Predominant clonal accumulation of CD8+ T cells with moderate avidity in the central nervous systems of Theiler's virus-infected C57BL/6 mice.

Induction of antigen-specific CD8(+) T cells bearing a high-avidity T-cell receptor (TCR) is thought to be an important factor in antiviral and antitumor immune responses. However, the relationship between TCR diversity and functional avidity of epitope-specific CD8(+) T cells accumulating in the central nervous system (CNS) during viral infection is unknown. Hence, analysis of T-cell diversity at the clonal level is important to understand the fate and function of virus-specific CD8(+) T cells. In this study, we examined the Vbeta diversity and avidity of CD8(+) T cells specific to the predominant epitope (VP2(121-130)) of Theiler's murine encephalomyelitis virus. We found that Vbeta6(+) CD8(+) T cells, associated with epitope specificity, predominantly expanded in the CNS during viral infection. Further investigations of antigen-specific Vbeta6(+) CD8(+) T cells by CDR3 spectratyping and sequencing indicated that distinct T-cell clonotypes are preferentially increased in the CNS compared to the periphery. Among the epitope-specific Vbeta6(+) CD8(+) T cells, MGX-Jbeta1.1 motif-bearing cells, which could be found at a high precursor frequency in naïve mice, were expanded in the CNS and tightly associated with gamma interferon production. These T cells displayed moderate avidity for the cognate epitope rather than the high avidity normally observed in memory/effector T cells. Therefore, our findings provide new insights into the CD8(+) T-cell repertoire during immune responses to viral infection in the CNS.

Unified immune modulation by 4-1BB triggering leads to diverse effects on disease progression in vivo.

4-1BB (CD137) is a powerful T-cell costimulatory molecule in the treatment of virus infections and tumors, but recent studies have also uncovered regulatory functions of 4-1BB signaling. Since 4-1BB triggering suppresses autoimmunity by accumulating indoleamine 2,3-dioxygenase (IDO) in dendritic cells (DCs) in an interferon (IFN)-γ-dependent manner, we asked whether similar molecular and cellular changes were induced by 4-1BB triggering in virus-infected mice. 4-1BB triggering increased IFN-γ and IDO, and suppressed CD4(+) T cells, in C57BL/6 mice infected with the type 1 KOS strain of Herpes simplex virus (HSV-1), as it does in an autoimmune disease model. Detailed analysis of the CD4(+) T suppression showed that freshly activated CD62L(high) T cells underwent apoptosis in the early phase of suppression, and CD62L(low) effector/memory T cells in the later phase. Although 4-1BB triggering resulted in similar cellular changes - increased CD8(+) T and decreased CD4(+) T cells, it had different effects on mortality in mice infected with HSV-1 RE, influenza, and Japanese encephalitis virus (JEV); it increased mortality in influenza-infected mice but decreased it in JEV-infected mice. Since the dominant type of immune cell generated to protect the host was different for each virus - CD4(+) T cells and neutrophils in HSV-1 RE infection, both CD4(+) T and CD8(+) T cells in influenza infection, and a crucial role for B cells in JEV infection, 4-1BB triggering resulted in different therapeutic outcomes. We conclude that the therapeutic outcome of 4-1BB triggering is determined by whether the protective immunity generated against the virus was beneficially altered by the 4-1BB triggering.

Inter-vendor variance of enteric eukaryotic DNA viruses in specific pathogen free C57BL/6N mice.

