CD8-positive lymphocytes in graft-versus-host disease of humanized NOD.Cg-Prkdc(scid)Il2rg(tm1Wjl)/SzJ mice.

Immunocompromised mice that can support a human immune system are an increasingly important model for the investigation of haemopoietic stem/progenitor cell (HSPC) development and human infectious disease. NOD-SCID IL-2Rγ(-/-) (NSG) mice engrafted with human fetal liver and thymus prior to HSPC engraftment, commonly known as NSG-bone marrow-liver-thymus (NSG-hu-BLT) mice, are one such model and have robust reconstitution of human leucocytes within the peripheral blood and tissues. Four NSG-hu-BLT mice were submitted for diagnostic necropsy examination following the development of alopecia, pruritus and lethargy after HSPC engraftment. Histopathology revealed multifocal to coalescing single keratinocyte cell death in the epidermis and follicles with dermatitis and mild dermal fibrosis. Single-cell hepatocyte cell death was present in three cases, with various degrees of portal fibrosis. In the skin and liver, cell death was associated with lymphocytes that reacted with anti-human CD45, CD3 and CD8 antibodies, consistent with a diagnosis of graft-versus-host disease (GvHD). This study expands on recently reported microscopical features of GvHD in NSG-hu-BLT mice and suggests a role for CD8(+) T lymphocytes in the progression of the disease. NSG-hu-BLT mice represent an excellent model of GvHD, but its prevalence may compromise their use in other fields of biomedical research.

SCH-C (SCH 351125), an orally bioavailable, small molecule antagonist of the chemokine receptor CCR5, is a potent inhibitor of HIV-1 infection in vitro and in vivo.

We describe here the identification and properties of SCH-C (SCH 351125), a small molecule inhibitor of HIV-1 entry via the CCR5 coreceptor. SCH-C, an oxime-piperidine compound, is a specific CCR5 antagonist as determined in multiple receptor binding and signal transduction assays. This compound specifically inhibits HIV-1 infection mediated by CCR5 in U-87 astroglioma cells but has no effect on infection of CXCR4-expressing cells. SCH-C has broad and potent antiviral activity in vitro against primary HIV-1 isolates that use CCR5 as their entry coreceptor, with mean 50% inhibitory concentrations ranging between 0.4 and 9 nM. Moreover, SCH-C strongly inhibits the replication of an R5-using HIV-1 isolate in SCID-hu Thy/Liv mice. SCH-C has a favorable pharmacokinetic profile in rodents and primates with an oral bioavailability of 50-60% and a serum half-life of 5-6 h. On the basis of its novel mechanism of action, potent antiviral activity, and in vivo pharmacokinetic profile, SCH-C is a promising new candidate for therapeutic intervention of HIV infection.

Impaired replication of protease inhibitor-resistant HIV-1 in human thymus.

Many HIV-1-infected patients treated with protease inhibitors (PI) develop PI-resistant HIV-1 variants and rebounds in viremia, but their CD4+ T-cell counts often do not fall. We hypothesized that in these patients, T-cell counts remain elevated because PI-resistant virus spares intrathymic T-cell production. To test this, we studied recombinant HIV-1 clones containing wild-type or PI-resistant protease domains, as well as uncloned isolates from patients, in activated peripheral blood mononuclear cells, human thymic organ cultures and human thymus implants in SCID-hu Thy/Liv mice. In most cases, wild-type and PI-resistant HIV-1 isolates replicated to similar degrees in peripheral blood mononuclear cells. However, the replication of PI-resistant but not wild-type HIV-1 isolates was highly impaired in thymocytes. In addition, patients who had PI-resistant HIV-1 had abundant thymus tissue as assessed by computed tomography. We propose that the inability of PI-resistant HIV-1 to replicate efficiently in thymus contributes to the preservation of CD4+ T-cell counts in patients showing virologic rebound on PI therapy.

Coinfection of SCID-hu Thy/Liv mice with human herpesvirus 6 and human immunodeficiency virus type 1.

Human herpesvirus 6 (HHV-6) has been proposed as a potential cofactor in the progression of human immunodeficiency virus type 1 (HIV-1) disease. We used the SCID-hu Thy/Liv mouse model to evaluate the in vivo interactions between HHV-6 and HIV-1. Our results demonstrate that HHV-6 and HIV-1 can simultaneously replicate in the human thymus in vivo. In this model, however, the presence of one virus appears not to modify the replication or cytopathicity of the other.

Antiviral activity of 2'-deoxy-3'-oxa-4'-thiocytidine (BCH-10652) against lamivudine-resistant human immunodeficiency virus type 1 in SCID-hu Thy/Liv mice.

