infections, which affected 10 of 18 animals (56%, Table 1), including all of the animals allowed to progress to terminal disease. The most common opportunistic infection in SIV-infected rhesus neonates was adenovirus, which was detected in 3/13 SIVmac239-, 2/3 SIVmac251-, and 2/2 SIVmac239/316-infected neonates. Other lesions caused by opportunists included hepatitis and/or typhlocolitis, caused by Clostridium pilliforme, in four SIVmac239-infected neonates at 14 and 21 dpi, and pneumonia, caused by Pneumocystis carinii in one SIVmac239-infected infant at 209 dpi. None of the opportunistic infections involved the CNS. Maximum growth rate for body weight, brain weight, and head size in normal infant rhesus macaques is from birth to 6 months of age with continued gradual increases in these parameters until maturity.33Saxton JL Lotz WG Growth of rhesus monkeys during the first 54 months of life.J Med Primatol. 1990; 19: 119-136PubMed Google Scholar, 34Wagman IH Loeffler JR McMillan JA Relationship between growth of brain and skull of Macaca mulatta and its importance for the stereotaxic technique.Brain Be
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