Induction of Mucosal B-Cell Memory by Intramuscular Inoculation of Mice with Rotavirus

We investigated the capacity of intramuscular (i.m.) immunization with heterologous-host rotavirus (simian strain RRV) to induce mucosal virus-specific memory B cells in mice. We found that prior i.m. immunization enhanced the magnitude of mucosal virus-specific immunoglobulin A (IgA) production but did not alter the site and timing of induction of virus-specific IgA responses after challenge.     

Rotavirus Infection Induces Transient Pancreatic Involution and Hyperglycemia in Weanling Mice

Rotavirus is a ubiquitous double-stranded RNA virus responsible for most cases of infantile gastroenteritis. It infects pancreatic islets in vitro and is implicated as a trigger of autoimmune destruction of islet beta cells leading to type 1 diabetes, but pancreatic pathology secondary to rotavirus infection in vivo has not been documented. To address this issue, we inoculated 3 week-old C57Bl/6 mice at weaning with rhesus rotavirus, which is closely related to human rotaviruses and known to infect mouse islets in vitro. Virus was quantified in tissues by culture-isolation and enzyme-linked immunosorbent assay. A requirement for viral double stranded RNA was investigated in toll-like receptor 3 (TLR3)-deficient mice. Cell proliferation and apoptosis, and insulin expression, were analyzed by immunohistochemistry. Following rotavirus inoculation by gavage, two phases of mild, transient hyperglycemia were observed beginning after 2 and 8 days. In the first phase, widespread apoptosis of pancreatic cells was associated with a decrease in pancreas mass and insulin production, without detectable virus in the pancreas. These effects were mimicked by injection of the double-stranded RNA mimic, polyinosinic-polycytidylic acid, and were TLR3-dependent. By the second phase, the pancreas had regenerated but islets were smaller than normal and viral antigen was then detected in the pancreas for several days. These findings directly demonstrate pathogenic effects of rotavirus infection on the pancreas in vivo, mediated initially by the interaction of rotavirus double-stranded RNA with TLR3.     

Transgenic Tobacco Expressing a Modified VP6 Gene Protects Mice Against Rotavirus Infection

Elevated expression of the rotavirus VP6 antigen in transgenic plants is a critical factor in the development of a safe and effective rotavirus vaccine. Using codon optimization, a gene that encodes the inner capsid protein VP6 of the human group A rotavirus was synthesized （sVP6）. The VP6 and sVp6 genes were transformed into tobacco （Nicotiana tabacum L.） plants using Agrobacterium tumefaciens. The expression level of the sVP6 gene in transgenic plants was 3.8-34-fold higher than that of controls containing the non-modified VP6 gene, accounting for up to 0.34% of the total soluble protein （TSP）. Then, BALB/ c female mice that had been gavaged weekly with 10 mg TSP containing 34 p.g VP6 protein, in which VP6-specific serum IgG and mucosal IgA antibodies were investigated. The severity and duration of diarrhea caused by simian rotavirus SA-11 challenge were reduced significantly in passively immunized pups, which indicates that anti-VP6 antibodies generated in orally immunized female mice can be passed onto pups and provide heterotypic protection. An edible vaccine based on the VP6 of human rotavirus group A could provide a means to protect children and young animals from severe acute diarrhea.     

A Hamster-Derived West Nile Virus Isolate Induces Persistent Renal Infection in Mice

Background

West Nile virus (WNV) can persist long term in the brain and kidney tissues of humans, non-human primates, and hamsters. In this study, mice were infected with WNV strain H8912, previously cultured from the urine of a persistently infected hamster, to determine its pathogenesis in a murine host.

