Murine Norovirus Transcytosis across an In Vitro Polarized Murine Intestinal Epithelial Monolayer Is Mediated by M-Like Cells

Noroviruses (NoVs) are the causative agent of the vast majority of nonbacterial gastroenteritis worldwide. Due to the inability to culture human NoVs and the inability to orally infect a small animal model, little is known about the initial steps of viral entry. One particular step that is not understood is how NoVs breach the intestinal epithelial barrier. Murine NoV (MNV) is the only NoV that can be propagated in vitro by infecting murine macrophages and dendritic cells, making this virus an attractive model for studies of different aspects of NoV biology. Polarized murine intestinal epithelial mIC_cl2 cells were used to investigate how MNV interacts with and crosses the intestinal epithelium. In this in vitro model of the follicle-associated epithelium (FAE), MNV is transported across the polarized cell monolayer in the absence of viral replication or disruption of tight junctions by a distinct epithelial cell with microfold (M) cell properties. In addition to transporting MNV, these M-like cells also transcytose microbeads and express an IgA receptor. Interestingly, B myeloma cells cultured in the basolateral compartment underlying the epithelial monolayer did not alter the number of M-like cells but increased their transcytotic activity. Our data demonstrate that MNV can cross an intact intestinal epithelial monolayer in vitro by hijacking the M-like cells'' intrinsic transcytotic pathway and suggest a potential mechanism for MNV entry into the host.     

Effect of Hypochlorite-Based Disinfectants on Inactivation of Murine Norovirus and Attempt to Eliminate or Prevent Infection in Mice by Additionto Drinking Water

We evaluated the in vitro efficacy of weak acid hypochlorous solution (WAHS) against murine norovirus (MNV) by plaque assay and compared the efficacy with diluted NaOCl (Purelox) and 70% ethanol. WAHS was as effective as 70% ethanol and diluted Purelox for 0.5-min reactions. For 0.5-min reactions in the presence of mouse feces emulsion, the efficacy of WHAS and 1:600 diluted Purelox was decreased, reducing the virus titers by 2.3 and 2.6 log₁₀, respectively, while 70% ethanol reduced the titer by more than 5 log₁₀. However, WAHS showed more than 5 log₁₀ reductions for the 5-min reaction even in the presence of feces emulsion. Since WAHS showed enough efficacy in inactivating MNV in vitro, we tried to eliminate MNV from MNV-infected mice by substituting WAHS for their drinking water. However, MNV was found to be positive in feces of mice drinking WAHS by an RT-nested PCR and plaque assay. To investigate whether hypochlorite-based disinfectants could prevent infection of a mouse with MNV, WAHS or 1:6,000 diluted Purelox was substituted for the drinking water of mice for 2 or 4 weeks, and then the mice were placed in a cage with an MNV-infected mouse. The supply of disinfectants was continued after cohabitation, but MNV was detected in the feces of all the mice at 1 week after cohabitation. In this study, we tried to eliminate and prevent MNV infection from mice by supplying hypochlorite-based disinfectants as an easy and low-cost method. Unfortunately, drinking disinfectants was ineffective, so it is important to keep the facility environment clean by use of effective disinfectants. Also, animals introduced into facilities should be tested as MNV free by quarantine and periodically confirmed as MNV free by microbiological monitoring.     

A prospective observational study of community-acquired bacterial bloodstream infections in Metro Manila,the Philippines

