Acute hepatitis B virus infection in humanized chimeric mice has multiphasic viral kinetics.

Chimeric urokinase type plasminogen activator (uPA)/severely severe combined immunodeficiency (SCID) mice reconstituted with humanized livers are useful for studying hepatitis B virus (HBV) infection in the absence of an adaptive immune response. However, the detailed characterization of HBV infection kinetics necessary to enable in-depth mechanistic studies in this in vivo HBV infection model is lacking. To characterize HBV kinetics post-inoculation (p.i.) to steady state, 42 mice were inoculated with HBV. Serum HBV DNA was frequently measured from 1 minute to 63 days p.i. Total intrahepatic HBV DNA, HBV covalently closed circular DNA (cccDNA), and HBV RNA was measured in a subset of mice at 2, 4, 6, 10, and 13 weeks p.i. HBV half-life (t1/2 ) was estimated using a linear mixed-effects model. During the first 6 hours p.i., serum HBV declined in repopulated uPA/SCID mice with a t1/2 = 62 minutes (95% confidence interval [CI] = 59-67). Thereafter, viral decline slowed followed by a 2-day lower plateau. Subsequent viral amplification was multiphasic with an initial mean doubling time of t2 = 8 ± 3 hours followed by an interim plateau before prolonged amplification (t2 = 2 ± 0.5 days) to a final HBV steady state of 9.3 ± 0.3 log copies (cps)/mL. Serum HBV and intrahepatic HBV DNA were positively correlated (R2 = 0.98). CONCLUSION: HBV infection in uPA/SCID chimeric mice is highly dynamic despite the absence of an adaptive immune response. Serum HBV t1/2 in humanized uPA/SCID mice was estimated to be ∼1 hour regardless of inoculum size. The HBV acute infection kinetics presented here is an important step in characterizing this experimental model system so that it can be effectively used to elucidate the dynamics of the HBV life cycle and thus possibly reveal effective antiviral drug targets. (Hepatology 2018).

Frequent Zika Virus Sexual Transmission and Prolonged Viral RNA Shedding in an Immunodeficient Mouse Model.

Circulation of Zika virus (ZIKV) was first identified in the Western hemisphere in late 2014. Primarily transmitted through mosquito bite, ZIKV can also be transmitted through sex and from mother to fetus, and maternal ZIKV infection has been associated with fetal malformations. We assessed immunodeficient AG129 mice for their capacity to shed ZIKV in semen and to infect female mice via sexual transmission. Infectious virus was detected in semen between 7 and 21 days post-inoculation, and ZIKV RNA was detected in semen through 58 days post-inoculation. During mating, 73% of infected males transmitted ZIKV to uninfected females, and 50% of females became infected, with evidence of fetal infection in resulting pregnancies. Semen from vasectomized mice contained significantly lower levels of infectious virus, though sexual transmission still occurred. This model provides a platform for studying the kinetics of ZIKV sexual transmission and prolonged RNA shedding also observed in human semen.

The extent of inflammatory infiltration in primary cancer tissues is associated with lymphomagenesis in immunodeficient mice.

Xenotransplantation of human cancers into immunodeficient mice is a very useful approach for studying human tumor biology. However, the occasional occurrence of lymphomagenesis in some mice can spoil the model and must be investigated in detail. We found that a high percentage (32.5%, 26/80) of cancer patient-derived xenografts (PDXs) resembled lymphoma in NOD/SCID mice. Of the 26 xenografts, 23 were human-derived expressing human CD45 (hCD45+) and proved to be of the B-cell subtype (CD3-/CD20+), and they were all positive for Epstein-Barr virus (EBV). The remaining 3 xenografts proved to be mouse-derived for both hCD45- and negative amplification of a human gene. The most interesting finding is that gastric cancer had much higher rates (24/126, 19.0%) of lymphoma formation in the PDX model than did colorectal cancer (1/43, 2.3%). Statistical analysis revealed that cancer type and inflammation in the parent tumor are significantly associated with lymphomagenesis. Further validation discovered lymphomagenesis by inoculating only gastritis mucosa. Therefore, our findings suggest that it is necessary to take precautions when directly xenografting cancer tissues with remarkable baseline inflammation, such as gastric cancer into immunodeficient NOD/SCID strains. Further, the established xenograft models should be validated by both leukocyte markers and human gene signatures.