The laboratory mouse strain C57BL/6 is widely used as an animal model for various applications. It is becoming increasingly clear that the bacterial enteric community highly influences the phenotype. Eukaryotic viruses represent a sparsely investigated member of the enteric microbiome that might also affect the phenotype. We here investigated the presence of enteric eukaryotic DNA viruses (EDVs) in specific pathogen-free (SPF) C57BL/6N mice purchased from three vendors upon arrival and after being fed a low-fat diet (LFD) or high-fat diet (HFD). We detected genetic fragments of EDVs belonging to the viral families of Herpes-, Mimi-, Baculo- and Phycodnaviridae represented by two genera; Chlorovirus and Prasinovirus. The EDVs were detected in the mice upon arrival and persisted for 13 weeks. However, these signals of EDVs were only detected at notable levels in mice fed LFD from 2 out of 3 vendors, which suggested that the enteric composition of these EDVs were affected by both vendor (p < 0.003) and different dietary regimes (p < 0.013). This highlights the need of additional studies assessing the potential function of these EDVs that may influence the mouse phenotype and the reproducibility of animal studies using this C57BL/6N substrain.

Host genetic variation affects resistance to infection with a highly pathogenic H5N1 influenza A virus in mice.

Despite the prevalence of H5N1 influenza viruses in global avian populations, comparatively few cases have been diagnosed in humans. Although viral factors almost certainly play a role in limiting human infection and disease, host genetics most likely contribute substantially. To model host factors in the context of influenza virus infection, we determined the lethal dose of a highly pathogenic H5N1 virus (A/Hong Kong/213/03) in C57BL/6J and DBA/2J mice and identified genetic elements associated with survival after infection. The lethal dose in these hosts varied by 4 logs and was associated with differences in replication kinetics and increased production of proinflammatory cytokines CCL2 and tumor necrosis factor alpha in susceptible DBA/2J mice. Gene mapping with recombinant inbred BXD strains revealed five loci or Qivr (quantitative trait loci for influenza virus resistance) located on chromosomes 2, 7, 11, 15, and 17 associated with resistance to H5N1 virus. In conjunction with gene expression profiling, we identified a number of candidate susceptibility genes. One of the validated genes, the hemolytic complement gene, affected virus titer 7 days after infection. We conclude that H5N1 influenza virus-induced pathology is affected by a complex and multigenic host component.

In vivo imaging of Zika virus reveals dynamics of viral invasion in immune-sheltered tissues and vertical propagation during pregnancy.

Rationale: Zika virus (ZIKV) is a pathogenic virus known to cause a wide range of congenital abnormalities, including microcephaly, Guillain-Barre syndrome, meningoencephalitis, and other neurological complications, in humans. This study investigated the noninvasive detection of ZIKV infection in vivo, which is necessary for elucidating the virus's mechanisms of viral replication and pathogenesis, as well as to accelerate the development of anti-ZIKV therapeutic strategies. Methods: In this study, a recombinant ZIKV harbouring Nluc gene (ZIKV-Nluc) was designed, recovered, and purified. The levels of bioluminescence were directly correlated with viral loads in vitro and in vivo. The dynamics of ZIKV infection in A129 (interferon (IFN)-α/ß receptor deficient), AG6 (IFN-α/ß and IFN-γ receptor deficient), and C57BL/6 mice were characterized. Pregnant dams were infected with ZIKV-Nluc at E10 via intra footpad injection. Then, the pooled immune sera (anti-ZIKV neutralizing antibodies) #22-1 in ZIKV-Nluc virus-infected mice were visualized. Results: ZIKV-Nluc showed a high genetic stability and replicated well in cells with similar properties to the wild-type ZIKV (ZIKVwt). Striking bioluminescence signals were consistently observed in animal organs, including spleen, intestine, testis, uterus/ovary, and kidney. The ileocecal junction was found to be the crucial visceral target. Infection of pregnant dams with ZIKV-Nluc showed that ZIKV was capable of crossing the maternal-fetal barrier to infect the fetuses via vertical transmission. Furthermore, it was visualized that treatment with the pooled immune sera was found to greatly restrict the spread of the ZIKV-Nluc virus in mice. Conclusions: This study is the first to report the real-time noninvasive tracking of the progression of ZIKV invading immune-sheltered tissues and propagating vertically during pregnancy. The results demonstrate that ZIKV-Nluc represents a powerful tool for the study of the replication, dissemination, pathogenesis, and treatment of ZIKV in vitro and in vivo.