Community-acquired bacterial bloodstream infections are caused by diverse pathogens with changing antimicrobial-resistance patterns. In low-middle income countries in Southeast Asia, where dengue fever is endemic and a leading cause of fever, limited information is available about bacterial bloodstream infections due to challenges of implementing a blood culture service. This study describes bacterial bloodstream pathogens and antimicrobial-resistance patterns in Metro Manila, the Philippines. We aimed to identify the proportion of patients with a positive blood culture, the bacteria isolated and their antimicrobial resistance patterns, and the clinical characteristics of these patients, in this dengue endemic area. We conducted a prospective observational study in a single hospital enrolling febrile patients clinically suspected of having a community-acquired bacterial bloodstream infection between 1^st July 2015 and 30^th June 2019. Each patient had a blood culture and additional diagnostic tests according to their clinical presentation. We enrolled 1315 patients and a significant positive blood culture was found in 77 (5.9%) including Staphylococcus aureus (n = 20), Salmonella enterica Typhi (n = 18), Escherichia coli (n = 16), Streptococcus pneumoniae (n = 3) and Burkholderia pseudomallei (n = 2). Thirty-four patients had meningococcal disease diagnosed by culture (n = 8) or blood PCR (n = 26). Additional confirmed diagnoses included leptospirosis (n = 177), dengue virus infection (n = 159) and respiratory diphtheria (n = 50). There were 79 (6.0%, 95%CI 4.8%−7.4%) patients who died within 28 days of enrollment. Patients with a positive blood culture were significantly more likely to die than patients with negative culture (15.2% vs 4.4%, P<0.01). Among S. aureus isolates, 11/20 (55%) were methicillin-resistant (MRSA) and ST30: USA1100 was dominant sequence type (88.9%). Antimicrobial-susceptibility was well preserved in S. enterica Typhi. Among hospitalized patients with clinically suspected community-acquired bacterial bloodstream infection in Metro Manila, the Philippines, 5.9% had a blood culture confirmed infection of whom 15.6% died. S. aureus, including a significant number of MRSA (USA1100 clones), S. enterica Typhi, E.coli and Neisseria meningitidis were frequently identified pathogens.     

The Viral Polymerase Inhibitor 2′-C-Methylcytidine Inhibits Norwalk Virus Replication and Protects against Norovirus-Induced Diarrhea and Mortality in a Mouse Model

Human noroviruses are a major cause of food-borne illness, accountable for 50% of all-etiologies outbreaks of acute gastroenteritis (in both developing and developed countries). There is no vaccine or antiviral drug for the prophylaxis or treatment of norovirus-induced gastroenteritis. We recently reported the inhibitory effect of 2′-C-methylcytidine (2CMC), a hepatitis C virus polymerase inhibitor, on the in vitro replication of murine norovirus (MNV). Here we evaluated the inhibitory effect of 2CMC on in vitro human norovirus replication through a Norwalk virus replicon model and in a mouse model by using AG129 mice orally infected with MNV. Survival, weight, and fecal consistency were monitored, and viral loads in stool samples and organs were quantified. Intestines were examined histologically. 2CMC reduced Norwalk virus replicon replication in a dose-dependent manner and was able to clear cells of the replicon. Treatment of MNV-infected AG129 mice with 2CMC (i) prevented norovirus-induced diarrhea; (ii) markedly delayed the appearance of viral RNA and reduced viral RNA titers in the intestine, mesenteric lymph nodes, spleen, lungs, and stool; (iii) completely prevented virus-induced mortality; and (iv) resulted in protective immunity against a rechallenge. We demonstrate for the first time that a small-molecule inhibitor of norovirus replication protects from virus-induced disease and mortality in a relevant animal model. These findings pave the way for the development of potent and safe antivirals as prophylaxis and therapy of norovirus infection.     

Influence of genome-scale RNA structure disruption on the replication of murine norovirus—similar replication kinetics in cell culture but attenuation of viral fitness in vivo