Oral administration of 2'-deoxy-3'-oxa-4'-thiocytidine (BCH-10652), a nucleoside analog structurally similar to lamivudine (3TC), caused dose-dependent inhibition of viral replication in SCID-hu Thy/Liv mice infected with human immunodeficiency virus type 1 NL4-3 and with an NL4-3 clone containing the M184V mutation in reverse transcriptase that confers resistance to 3TC. These experiments demonstrate the utility of this mouse model for evaluating drug resistance and for performing direct comparisons between antiviral compounds in vivo.

R5 strains of human immunodeficiency virus type 1 from rapid progressors lacking X4 strains do not possess X4-type pathogenicity in human thymus.

Some individuals infected with only R5 strains of human immunodeficiency virus type 1 progress to AIDS as quickly as individuals harboring X4 strains. We determined that three R5 viruses were much less pathogenic than an X4 virus in SCID-hu Thy/Liv mice, suggesting that R5 virus-mediated rapid disease progression is associated with host, not viral, factors.

Human herpesvirus 6 (HHV-6) causes severe thymocyte depletion in SCID-hu Thy/Liv mice.

Human herpesvirus 6 (HHV-6) is a potentially immunosuppressive agent that may act as a cofactor in the progression of AIDS. Here, we describe the first small animal model of HHV-6 infection. HHV-6 subgroup A, strain GS, efficiently infected the human thymic tissue implanted in SCID-hu Thy/Liv mice, leading to the destruction of the graft. Viral DNA was detected in Thy/Liv implants by quantitative polymerase chain reaction (PCR) as early as 4 d after inoculation and peaked at day 14. The productive nature of the infection was confirmed by electron microscopy and immunohistochemical staining. Atypical thymocytes with prominent nuclear inclusions were detected by histopathology. HHV-6 replication was associated with severe, progressive thymocyte depletion involving all major cellular subsets. However, intrathymic T progenitor cells (ITTPs) appeared to be more severely depleted than the other subpopulations, and a preferred tropism of HHV-6 for ITTPs was demonstrated by quantitative PCR on purified thymocyte subsets. These findings suggest that thymocyte depletion by HHV-6 may be due to infection and destruction of these immature T cell precursors. Similar results were obtained with strain PL-1, a primary isolate belonging to subgroup B. The severity of the lesions observed in this animal model underscores the possibility that HHV-6 may indeed be immunosuppressive in humans.

CCR5- and CXCR4-utilizing strains of human immunodeficiency virus type 1 exhibit differential tropism and pathogenesis in vivo.

CCR5-utilizing (R5) and CXCR4-utilizing (X4) strains of human immunodeficiency virus type 1 (HIV-1) have been studied intensively in vitro, but the pathologic correlates of such differential tropism in vivo remain incompletely defined. In this study, X4 and R5 strains of HIV-1 were compared for tropism and pathogenesis in SCID-hu Thy/Liv mice, an in vivo model of human thymopoiesis. The X4 strain NL4-3 replicates quickly and extensively in thymocytes in the cortex and medulla, causing significant depletion. In contrast, the R5 strain Ba-L initially infects stromal cells including macrophages in the thymic medulla, without any obvious pathologic consequence. After a period of 3 to 4 weeks, Ba-L infection slowly spreads through the thymocyte populations, occasionally culminating in thymocyte depletion after week 6 of infection. During the entire time of infection, Ba-L did not mutate into variants capable of utilizing CXCR4. Therefore, X4 strains are highly cytopathic after infection of the human thymus. In contrast, infection with R5 strains of HIV-1 can result in a two-phase process in vivo, involving apparently nonpathogenic replication in medullary stromal cells followed by cytopathic replication in thymocytes.

New iridoids from the medicinal plant Barleria prionitis with potent activity against respiratory syncytial virus.

Two new iridoid glycosides (1 and 2), together with the known compounds barlerin (3) and verbascoside (4), were isolated from Barleria prionitis. The new iridoid glycosides were determined to be 6-O-trans-p-coumaroyl-8-O-acetylshanzhiside methyl ester (1) and its cis isomer (2) by using spectroscopic, especially 2D NMR, data. A 3:1 mixture of 1 and 2 was shown to have potent in vitro activity against respiratory syncytial virus (EC50 2.46 microgram/mL, IC50 42.2 microgram/mL).

Inhibition of human immunodeficiency virus type 1 infection in SCID-hu Thy/Liv mice by the G-quartet-forming oligonucleotide, ISIS 5320.

Viral replication was inhibited in a dose-dependent manner after administration of the phosphorothioate oligonucleotide TTGGGGTT (ISIS 5320) to human immunodeficiency virus type 1 (HIV-1)-infected SCID-hu Thy/Liv mice. Potent in vivo antiviral activity was observed against the T-cell-tropic molecular clone NL4-3; the agent was found to have weak activity against one primary HIV-1 isolate, and the agent was inactive against a second primary isolate.