African and Asian Zika Virus Isolates Display Phenotypic Differences Both In Vitro and In Vivo.

Zika virus (ZIKV) is a mosquito-borne member of the genus Flavivirus that has emerged since 2007 to cause outbreaks in Africa, Asia, Oceania, and most recently, in the Americas. Here, we used an isolate history as well as genetic and phylogenetic analyses to characterize three low-passage isolates representing African (ArD 41525) and Asian (CPC-0740, SV0127-14) lineages to investigate the potential phenotypic differences in vitro and in vivo. The African isolate displayed a large plaque phenotype (∼3-4 mm) on Vero and HEK-293 cells, whereas the Asian isolates either exhibited a small plaque phenotype (∼1-2 mm) or did not produce any plaques. In multistep replication kinetics in nine different vertebrate and insect cell lines, the African isolate consistently displayed faster replication kinetics and yielded ∼10- to 10,000-fold higher peak virus titers (infectious or RNA copies) compared with the Asian isolates. Oral exposure of Aedes aegypti mosquitoes with the African isolate yielded higher infection and dissemination rates compared with the Asian isolates. Infection of Ifnar1-/- mice with the African isolate produced a uniformly fatal disease, whereas infection with the Asian isolates produced either a delay in time-to-death or a significantly lower mortality rate. Last, the African isolate was > 10,000-fold more virulent than the Asian isolates in an interferon type I antibody blockade mouse model. These data demonstrate substantial phenotypic differences between low-passage African and Asian isolates both in vitro and in vivo and warrant further investigation. They also highlight the need for basic characterization of ZIKV isolates, as the utilization of the uncharacterized isolates could have consequences for animal model and therapeutic/vaccine development.

Lack of Effect of Murine Astrovirus Infection on Dextran Sulfate-induced Colitis in NLRP3-deficient Mice.

Murine astrovirus (MuAstV) is a recently identified, widespread infection among laboratory mice. MuAstV is found predominantly in the gastrointestinal tract of mice. Human and turkey astroviruses have been shown to disrupt tight junctions in the intestinal epithelium. The potential of MuAstV to alter research results was tested in a well-established dextran sodium sulfate (DSS)-induced colitis model in Nod-like receptor 3 (NLRP3)-deficient mice. This model offers a direct approach to determine whether MuAstV, as a component of the mouse microbiome, contributes to the issue of poor reproducibility in murine inflammatory bowel disease research. In this model, defective inflammasome activation causes loss of epithelial integrity, resulting in leakage of intestinal bacteria and colitis. Our goal was to determine whether MuAstV, which also may affect intestinal permeability, altered the onset or severity of colitis. Male and female mice (age, 8 to 12 wk) homozygous or heterozygous for an NLRP3 mutation were inoculated orally with MuAstV or mock-inoculated with media 3 or 20 d prior to being exposed to 2% DSS in their drinking water for 9 d. MuAstV infection alone did not cause clinical signs or histopathologic changes in NLRP3-/- or NLRP3+/- mice. No significant difference was seen in weight loss, clinical disease, intestinal inflammation, edema, hyperplasia, or mucosal ulceration between MuAstV- infected and mock-infected mice that received 2% DSS for 9 d. Therefore, MuAstV does not appear to be a confounding variable in the DSS colitis model in NLRP3 mice.

Successful rederivation of contaminated immunocompetent mice using neonatal transfer with iodine immersion.