Methodology/Principal Findings

We found that WNV H8912 was highly attenuated for neuroinvasiveness in mice. Following a systemic infection, viral RNA could be detected quickly in blood and spleen and much later in kidneys. WNV H8912 induced constitutive IL-10 production, upregulation of IFN-β and IL-1β expression, and a specific IgM response on day 10 post-infection. WNV H8912 persisted preferentially in kidneys with mild renal inflammation, and less frequently in spleen for up to 2.5 months post infection. This was concurrent with detectable serum WNV-specific IgM and IgG production. There were also significantly fewer WNV- specific T cells and lower inflammatory responses in kidneys than in spleen. Previous studies have shown that systemic wild-type WNV NY99 infection induced virus persistence preferentially in spleen than in mouse kidneys. Here, we noted that splenocytes of WNV H8912-infected mice produced significantly less IL-10 than those of WNV NY99-infected mice. Finally, WNV H8912 was also attenuated in neurovirulence. Following intracranial inoculation, WNV persisted in the brain at a low frequency, concurrent with neither inflammatory responses nor neuronal damage in the brain.

Conclusions

WNV H8912 is highly attenuated in both neuroinvasiveness and neurovirulence in mice. It induces a low and delayed anti-viral response in mice and preferentially persists in the kidneys.     

轮状病毒感染成年小鼠的研究

目的研究成年昆明种小鼠对实验感染人轮状病毒(rotavirus,RV)的敏感性。方法在实验条件下,用A组人Wa和恒河猴SA11株RV感染成年昆明种小鼠,观察小鼠的临床反应和排毒情况。结果成年昆明种小鼠感染Wa和SA11RV第二天后出现明显的临床腹泻症状,第四天达到高峰;至少在感染后连续6天的动物大便中可检测到较高滴度的RV抗原。结论成年昆明种小鼠对RV感染有很高的敏感性,可做为动物模型,在RV感染的药物治疗效果评价和疫苗保护性效果评价中具有重要价值。     

Systemic and Mucosal Immune Reactivity upon Mycobacterium avium ssp. paratuberculosis Infection in Mice

Mycobacterium avium ssp. paratuberculosis (MAP) is the cause of Johne''s disease, an inflammatory bowel disorder of ruminants. Due to the similar pathology, MAP was also suggested to cause Crohn''s disease (CD). Despite of intensive research, this question is still not settled, possibly due to the lack of versatile mouse models. The aim of this study was to identify basic immunologic mechanisms in response to MAP infection. Immune compromised C57BL/6 Rag2 ^−/− mice were infected with MAP intraperitoneally. Such chronically infected mice were then reconstituted with CD4⁺ and CD8⁺ T cells 28 days after infection. A systemic inflammatory response, detected as enlargement of the spleen and granuloma formation in the liver, was observed in mice infected and reconstituted with CD4⁺ T cells. Whereby inflammation in infected and CD4⁺CD45RB^hi T cell reconstituted animals was always higher than in the other groups. Reconstitution of infected animals with CD8⁺ T cells did not result in any inflammatory signs. Interestingly, various markers of inflammation were strongly up-regulated in the colon of infected mice reconstituted with CD4⁺CD45RB^lo/int T cells. We propose, the usual non-colitogenic CD4⁺CD45RB^lo/int T cells were converted into inflammatory T cells by the interaction with MAP. However, the power of such cells might be not sufficient for a fully established inflammatory response in the colon. Nevertheless, our model system appears to mirror aspects of an inflammatory bowel disease (IBD) like CD and Johne''s diseases. Thus, it will provide an experimental platform on which further knowledge on IBD and the involvement of MAP in the induction of CD could be acquired.     

Diabetes in Mice after Coxsackie B4 Virus Infection

The intraperitoneal inoculation of CD₁ mice with Coxsackie virus B₄ resulted in the raising of blood sugar levels to diabetic values 12 days after the administration of the virus. Serum insulin remained inappropriately low. Light microscopy changes in the islets of Langerhans showed mononuclear cell infiltration of the islets and marked degranulation of the β cells. The acinar tissue appeared to be little changed. It is concluded that Coxsackie B₄ virus may cause a diabetic state compatible with islet cell damage.     