Antiviral phenylpropanoid glycosides from the medicinal plant Markhamia lutea.

Three new phenylpropanoid glycosides, named luteoside A (3), luteoside B (4), and luteoside C (5), were isolated together with the known compounds verbascoside (1) and isoverbascoside (2) from the roots of the medicinal plant Markhamia lutea. The structures of the new compounds were determined to be 1-O-(3, 4-dihydroxyphenyl)ethyl beta-D-apiofuranosyl(1-->2)-alpha-l-rhamnopyranosyl(1-->3)-4-O- caffeo yl-6-acetyl-beta-d-glucopyranoside, 1-O-(3,4-dihydroxyphenyl)ethyl beta-d-apiofuranosyl(1-->2)-alpha-l-rhamnopyranosyl(1-->3)-6-O- caffeo yl-beta-d-glucopyranoside, and 1-O-(3,4-dihydroxyphenyl)ethyl beta-D-apiofuranosyl(1-->2)-alpha-l-rhamnopyranosyl(1-->3)-6-O- ferulo yl-beta-d-glucopyranoside, respectively, on the basis of chemical and spectroscopic data. All five phenylpropanoid glycosides exhibited potent in vitro activity against respiratory syncytial virus.

Two new lignans with activity against influenza virus from the medicinal plant Rhinacanthus nasutus.

Two new lignans, rhinacanthin E (1) and rhinacanthin F (2), were isolated from the aerial parts of the plant Rhinacanthus nasutus. Their structures were established by detailed spectroscopic analysis. These compounds show significant antiviral activity against influenza virus type A.

Murine cytomegalovirus IE2, an activator of gene expression, is dispensable for growth and latency in mice.

The murine cytomegalovirus alpha (immediate-early) gene product, IE2(391aa), a protein that is related to the human cytomegalovirus US22 protein family, had previously been shown to be dispensable for viral growth in cell culture. In transient assays, however, this protein was found to transactivate the murine CMV ie1/ie3 and ie2 promoters, as well as a number of other promoters. Transactivation was mediated via promoter-proximal elements rather than through elements located upstream in the enhancer region. This activation predicted that ie2 would play a role in regulating gene expression; however, ie2 mutants did not exhibit altered growth or latency in the mouse. ie2-deficient viruses reached peak titers in spleen, salivary glands, lungs, liver, kidneys, pancreas, peripheral blood leukocytes, and adrenal glands that were comparable to wild-type virus. When assayed by spleen explant culture, ie2-deficient viruses yielded reactivation levels similar to wild type. Thus, the murine CMV ie2 gene encodes a regulatory protein that is dispensable for viral infection of cells in culture as well as for interaction with tissues in the infected BALB/c mouse.

Peripheral blood mononuclear phagocytes mediate dissemination of murine cytomegalovirus.

Cytomegalovirus is transmitted with blood and organs from seropositive individuals, although the particular leukocyte population harboring latent or persistent virus remains poorly characterized. Murine cytomegalovirus, tagged with the Escherichia coli lacZ gene, was used to identify cells in which virus replicates during acute infection of immunocompetent mice. Recombinant murine cytomegaloviruses, RM461, RM460, and RM427, were constructed to express beta-galactosidase under control of the human cytomegalovirus ie1/ie2 promoter/enhancer. The lacZ gene was inserted between the ie2 and sgg1 genes in RM461 and RM460, disrupting a 0.85-kb late transcript that was found to be dispensable for replication in cultured cells as well as for infection of mice. In BALB/c mice, lacZ-tagged and wild-type viruses exhibited a similar 50% lethal dose and all had the capacity to latently infect the spleen. Peripheral blood mononuclear phagocytes were the major infected leukocyte cell type, as demonstrated by the ability of infected cells to adhere to glass and to phagocytize latex beads; however, these cells did not exhibit typical monocyte markers. Plaque assay for virus and 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal) staining of frozen sections of organs from infected mice revealed that the major target organs included the spleen, adrenal glands, liver, and salivary glands, although tissues as diverse as brown fat and lungs were also involved. Individual blue-staining cells were readily identified in all infected tissues. These studies identified a mononuclear phagocyte, possibly a macrophage or dendritic cell precursor, as the vehicle of virus dissemination during acute infection, and demonstrate the utility of using lacZ-tagged murine cytomegalovirus for tropism, pathogenesis, and latency studies.

SP-303, an antiviral oligomeric proanthocyanidin from the latex of Croton lechleri (Sangre de Drago).