There is an ongoing need to eradicate intercurrent disease from research mouse colonies. Commonly used surgical methods, however, are expensive and time-consuming. The purpose of this study was to determine the percentage of litters that could be rederived from infected mouse colonies by neonatal transfer. We immersed neonatal mice in a dilute iodine solution and transferred them to disease-free foster mothers within 48 h of birth. Donor and foster mothers were evaluated for pathogens by serology and fecal polymerase chain reaction (PCR) assay. Of 55 donor mothers, 100% were positive serologically and 59% were positive by fecal PCR for one or more tested organisms, including mouse hepatitis virus, Theiler's murine encephalomyelitis virus, mouse rotavirus, and Helicobacter hepaticus. At 4 to 6 weeks after neonatal transfer, 95% of foster mothers (which served as sentinels for the transferred pups) tested free of pathogens, the exceptions being one case of mouse parvovirus 1 and two of Helicobacter spp. We suggest that cross-fostering is a viable low-cost method for rederivation of mouse colonies contaminated with pathogens such as mouse hepatitis virus, Theiler's murine encephalomyelitis virus, mouse rotavirus, and H. hepaticus.

A comparison of mouse parvovirus 1 infection in BALB/c and C57BL/6 mice: susceptibility, replication, shedding, and seroconversion.

This study characterized the effects of challenge with a field isolate of mouse parvovirus 1 (MPV1e) in C57BL/6NCrl (B6) and BALB/cAnNCrl (C) mice. We found that C mice were more susceptible to MPV1e infection than were B6 mice; ID50 were 50 to 100 times higher after gavage and 10-fold higher after intraperitoneal injection in B6 as compared with C mice. To evaluate the host strain effect on the pathogenesis of MPV1e, B6 and C mice were inoculated by gavage. Feces and tissues, including mesenteric lymph nodes (MLN), ileum, spleen and blood, were collected for analysis by quantitative PCR (qPCR) to assess infection and fecal shedding and by RT-qPCR to evaluate replication. Peak levels of MPV1e shedding, infection, and replication were on average 3.4, 4.3, and 6.2 times higher, respectively, in C than in B6 mice. Peaks occurred between 3 and 10 d after inoculation in C mice but between 5 and 14 d in B6 mice. Multiplexed fluorometric immunoassays detected seroconversion in 2 of 3 C mice at 7 d after inoculation and in all 3 B6 mice at 10 d. By 56 d after inoculation, viral replication was no longer detectable, and fecal shedding was very low; infection persisted in ileum, spleen, and MLN, with levels higher in C than B6 mice and highest in MLN. Therefore, the lower susceptibility of B6 mice, as compared with C mice, to MPV1e infection was associated with lower levels of infection, replication, and shedding and delayed seroconversion.

Effect of mouse strain and age on detection of mouse parvovirus 1 by use of serologic testing and polymerase chain reaction analysis.

BACKGROUND AND PURPOSE: Detection of mouse parvovirus 1 (MPV) depends on use of serologic and polymerase chain reaction (PCR) assays. These assays were evaluated for their ability to detect virus-specific antibodies or viral DNA in multiple strains and ages of mice inoculated with MPV. METHODS: Twelve-week-old ICR, BALB/c, C3H, C57BL/6, and DBA/2 mice and four- and eight-week-old ICR mice were inoculated with MPV. Serum was harvested four weeks after inoculation and analyzed by use of recombinant non structural protein 1 (rNS1) enzyme-linked immunosorbent assay (ELISA), minute virus of mice (MVM) ELISA, and MPV indirect fluorescent antibody (IFA), MVM IFA, and MPV hemagglutination inhibition (HAI) assays. Select tissues were harvested and analyzed by use of an MPV-specific PCR assay. RESULTS: The number of mice in each group with detectable MPV-specific antibodies or MPV DNA varied with mouse strain, mouse age when inoculated, and viral dose. Seroconversion in mice inoculated at 12 weeks of age was detected almost exclusively by use of the MPV IFA and MPV HAI assays, whereas seroconversion in almost all mice inoculated at 4 and 8 weeks of age was detected by use of all immunoassays except the MVM ELISA. Viral DNA was detected by use of PCR analysis in all strains and ages of mice except DBA/2 mice. CONCLUSIONS: Mouse strain and age have important roles in seroconversion to nonstructural and structural MPV antigens and persistence of viral DNA in mouse tissues. Therefore, diagnostic serologic testing and PCR analysis should be considered within the context of mouse strain and age at the time of MPV exposure, especially when sentinel mice are used for surveillance.