Expression of the Mucosal Homing Receptor α4β7 Correlates with the Ability of CD8+ Memory T Cells To Clear Rotavirus Infection

The integrin α₄β₇ plays an important role in lymphocyte homing to mucosal lymphoid tissues and has been shown to define a subpopulation of memory T cells capable of homing to intestinal sites. Here we have used a well-characterized intestinal virus, murine rotavirus, to investigate whether memory/effector function for an intestinal pathogen is associated with α₄β₇ expression. α₄β₇^hi memory phenotype (CD44^hi), α₄β₇⁻ memory phenotype, and presumptively naive (CD44^lo) CD8⁺ T lymphocytes from rotavirus-infected mice were sorted and transferred into Rag-2 (T- and B-cell-deficient) recipients that were chronically infected with murine rotavirus. α₄β₇^hi memory phenotype CD8⁺ cells were highly efficient at clearing rotavirus infection, α₄β₇⁻ memory cells were inefficient or ineffective, depending on the cell numbers transferred, and CD44^lo cells were completely unable to clear chronic rotavirus infection. These data demonstrate that functional memory for rotavirus resides primarily in memory phenotype cells that display the mucosal homing receptor α₄β₇.     

Antibody-Independent Protection against Rotavirus Infection of Mice Stimulated by Intranasal Immunization with Chimeric VP4 or VP6 Protein

This study was to determine whether individual rotavirus capsid proteins could stimulate protection against rotavirus shedding in an adult mouse model. BALB/c mice were intranasally or intramuscularly administered purified Escherichia coli-expressed murine rotavirus strain EDIM VP4, VP6, or truncated VP7 (TrVP7) protein fused to the 42.7-kDa maltose-binding protein (MBP). One month after the last immunization, mice were challenged with EDIM and shedding of rotavirus antigen was measured. When three 9-microg doses of one of the three rotavirus proteins fused to MBP were administered intramuscularly with the saponin adjuvant QS-21, serum rotavirus immunoglobulin G (IgG) was induced by each protein. Following EDIM challenge, shedding was significantly (P = 0.02) reduced (i.e., 38%) in MBP::VP6-immunized mice only. Three 9-micrograms doses of chimeric MBP::VP6 or MBP::TrVP7 administered intranasally with attenuated E. coli heat-labile toxin LT(R192G) also induced serum rotavirus IgG, but MBP::VP4 immunization stimulated no detectable rotavirus antibody. No protection against EDIM shedding was observed in the MBP::TrVP7-immunized mice. However, shedding was reduced 93 to 100% following MBP::VP6 inoculation and 56% following MBP::VP4 immunization relative to that of controls (P = <0.001). Substitution of cholera toxin for LT(R192G) as the adjuvant, reduction of the number of doses to 1, and challenge of the mice 3 months after the last immunization did not reduce the level of protection stimulated by intranasal administration of MBP::VP6. When MBP::VP6 was administered intranasally to B-cell-deficient microMt mice that made no rotavirus antibody, shedding was still reduced to <1% of that of controls. These results show that mice can be protected against rotavirus shedding by intranasal administration of individual rotavirus proteins and that this protection can occur independently of rotavirus antibody.     

Group A Rotavirus Infection and Age-Dependent Diarrheal Disease in Rats: a New Animal Model To Study the Pathophysiology of Rotavirus Infection