SP-303, a large proanthocyanidin oligomer isolated from the latex of the plant species Croton lechleri (Eupborbiaceae) has demonstrated broad activity against a variety of DNA and RNA viruses. In cell culture, SP-303 exhibits potent activity against isolates and laboratory strains of respiratory syncytial virus (RSV), influenza A virus (FLU-A) and parainfluenza virus (PIV). Parallel assays of SP-303 and ribavirin showed comparable activity against these viruses. SP-303 also exhibits significant inhibitory activity against herpesvirus (HSV) types 1 and 2, including herpesviruses resistant to acyclovir and foscarnet. Inhibition was also observed against hepatitis A and B viruses. The antiviral mechanism of SP-303 seems to derive from its direct binding to components of the viral envelope, resulting in inhibition of viral attachment and penetration of the plasma membrane. Antiviral effects of SP-303 were measured by three distinct methods: CPE, MTT and precursor uptake/incorporation. Cytotoxicity endpoints were markedly greater than the respective antiviral endpoints. SP-303 exhibited activity in RSV-infected cotton rats and African green monkeys, PIV-3-infected cotton rats, HSV-2 infected mice and guinea pigs and FLU-A-infected mice. The most successful routes of SP-303 administration for producing efficacy were: topical application to HSV-2- genital lesions in mice and guinea pigs, aerosol inhalation to FLU-A-infected mice and PIV-3-infected cotton rats, and oral dosage to RSV-infected cotton rats. A variety of toxicological evaluations demonstrated the safety of SP-303, particularly orally, which was predictable, since condensed tannins are a common dietary component. It is notable that the larger proanthocyanidins as a class have high antiviral activity, whereas most of the monomers are inactive. Clinical trials are ongoing to evaluate SP-303 as a therapeutic antiviral agent.

Cytomegalovirus determinant of replication in salivary glands.

Murine cytomegalovirus carrying a deletion mutation disrupting the expression of a gene dispensable for growth in cultured cells was found to disseminate poorly in the mouse. The mutation resulted in a dramatic decrease in the expression of a 1.5-kb major and a 1.8-kb minor beta transcript from a region adjacent to the ie2 gene in the viral genome. Nucleotide sequence determination indicated that 323 bp, including a predicted polyadenylation signal, was deleted from this beta gene. In cultured cells, the plaque morphology and growth characteristics of the mutant were similar to those of parental or rescued wild-type viruses. Following intraperitoneal inoculation of BALB/c mice, growth of the mutant in the salivary gland was dramatically reduced 10,000-fold, while growth in the liver and spleen was not dramatically affected. The beta gene was thus denoted sgg1 (salivary gland growth gene 1). Neither intranasal infection nor direct inoculation into the salivary glands completely overcame the restriction of growth in this organ, suggesting that the sgg1 gene encoded a determinant of tissue tropism. To investigate the impact of the sgg1 mutation on virus dissemination via the blood, the virus titer in peripheral blood leukocytes was determined. No difference was found between the sgg1 mutant and rescued wild-type virus. Thus, murine cytomegalovirus sgg1 gene products appear to be involved in entry or replication of virus in salivary gland cells.

Intrinsic resistance of feline peritoneal macrophages to coronavirus infection correlates with in vivo virulence.

Cats infected with virulent feline coronavirus strains develop feline infectious peritonitis, an invariably fatal, immunologically mediated disease; avirulent strains cause either clinically inapparent infection or mild enteritis. Four virulent coronavirus isolates and five avirulent isolates were assessed by immunofluorescence and virus titration for their ability to infect and replicate in feline peritoneal macrophages in vitro. The avirulent coronaviruses infected fewer macrophages, produced lower virus titers, were less able to sustain viral replication, and spread less efficiently to other susceptible macrophages than the virulent coronaviruses. Thus, the intrinsic resistance of feline macrophages may play a pivotal role in the outcome of coronavirus infection in vivo.

Isolation and identification of feline peritoneal macrophages for in vitro studies of coronavirus-macrophage interactions.

Feline peritoneal cells were collected by lavage with isotonic saline without the use of irritants or need for euthanasia of the cats. Macrophages were purified by centrifugation on Percoll followed by selective adherence. Although few macrophages could be obtained from an initial lavage, a second lavage performed on the same cat 9-11 days later yielded six times as many macrophages as the first lavage, providing sufficient numbers of cells for characterization and infection experiments. Macrophages from these subsequent lavages were not more functionally activated in phagocytosis assays than the resident macrophages from the initial lavage, and they were equally susceptible to infection with feline infectious peritonitis virus (FIPV). Infected cultures produced peak titers of 10(5.0) TCID50 per ml, and FIPV antigen was detected in a small subset (0.1-1.0%) of cells by indirect immunofluorescence. The FIPV-infected cells were identified as macrophages by their characteristic morphology and ability to phagocytize rhodamine-labeled latex beads. The successful isolation of large numbers of unactivated feline macrophages will permit in vitro studies of feline coronavirus-macrophage interactions that otherwise would not have been possible. Such studies will undoubtedly provide valuable insights into the pathogenesis of feline infectious peritonitis, an invariably fatal disease of domestic and exotic cats.