Neutrophil depletion suppresses pulmonary vascular hyperpermeability and occurrence of pulmonary edema caused by hantavirus infection in C.B-17 SCID mice.

UNLABELLED: Hantavirus infections are characterized by vascular hyperpermeability and neutrophilia. However, the pathogenesis of this disease is poorly understood. Here, we demonstrate for the first time that pulmonary vascular permeability is increased by Hantaan virus infection and results in the development of pulmonary edema in C.B-17 severe combined immunodeficiency (SCID) mice lacking functional T cells and B cells. Increases in neutrophils in the lung and blood were observed when pulmonary edema began to be observed in the infected SCID mice. The occurrence of pulmonary edema was inhibited by neutrophil depletion. Moreover, the pulmonary vascular permeability was also significantly suppressed by neutrophil depletion in the infected mice. Taken together, the results suggest that neutrophils play an important role in pulmonary vascular hyperpermeability and the occurrence of pulmonary edema after hantavirus infection in SCID mice. IMPORTANCE: Although hantavirus infections are characterized by the occurrence of pulmonary edema, the pathogenic mechanism remains largely unknown. In this study, we demonstrated for the first time in vivo that hantavirus infection increases pulmonary vascular permeability and results in the development of pulmonary edema in SCID mice. This novel mouse model for human hantavirus infection will be a valuable tool and will contribute to elucidation of the pathogenetic mechanisms. Although the involvement of neutrophils in the pathogenesis of hantavirus infection has largely been ignored, the results of this study using the mouse model suggest that neutrophils are involved in the vascular hyperpermeability and development of pulmonary edema in hantavirus infection. Further study of the mechanisms could lead to the development of specific treatment for hantavirus infection.

Challenge pools of hepatitis C virus genotypes 1-6 prototype strains: replication fitness and pathogenicity in chimpanzees and human liver-chimeric mouse models.

Chimpanzees represent the only animal model for studies of the natural history of hepatitis C virus (HCV). To generate virus stocks of important HCV variants, we infected chimpanzees with HCV strains of genotypes 1-6 and determined the infectivity titer of acute-phase plasma pools in additional animals. The courses of first- and second-passage infections were similar, with early appearance of viremia, HCV RNA titers of >10(4.7) IU/mL, and development of acute hepatitis; the chronicity rate was 56%. The challenge pools had titers of 10(3)-10(5) chimpanzee infectious doses/mL. Human liver-chimeric mice developed high-titer infections after inoculation with the challenge viruses of genotypes 1-6. Inoculation studies with different doses of the genotype 1b pool suggested that a relatively high virus dose is required to consistently infect chimeric mice. The challenge pools represent a unique resource for studies of HCV molecular virology and for studies of pathogenesis, protective immunity, and vaccine efficacy in vivo.

Experimental infection of mice with hamster parvovirus: evidence for interspecies transmission of mouse parvovirus 3.

Hamster parvovirus (HaPV) was isolated 2 decades ago from hamsters with clinical signs similar to those induced in hamsters experimentally infected with other rodent parvoviruses. Genetically, HaPV is most closely related to mouse parvovirus (MPV), which induces subclinical infection in mice. A novel MPV strain, MPV3, was detected recently in naturally infected mice, and genomic sequence analysis indicates that MPV3 is almost identical to HaPV. The goal of the present studies was to examine the infectivity of HaPV in mice. Neonatal and weanling mice of several mouse strains were inoculated with HaPV. Tissues, excretions, and sera were harvested at 1, 2, 4, and 8 wk after inoculation and evaluated by quantitative PCR and serologic assays specific for HaPV. Quantitative PCR detected viral DNA quantities that greatly exceeded the quantity of virus in inocula in multiple tissues of infected mice. Seroconversion to both nonstructural and structural viral proteins was detected in most immunocompetent mice 2 or more weeks after inoculation with HaPV. In neonatal SCID mice, viral transcripts were detected in lymphoid tissues by RT-PCR and viral DNA was detected in feces by quantitative PCR at 8 wk after inoculation. No clinical signs, gross, or histologic lesions were observed. These findings are similar to those observed in mice infected with MPV. These data support the hypothesis that HaPV and MPV3 are likely variants of the same viral species, for which the mouse is the natural rodent host with rare interspecies transmission to the hamster.