Mechanisms by which certain RNA viruses, such as hepatitis C virus, establish persistent infections and cause chronic disease are of fundamental importance in viral pathogenesis. Mammalian positive-stranded RNA viruses establishing persistence typically possess genome-scale ordered RNA secondary structure (GORS) in their genomes. Murine norovirus (MNV) persists in immunocompetent mice and provides an experimental model to functionally characterize GORS. Substitution mutants were constructed with coding sequences in NS3/4- and NS6/7-coding regions replaced with sequences with identical coding and (di-)nucleotide composition but disrupted RNA secondary structure (F1, F2, F1/F2 mutants). Mutants replicated with similar kinetics to wild-type (WT) MNV3 in RAW264.7 cells and primary macrophages, exhibited similar (highly restricted) induction and susceptibility to interferon-coupled cellular responses and equal replication fitness by serial passaging of co-cultures. In vivo, both WT and F1/F2 mutant viruses persistently infected mice, although F1, F2 and F1/F2 mutant viruses were rapidly eliminated 1–7 days post-inoculation in competition experiments with WT. F1/F2 mutants recovered from tissues at 9 months showed higher synonymous substitution rates than WT and nucleotide substitutions that potentially restored of RNA secondary structure. GORS plays no role in basic replication of MNV but potentially contributes to viral fitness and persistence in vivo.     

Plaque Assay for Murine Norovirus

Murine norovirus (MNV) is the only member of the Norovirus genus that efficiently grows in tissue culture ^{1, 2}. Cell lysis and cytopathic effect (CPE) are observed during MNV-1 infection of murine dendritic cells or macrophages ¹. This property of MNV-1 can be used to quantify the number of infectious particles in a given sample by performing a plaque assay ¹. The plaque assay relies on the ability of MNV-1 to lyse cells and to form holes in a confluent cell monolayer, which are called plaques ³.Multiple techniques can be used to detect viral infections in tissue culture, harvested tissue, clinical, and environmental samples, but not all measure the number of infectious particles (e.g. qRT-PCR). One way to quantify infectious viral particles is to perform a plaque assay ³, which will be described in detail below. A variation on the MNV plaque assay is the fluorescent focus assay, where MNV antigen is immunostained in cell monolayers ⁴. This assay can be faster, since viral antigen expression precedes plaque formation. It is also useful for titrating viruses unable to form plaques. However, the fluorescent focus assay requires additional resources beyond those of the plaque assay, such as antibodies and a microscope to count focus-forming units. Infectious MNV can also be quantified by determining the 50% Tissue Culture Infective Dose (TCID₅₀) ³. This assay measures the amount of virus required to produce CPE in 50% of inoculated tissue culture cells by endpoint titration ⁵. However, its limit of detection is higher compared to a plaque assay ⁴.In this article, we describe a plaque assay protocol that can be used to effectively determine the number of infectious MNV particles present in biological or environmental samples ^{1, 4, 6}. This method is based on the preparation of 10-fold serial dilutions of MNV-containing samples, which are used to inoculate a monolayer of permissive cells (RAW 264.7 murine macrophage cells). Virus is allowed to attach to the cell monolayer for a given period of time and then aspirated before covering cells with a mixture of agarose and cell culture media. The agar enables the spread of viral progeny to neighboring cells while limiting spread to distantly located cells. Consequently, infected cells are lysed and form holes in the monolayer known as plaques. Upon sufficient spread of virus, plaques become visible following staining of cells with dyes, like neutral red, methylene blue, or crystal violet. At low dilutions, each plaque originates from one infectious viral particle and its progeny, which spread to neighboring cells. Thus, counting the number of plaques allows one to calculate plaque-forming units (PFU) present in the undiluted sample ³.     

Physiologic Stresses Reveal a Salmonella Persister State and TA Family Toxins Modulate Tolerance to These Stresses