Group A rotaviruses are major pathogens causing acute gastroenteritis in children and animals. To determine if group A rotavirus replicates and induces disease in rats, antibody-negative Lewis neonatal or adult rats were inoculated orally with tissue culture-adapted human (Wa, WI61, and HAL1166), simian (rhesus rotavirus [RRV] and SA11), bovine (WC3), lapine (ALA), or porcine (OSU) rotavirus strains, wild-type murine (EC(wt)) rotavirus strain, or phosphate-buffered saline (PBS). Rotavirus infection in rats was evaluated by (i) clinical findings, (ii) virus antigen shedding or infectious virus titers in the feces or intestinal contents measured by enzyme-linked immunosorbent assay or fluorescent-focus assay, (iii) histopathological changes in the small intestine, (iv) distribution of rotavirus antigen in small-intestine sections by immunofluorescence, and (v) growth rate. Rotavirus infection of 5-day-old but not > or =21-day-old rats resulted in diarrhea that lasted from 1 to 10 days postinoculation. The severity of disease and spread of infection to naIve littermates differed depending on the virus strain used for inoculation. The duration of virus antigen shedding following infection was considerably prolonged (up to 10 days) in neonatal rats compared to that in 21-day-old rats (1 or 2 days). Based on lack of virus antigen shedding and disease induction, the murine EC(wt) rotavirus was the only strain tested that did not infect rats. Histopathological changes in the small-intestine mucosa of 5-day-old RRV-inoculated rats but not of PBS-inoculated rats was limited to extensive enterocyte vacuolation in the ileum. In RRV-inoculated neonatal rats, rotavirus antigen was detected in the epithelial cells on the upper half of the intestinal villi of the jejunum and ileum. In addition, infection of neonatal rats with RRV but not with PBS resulted in reduced weight gain. Rats infected with group A rotaviruses provide a new animal model with unique features amenable to investigate rotavirus pathogenesis and the molecular mechanisms of intestinal development, including physiological factors that may regulate age-dependent rotavirus-induced diarrhea.     

Lymph Node Colonization Dynamics after Oral Salmonella Typhimurium Infection in Mice

An understanding of how pathogens colonize their hosts is crucial for the rational design of vaccines or therapy. While the molecular factors facilitating the invasion and systemic infection by pathogens are a central focus of research in microbiology, the population biological aspects of colonization are still poorly understood. Here, we investigated the early colonization dynamics of Salmonella enterica subspecies 1 serovar Typhimurium (S. Tm) in the streptomycin mouse model for diarrhea. We focused on the first step on the way to systemic infection — the colonization of the cecal lymph node (cLN) from the gut — and studied roles of inflammation, dendritic cells and innate immune effectors in the colonization process. To this end, we inoculated mice with mixtures of seven wild type isogenic tagged strains (WITS) of S. Tm. The experimental data were analyzed with a newly developed mathematical model describing the stochastic immigration, replication and clearance of bacteria in the cLN. We estimated that in the beginning of infection only 300 bacterial cells arrive in the cLN per day. We further found that inflammation decreases the net replication rate in the cLN by 23%. In mice, in which dendritic cell movement is impaired, the bacterial migration rate was reduced 10-fold. In contrast, mice that cannot generate toxic reactive oxygen species displayed a 4-fold higher migration rate from gut to cLN than wild type mice. Thus, combining infections with mixed inocula of barcoded strains and mathematical analysis represents a powerful method for disentangling immigration into the cLN from replication in this compartment. The estimated parameters provide an important baseline to assess and predict the efficacy of interventions.     

人轮状病毒感染昆明小鼠乳鼠模型的建立

目的建立人轮状病毒G3型709株感染4d龄昆明小鼠乳鼠模型。方法通过灌胃病毒的方式造模,观察乳鼠被病毒攻击后不同时间其临床表现、小肠组织病理改变、小肠组织上皮细胞超微结构改变。酶联免疫吸附法检测轮状病毒抗原在乳鼠粪便中的表达,免疫荧光法检测轮状病毒在乳鼠小肠组织中的表达。结果4d龄昆明小鼠乳鼠被轮状病毒攻击24h后出现腹泻表现和小肠组织病理改变,72h最严重,之后腹泻率下降,病理改变减轻,第7天腹泻停止,病理改变消失。乳鼠小肠上皮细胞出现糖、脂肪代谢紊乱,其粪便和小肠组织中都可以检测出轮状病毒抗原表达。结论4d龄昆明小鼠乳鼠被人轮状病毒A组G3型709株经口攻击后病毒能够在其体内复制,出现腹泻表现。该病毒感染腹泻过程具有自愈特点。     