Embryo transfer rederivation of C.B-17/Icr-Prkdc(scid) mice experimentally infected with mouse parvovirus 1.

We determined whether embryos derived from C.B-17/Icr-Prkdc(scid) (SCID) mice infected with mouse parvovirus (MPV) 1b and mated to MPV-naive B6C3F1 mice would transmit virus to naive recipient female mice and rederived progeny. Viral DNA was detected by quantitative PCR (qPCR) in lymphoid tissues, gonad, sperm, and feces of all MPV1b-inoculated SCID mice. Viral DNA was detected in 1 of 16 aliquots of embryos from infected male SCID mice and in 12 of 18 aliquots of embryos from infected female SCID mice. All recipient female mice implanted with embryos from infected SCID male mice and their progeny were negative by serology and qPCR. In contrast, 3 of 5 recipient female mice implanted with embryos from infected SCID female mice and 14 of 15 progeny mice from these recipients were seropositive by multiplex fluorescent immunoassay (MFI) for MPV capsid antigen (rVP2). All of these mice were negative by MFI for parvovirus nonstructural protein antigen (rNS1) and by qPCR, with the exception of 1 recipient female mouse that displayed weak rNS1 seroreactivity and low levels of MPV DNA in lymphoid tissues. Seroreactivity to rVP2 declined over time in all progeny mice from infected SCID female mice until all were seronegative by 20 wk of age, consistent with maternal antibody transfer. Given that the high levels of MPV contamination detected in our experimentally infected SCID mice are unlikely in naturally infected immunocompetent mice, these data indicate that embryo transfer rederivation is effective for the eradication of MPV from infected colonies.

Dengue fever in humanized NOD/SCID mice.

The increased transmission and geographic spread of dengue fever (DF) and its more severe presentation, dengue hemorrhagic fever (DHF), make it the most important mosquito-borne viral disease of humans (50 to 100 million infections/year) (World Health Organization, Fact sheet 117, 2002). There are no vaccines or treatment for DF or DHF because there are no animal or other models of human disease; even higher primates do not show symptoms after infection (W. F. Scherer, P. K. Russell, L. Rosen, J. Casals, and R. W. Dickerman, Am. J. Trop. Med. Hyg. 27:590-599, 1978). We demonstrate that nonobese diabetic/severely compromised immunodeficient (NOD/SCID) mice xenografted with human CD34+ cells develop clinical signs of DF as in humans (fever, rash, and thrombocytopenia), when infected in a manner mimicking mosquito transmission (dose and mode). These results suggest this is a valuable model with which to study pathogenesis and test antidengue products.

Hippocampal synaptic dysfunction in a murine model of human immunodeficiency virus type 1 encephalitis.

Alterations in hippocampal physiology affect cognition in human immunodeficiency virus type 1 (HIV-1)-associated dementia (HAD). The mechanism for how this occurs is not well understood. To address this, we investigated how changes in synaptic transmission and plasticity are affected by viral infection and macrophage activation using a severe combined immunodeficiency mouse model of human HIV-1 encephalitis (HIVE). HIVE was induced in mice by stereotactic injection of HIV-1-infected human monocyte-derived macrophages (MDM) into the striatum. Animals were sacrificed after 3, 7 and 15 days. Hippocampal slices were prepared from HIV-1, MDM- and sham-injected animals. Electrically evoked field excitatory postsynaptic potentials were recorded in the CA1 region of the hippocampus. Neuronal physiology was assessed by input-output and by long-term potentiation (LTP) assays. We observed that a higher stimulation intensity (mA) was required to induce a 1-mV response in the HIVE mice (0.32+/-0.06) compared with shams (0.17+/-0.01) at day 7. The stimulation intensities at day 15 were 0.44+/-0.07 and 0.23+/-0.05 in the HIVE and shams, respectively. An impairment of synaptic function was detected through measuring synaptic responses induced by stimuli with different intensities. Paired-pulse facilitation (PPF) showed deficits in HIVE mice at days 3, 7, and 15. At day 3, PPF ratios were 1.13+/-0.02 and 1.24+/-0.04 in HIVE and sham. The induction and maintenance of LTP was also impaired in HIVE mice. The average magnitude of LTP was 131.23+/-15.26% of basal in HIVE as compared with sham animals of 232.63+/-24.18%. MDM-injected mice showed an intermediate response. Taken together, the results show a range of neuronal synaptic transmission and plasticity changes in HIVE mice that may reflect the mechanisms of cognitive dysfunction in human HAD.