Differences between BALB/c and C57BL/6 mice in mouse hepatitis virus replication in primary hepatocyte culture.

We previously showed that an intraperitoneal infection with mouse hepatitis virus (MHV) resulted in acute hepatic failure accompanying extremely elevated viral growth in the liver in interferon-gamma-deficient BALB/c (BALB-GKO), but not C57BL/6 (B6-GKO) mice. To examine the basis of the strain difference against MHV infection in interferon-gamma-deficient mice, viral replication in primary hepatocyte cultures from BALB/c and B6 mice with or without the IFN-gamma gene was compared in vitro. The MHV replication in BALB/c hepatocytes with or without the IFN-gamma gene was significantly higher than that in B6 hepatocytes with or without the IFN-gamma gene, suggesting that there is a strain difference in MHV replication in hepatocytes. Since a significant difference in MHV replication in hepatocytes was not observed between wild type and IFN-gamma-deficient mice of the same genetic background, the phenomenon is thought to be independent of IFN-gamma. However, pretreatment of hepatocytes with recombinant mouse interferon-gamma inhibited MHV replication in a dose-dependent fashion. The results are discussed with respect to the pathology of MHV infection in mice with or without the IFN-gamma gene.

Heterologous protection against lethal A/HongKong/156/97 (H5N1) influenza virus infection in C57BL/6 mice.

The continual threat posed by newly emerging influenza virus strains is demonstrated by the recent outbreak of H5N1 influenza virus in Hong Kong. Currently, immunization against influenza virus infection is fairly adequate, but it is imperative that improved vaccines are developed that can protect against a variety of strains and be generated rapidly. Since humoral immunity is ineffective against serologically distinct viruses, one strategy would be to develop vaccines that emphasize cellular immunity. Here we report the successful protection of C57BL/6 mice from a lethal A/HK/156/97 (HK156) infection by immunizing first with an H9N2 isolate, A/Quail/HK/G1/97 (QHKG1), that harbours internal genes 98% homologous to HK156. This strategy also protected mice that are deficient in antibody production, indicating that the immunity is T-cell-mediated. In the course of these studies, we generated a highly pathogenic H5N1 reassortant which implicated NP and PB2 as having an important contribution to pathogenesis when present with a highly cleavable H5. These results provide the first demonstration that protective cell-mediated immunity can be established against the highly virulent HK156 virus and have important implications for the development of novel strategies for the prevention and treatment of HK156 infection and the design of future influenza vaccines.

Sendai viruses with altered P, V, and W protein expression.

Wild-type Sendai virus expresses three proteins containing the N-terminal half of the P protein open reading frame due to mRNA editing; a full-length P protein (ca. 70% of the total), a V protein with the N-terminal half fused to a Cys-rich Zn(2+)-binding domain (ca. 25% of the total), and a W protein representing the N-terminal half alone (ca. 5% of the total). To examine the role of these proteins in the virus life cycle, we have prepared recombinant viruses in which the normal V mRNA expresses a W protein (V-stop; 70% P, 30% W), one which cannot edit its P gene mRNA (delta 6A; 100% P), and one which overedits its mRNA like parainfluenza virus type 3 (swap/8;20-40% P, 30% V, 30% W). All these viruses were readily recovered and grew to similar titers in eggs, and except for the P gene products, cell lines individually infected with these viruses accumulated similar amounts of viral macromolecules. The relative competitive advantage of each virus was determined by multiple cycle coinfections of eggs and found to be rSeV-Vstop = rSeV-wt >> rSeV-delta 6A > rSeV-swap/8. On the other hand, rSeV-swap/8 underwent multiple cycles of replication in C57BI/6 mouse lungs and was highly virulent for these animals, whereas rSeV-delta 6A was avirulent in mice and this infection was quickly cleared. Remarkably, rSeV-Vstop appeared to be more virulent for inbred C57BI/6 mice than rSeV-wt, but was partially attenuated in infections of outbred ICR mice. Thus, the expression of either the V or the W proteins is sufficient for multiple cycles of infection and pathogenesis in C57BI/6 mice, whereas W can only partially substitute for V for pathogenesis in ICR mice.