Mx—cre转基因小鼠品系的建立及其培育

将携带MX启动子调控的Cre基因真核表达载体pMx-cre线性化后,通过受卵显微注射途径制备转基因小鼠,共注射99个卵,产佴0只,利用PCR对小鼠进行筛选,以基因组Southern blot确证,最后得到一个阳性的小鼠品系,进而将其保护和扩大繁殖。     

High Numbers of Viral RNA Copies in the Central Nervous System of Mice during Persistent Infection with Theiler's Virus

The low-neurovirulence Theiler's murine encephalomyelitis viruses (TMEV), such as BeAn virus, cause a persistent infection of the central nervous system (CNS) in susceptible mouse strains that results in inflammatory demyelination. The ability of TMEV to persist in the mouse CNS has traditionally been demonstrated by recovering infectious virus from the spinal cord. Results of infectivity assays led to the notion that TMEV persists at low levels. In the present study, we analyzed the copy number of TMEV genomes, plus- to minus-strand ratios, and full-length species in the spinal cords of infected mice and infected tissue culture cells by using Northern hybridization. Considering the low levels of infectious virus in the spinal cord, a surprisingly large number of viral genomes (mean of 3.0 x 10(9)) was detected in persistently infected mice. In the transition from the acute (approximately postinfection [p.i.] day 7) to the persistent (beginning on p.i. day 28) phase of infection, viral RNA copy numbers steadily increased, indicating that TMEV persistence involves active viral RNA replication. Further, BeAn viral genomes were full-length in size; i.e., no subgenomic species were detected and the ratio of BeAn virus plus- to minus-strand RNA indicated that viral RNA replication is unperturbed in the mouse spinal cord. Analysis of cultured macrophages and oligodendrocytes suggests that either of these cell types can potentially synthesize high numbers of viral RNA copies if infected in the spinal cord and therefore account for the heavy viral load. A scheme is presented for the direct isolation of both cell types directly from infected spinal cords for further viral analyses.     

Resistance to Infection,Early and Persistent Suppression of Simian Immunodeficiency Virus SIVmac251 Viremia,and Significant Reduction of Tissue Viral Burden after Mucosal Vaccination in Female Rhesus Macaques

The efficacy of oral, intestinal, nasal, and vaginal vaccinations with DNA simian immunodeficiency virus (SIV)/interleukin-2 (IL-2)/IL-15, SIV Gag/Pol/Env recombinant modified vaccinia virus Ankara (rMVA), and AT-2 SIV_mac239 inactivated particles was compared in rhesus macaques after low-dose vaginal challenge with SIV_mac251. Intestinal immunization provided better protection from infection, as a significantly greater median number of challenges was necessary in this group than in the others. Oral and nasal vaccinations provided the most significant control of disease progression. Fifty percent of the orally and nasally vaccinated animals suppressed viremia to undetectable levels, while this occurred to a significantly lower degree in intestinally and vaginally vaccinated animals and in controls. Viremia remained undetectable after CD8⁺ T-cell depletion in seven vaccinated animals that had suppressed viremia after infection, and tissue analysis for SIV DNA and RNA was negative, a result consistent with a significant reduction of viral activity. Regardless of the route of vaccination, mucosal vaccinations prevented loss of CD4⁺ central memory and CD4⁺/α4β7⁺ T-cell populations and reduced immune activation to different degrees. None of the orally vaccinated animals and only one of the nasally vaccinated animals developed AIDS after 72 to 84 weeks of infection, when the trial was closed. The levels of anti-SIV gamma interferon-positive, CD4⁺, and CD8⁺ T cells at the time of first challenge inversely correlated with viremia and directly correlated with protection from infection and longer survival.