Severe combined immunodeficient mice engrafted with macaque peripheral blood leukocytes support replication of SIVsmm.

Peripheral blood leukocytes (PBLs) from normal pigtail macaques were engrafted into severe combined immunodeficient C.B-17 scid/scid (SCID) mice to develop a small animal model in which to study and identify genetic determinants responsible for the acutely lethal disease syndrome induced by SIVsmmPBj14 (SIV-PBj14) in pigtail macaques. In vivo infection of macaques with SIV-PBj14 results in acute disease in all animals and death of most animals, depending on the route of infection, due to immune activation and production of inflammatory cytokines. A small animal model in which a similar acute disease syndrome was induced would facilitate screening of virus variants to identify regions of the SIV-PBj14 genome responsible for the unique phenotype. Although intraperitoneal inoculation of SCID mice with SIV-PBj14-infected PBLs or uninfected PBLs followed by cell-free SIV-PBj14 produced chimeric mac-PBL-SCID mice that supported SIV replication, obvious clinical signs of disease were not observed. SIV-infected macaque PBLs were recovered from spleen, bone marrow, peripheral blood, and the peritoneal cavity; cell-free SIV was recovered from peritoneal lavage fluid and serum or plasma. PBLs that were mitogen stimulated and SIV-PBj14 infected in vitro migrated rapidly and were recovered from the spleen and bone marrow as early as 1 day after inoculation of mice. The mac-PBL-SCID model may be useful for screening potential drug or immunomodulatory therapies before testing in macaques.

hu-PBL-SCID mice: an in vivo model of Epstein-Barr virus-dependent lymphoproliferative disease.

The Epstein-Barr virus (EBV) exists in most humans as a lifelong latent infection established in host B cells after a primary viral encounter. In immunosuppressed individuals, such as post-transplant patients, the presence of EBV-infected B cells may lead to lymphoproliferative disease. Injection of human peripheral blood lymphocytes from EBV-positive donors into mice with severe combined immunodeficiency induces human lymphoproliferative disease in the recipient closely resembling that of human post-transplant patients. This xenochimeric human-mouse model is increasingly being used to elucidate the mechanisms of EBV-specific lymphomagenesis and to assess novel therapeutic approaches.

Hantavirus infection in SCID mice.

Severe combined immunodeficiency (SCID) mice were inoculated with Hantaan virus strain 76-118 (HTN) or Seoul virus strain SR-11 (SR) of hantaviruses. Susceptibility of SCID mice was compared with those of immunocompetent adult mice, newborn mice and nude mice. SCID mice inoculated with HTN or SR died 32 to 35 days after infection. Unlike newborn mice which also died of hantavirus infection, SCID mice survived longer than newborn mice and showed typical wasting symptoms rather than nervous symptoms. Immunohistochemical staining and virus isolation indicated that both HTN and SR inoculated SCID and SR inoculated nude mice showed systemic infection, but nude mice inoculated with SR survived for longer than 8 weeks after inoculation. Passive transfer of spleen cells from immunocompetent BALB/c mice conferred protection on SCID mice within 2 weeks of HTN infection. Immune mediated pathologic mechanism was examined by transferring the spleen cells to SCID mice inoculated with HTN virus 3 weeks before the cell transfer. The recipient SCID mice showed an increase of serum BUN level coinciding with the appearance of serum antibody to HTN virus, suggesting the immune mediated pathogenicity.

Simple i.v. inoculation of HIV-1 to Thy/Liv SCID-hu mice induce reproducible HIV infection with narrowing of medulla in human thymic implant.

Human fetal thymus/liver engrafted SCID mice were constructed and studied for its susceptibility to HIVBRU infection by i.v. inoculation which seemed to represent an appropriate route of HIV infection in vivo. By the i.v. inoculation of HIV, the medulla in the engrafted thymus narrowed significantly when compared with that of the human thymic implant from virus-uninoculated mice. Further, immunohistochemical staining indicated the presence of HIV antigen predominantly in thymic epithelial cells in medulla of the engrafted thymus. Polymerase chain reaction (PCR) assays resulted in amplifications of HIV genome in the implanted grafts as well as in lymph nodes and PBMC. The virus infections to the implants were confirmed biologically by coculturing with PHA-stimulated human PBMC and the graft cells from the HIV-inoculated SCID-hu mice. Thus, the i.v. inoculation of HIV into Thy/Liv SCID-hu mice induce narrowing of medulla of the engrafted thymus and may become an efficient and useful tool for screening candidate anti-HIV agents.