Supplementation with Lactobacillus reuteri or L. acidophilus reduced intestinal shedding of cryptosporidium parvum oocysts in immunodeficient C57BL/6 mice.

The effect of L. acidophilus supplementation to reduce fecal shedding of Cryptosporidium parvum oocysts was compared to L. reuteri using C57BL/6 female mice immunosuppressed by murine leukemia virus (strain LP-BM5) inoculation. After 12 weeks post LP-BM5 inoculation, 15 immunosuppressed mice each were randomly assinged to one of the following treatment groups: historical control (group A), LP-BM5 control (group B), C. parvum (group C), L. reuteri plus C. parvum (group D) or L. acidophilus plus C. parvum (group E). Mice were pre-fed the L. reuteri or L. acidophilus bacteria strains daily for 13 days, challenged with C. parvum oocysts and thereafter fed the specified Lactobacillus regimens daily during the experimental period. Animals supplemented with L. reuteri shed fewer (p<0.05) oocysts on day-7 post C. parvum challenge compared to controls. Mice supplemented with L. acidophilus also shed fewer (p<0.05) oocysts on days 7 and 14 post-challenge compared to controls. Overall, Lactobacillus supplementation reduced C. parvum shedding in the feces but failed to suppress the production of T-helper type 2 cytokines [interleukin-4 (IL-4), IL-8)] which are associated with immunosuppression. Additionally, Lactobacillus supplementation did not restore T-helper type 1 cytokines (interleukin-2 (IL-2) and gamma interferon (IFN-gamma), which are required for recovery from parasitic infections. Altered T-helper types 1 and 2 cytokine production as a consequence of immunodysfunction permitted the development of persistent cryptosporidiosis while mice with intact immune system were refractory to infection with C. parvum. Reduction in shedding of oocysts observed in the Lactobacillus supplemented mice during deminished IL-2 and IFN-gamma production may be mediated by factors released into the intestinal lumen by the Lactobacillus and possibly other host cellular mechanisms. These observations suggest that L. reuteri or L. acidophilus can reduce C. parvum parasite burdens in the intestinal epithelium during cryptosporidiosis and may serve potential benefits as probiotics for host resistance to intestinal parasitic infections. L. acidophilus was more efficacious in reducing fecal shedding than L. reuteri and therefore may also have implication in the therapy of cryptosporidiosis during immunosuppressive states including human AIDS.

Viral abrogation of stem cell transplantation tolerance causes graft rejection and host death by different mechanisms.

Tolerance-based stem cell transplantation using sublethal conditioning is being considered for the treatment of human disease, but safety and efficacy remain to be established. We have shown that mouse bone marrow recipients treated with sublethal irradiation plus transient blockade of the CD40-CD154 costimulatory pathway develop permanent hematopoietic chimerism across allogeneic barriers. We now report that infection with lymphocytic choriomeningitis virus at the time of transplantation prevented engraftment of allogeneic, but not syngeneic, bone marrow in similarly treated mice. Infected allograft recipients also failed to clear the virus and died. Postmortem study revealed hypoplastic bone marrow and spleens. The cause of death was virus-induced IFN-alphabeta. The rejection of allogeneic bone marrow was mediated by a radioresistant CD8(+)TCR-alphabeta(+)NK1.1(-) T cell population. We conclude that a noncytopathic viral infection at the time of transplantation can prevent engraftment of allogeneic bone marrow and result in the death of sublethally irradiated mice treated with costimulation blockade. Clinical application of stem cell transplantation protocols based on costimulation blockade and tolerance induction may require patient isolation to facilitate the procedure and to protect recipients.