Severely combined immunodeficient (SCID) mice resist infection with bovine spongiform encephalopathy.

Following combined intraperitoneal and intracerebral injection with bovine spongiform encephalopathy (BSE) cow brain homogenate, SCID mice show a resistance to infection in comparison with immunocompetent CB20 mice. BSE occurred in only five out of 22 challenged SCID mice, with a mean incubation period of 573 days, whereas all the CB20 mice developed the disease with a mean incubation period of 456 days. In contrast, previous studies have shown that intracerebral infection of SCID mice with a mouse-passaged scrapie strain, ME7, produces 100% incidence of disease but no replication of infectivity in spleen. The results with BSE suggest that there is little or no direct infection of the CNS in interspecies transmissions, but that processing or replication of infectivity in peripheral lymphoid tissues may facilitate subsequent spread of infection to the CNS.

Dissociation of demyelination and viral clearance in congenitally immunodeficient mice infected with murine coronavirus JHM.

Infection of rodents with murine coronavirus JHM results in a subacute or chronic demyelinating disease which serves as a model for the human disease multiple sclerosis. Previous studies with JHMV have established a role for the immune system in both viral clearance and demyelination. To further clarify the role of the immune system in JHMV pathogenesis, several strains of congenitally immunodeficient mice were studied. Infection of immunocompetent C57BL/6 mice with JHMV resulted in severe paralysis and demyelination and complete clearance of infectious virus from the brain (C+D+ phenotype). In contrast, infected SCID mice showed little or no paralysis or demyelination and were unable to clear infectious virus (C-D- phenotype). Athymic nude mice and a proportion of mice lacking MHC Class I or II expression exhibited robust demyelination but did not completely clear infectious virus from the brain (C-D+ phenotype). These results are consistent with an immune-mediated mechanism for JHMV-induced demyelination, but indicate that the immune mechanisms which participate in demyelination and viral clearance are distinct. It may thus be possible to experimentally alter immunopathological responses without impairing antimicrobial immunity.

Use of standardized SCID-hu Thy/Liv mouse model for preclinical efficacy testing of anti-human immunodeficiency virus type 1 compounds.

We have developed standardized procedures and practices for infection of SCID-hu Thy/Liv mice with human immunodeficiency virus type 1 for the prophylactic administration of antiviral compounds and for evaluation of the antiviral effect in vivo. Endpoint analyses included quantitation of viral load by intracellular p24 enzyme-linked immunosorbent assay, DNA PCR for the presence of proviral genomes, flow cytometry to measure the representation of CD4+ and CD8+ cells, and cocultivation for the isolation of virus. Efficacy tests in this model are demonstrated with the nucleoside analogs zidovudine and dideoxyinosine and with the nonnucleoside reverse transcriptase inhibitor nevirapine. This small-animal model should be particularly useful in the preclinical prioritization of lead compounds within a common chemical class, in the evaluation of alternative in vivo dosing regimens, and in the determination of appropriate combination therapy in vivo.

Dominance of a single Epstein-Barr virus strain in SCID-mouse tumors induced by injection of peripheral blood mononuclear cells from healthy human donors.

Severe Combined Immune Deficiency mouse tumors, induced by inoculating peripheral blood mononuclear cells from 11 healthy human donors (hu-PBMC-SCID tumors), were used to analyse Epstein-Barr virus (EBV) type and strain variations. PCR analysis of EBNA 2- and EBNA 3C-specific sequences showed that EBV type A was present in SCID-mouse tumors induced by PBMC from all donors but one, while, using amplimers for a highly polymorphic region within the latent membrane protein (LMP) coding sequence, 5 different strains could be detected among the samples examined. The same LMP fragment was present in different tumors arising in the same animal, as well as in different mice injected with PBMC from any donor. Compared to B95.8 and AG876 prototype viruses, sequence analysis of LMP variants disclosed a higher homology to the latter, with 33 bp additional repetitions and a few point mutations in specific sites. This study confirms and extends previous data on the presence of a single EBV type and strain in the peripheral blood of most normal healthy subjects using the SCID-mouse system.