Bacterial persister cells are considered a basis for chronic infections and relapse caused by bacterial pathogens. Persisters are phenotypic variants characterized by low metabolic activity and slow or no replication. This low metabolic state increases pathogen tolerance to antibiotics and host immune defenses that target actively growing cells. In this study we demonstrate that within a population of Salmonella enterica serotype Typhimurium, a small percentage of bacteria are reversibly tolerant to specific stressors that mimic the macrophage host environment. Numerous studies show that Toxin-Antitoxin (TA) systems contribute to persister states, based on toxin inhibition of bacterial metabolism or growth. To identify toxins that may promote a persister state in response to host-associated stressors, we analyzed the six TA loci specific to S. enterica serotypes that cause systemic infection in mammals, including five RelBE family members and one VapBC member. Deletion of TA loci increased or decreased tolerance depending on the stress conditions. Similarly, exogenous expression of toxins had mixed effects on bacterial survival in response to stress. In macrophages, S. Typhimurium induced expression of three of the toxins examined. These observations indicate that distinct toxin family members have protective capabilities for specific stressors but also suggest that TA loci have both positive and negative effects on tolerance.     

Murine Norovirus Infection Has No Significant Effect on Adaptive Immunity to Vaccinia Virus or Influenza A Virus

Murine norovirus (MNV) is endemic in many research mouse colonies. Although MNV infections are typically asymptomatic in immunocompetent mice, the effects of MNV infection on subsequent experimental viral infections are poorly documented. Here, we infected C57BL/6 mice with MNV and then with either vaccinia virus or influenza A virus. MNV infection had no effect on CD8⁺ T-cell or antibody responses to secondary viruses or to secondary virus-induced morbidity or mortality. While our findings suggest that MNV has little influence on host immunity in immunocompetent mice, we would urge caution regarding the potential effects of MNV on immune responses to viruses and other pathogens, which must be determined on a system-by-system basis.Human norovirus (NoV) infections cause greater than 90% of nonbacterial gastroenteritis cases (4, 5) and are an important public health concern. Murine noroviruses (MNV) were recently identified (7) as highly pathogenic agents in immunocompromised mice, and serological studies indicate that over 20% of mice in research colonies are exposed to MNV (6). As with NoV, MNV is spread through the fecal-oral route. While NoV rapidly causes gastrointestinal symptoms and fever in healthy individuals, MNV is typically asymptomatic in immunocompetent mice.MNV isolates are both genetically and biologically diverse (13). In wild-type (wt) mice, some strains of MNV are rapidly cleared, while others persist (13). Controlling MNV infections requires elements of both innate and adaptive immunity. Mice with defects in interferon (IFN) signaling pathways demonstrate increased MNV lethality (7, 9). CD4⁺ and CD8⁺ T cells and B cells are all needed for complete MNV clearance (1, 2). Natural exposure of immunocompromised mice to MNV leads to inflammation of the liver, lungs, and peritoneal and pleural cavities (14).It is well established that infection with natural mouse viruses can greatly impact immune responses to infections with other viruses. The prevalence of MNV in research mouse colonies might therefore lead to irreproducible and variable results that significantly impact research efforts. Indeed, MNV was recently reported to alter disease progression in a mouse model of bacterium-induced inflammatory bowel disease (8). Concern over the potential effects of MNV on viral immunology research prompted a dedicated workshop at the 2008 Keystone Viral Immunity meeting (http://www.keystonesymposia.org). In the present study, we examined the effect of MNV infection on adaptive immune responses in wt mice to influenza A virus (IAV) and vaccinia virus (VV).We infected C57BL/6 mice perorally with a high dose (3 × 10⁷ PFU/mouse) of a plaque-purified MNV stock derived from MNV-CR6p2 (13). The capacity of this plaque-purified virus to persist in wt mice has been confirmed by quantitative PCR analysis and a plaque assay (D. Strong, L. Thackray, and H. Virgin, unpublished observation). We confirmed that the mice were infected by measuring anti-MNV antibodies (Abs) by using an enzyme-linked immunosorbent assay (ELISA) (data not shown). For all experiments, mice were infected with MNV at Washington University and shipped 4 to 5 days later to NIAID for further study. To contain MNV, infected mice were housed in microisolator cages in a quarantine room. In some experiments, control mice were housed in the same room as MNV-infected mice. Sera collected from control mice did not contain anti-MNV Abs as determined by ELISA (data not shown), confirming that transmission of MNV between mice housed in microisolator cages can be prevented by proper cage changing and aseptic handling of samples from infected mice.Upon intraperitoneal (i.p.) infection with either VV or IAV, mice mount robust CD8⁺ T-cell responses that peak, respectively, on day 6 or 7. Anti-VV and anti-IAV CD8⁺ T-cell responses in C57BL/6 mice conform to a well-established immunodominance hierarchy (3, 10). To determine to what extent MNV infection alters the magnitude and/or immunodominance hierarchy of CD8⁺ T-cell responses, we infected C57BL/6 mice i.p. with either VV or IAV 19 days following MNV infection. As controls, naïve mice (MNV negative) were infected with either virus. Lymphocytes were isolated from mice 6 days postinfection with VV and 7 days postinfection with IAV. The fraction of antigen-specific CD8⁺ T cells present in spleen and peritoneal exudate cells (PEC) was determined by intracellular IFN-γ staining after stimulation with synthetic peptides. MNV infection had little effect on the magnitude of splenic or PEC CD8⁺ T cells responding to VV (Fig. 1A and B) or IAV (Fig. 1C and D) infection. Regardless of MNV exposure history, splenic and PEC responses were dominated by B8R- and A8R-specific CD8⁺ T cells following VV infection (Fig. 1A and B) and by PA-specific and NP-specific CD8⁺ T cells following IAV infection (Fig. 1C and D).Open in a separate windowFIG. 1.MNV exposure does not alter CD8⁺ T-cell responses to VV or IAV. MNV-infected and naïve C57BL/6 mice were infected i.p. with ∼1 × 10⁶ PFU of VV (A and B) or ∼1 × 10⁷ 50% tissue culture infective dose units of IAV (C and D), and specific CD8⁺ T cells were determined by intracellular IFN-γ staining after restimulating lymphocytes with peptides. Lymphocytes isolated from the spleen (A and C) and peritoneal cavity (B and D) were tested. MNV infections were completed 19 days prior to VV or IAV infections. Means and SEM are shown in panels A and C. A two-way analysis of variance and Bonferroni statistical analysis were completed for these experiments. Cells were pooled for peritoneal lavage samples as shown in panels B and D. Four to five mice/group were used for each experiment; data are representative of two independent experiments.To examine the effect of MNV infection on antiviral Ab responses, MNV-infected and control C57BL/6 mice were infected intranasally (i.n.) with a sublethal dose of either VV or IAV. Three weeks later, levels of anti-VV and anti-IAV Abs were determined by ELISA and hemagglutination inhibition assays, respectively. MNV infection did not significantly modify the magnitude of Ab responses to VV (Fig. ​(Fig.2A)2A) or IAV (Fig. ​(Fig.2B).2B). Next, we determined the effect of MNV infection on heavy chain class switching of anti-VV or anti-IAV Ab responses. Anti-VV and anti-IAV Ab responses exhibited similar heavy chain profiles dominated by immunoglobulin G2b (IgG2b) Abs regardless of MNV status (Fig. 2C and D). Thus, the CD8⁺ T-cell and Ab response to both VV and IAV appears to be essentially unaffected by chronic MNV infection. Since IgG anti-VV or anti-IAV Ab responses are entirely dependent on CD4⁺ T-cell help (11, 12), we can also infer that MNV also does not significantly affect CD4⁺ T-cell responses to VV or IAV.Open in a separate windowFIG. 2.MNV exposure does not alter Ab responses to VV or IAV. MNV-infected and naïve C57BL/6 mice were infected i.n. with ∼1 × 10³ PFU of VV (A and C) or ∼50 50% tissue culture infective dose units of IAV (B and D), and virus-specific Abs were determined by ELISA (A, C, and D) or hemagglutination inhibition (B). The ELISA results shown in panel A measured the total IgG, while the ELISA results shown in panels C and D measured the individual isotype indicated. MNV infections were completed 19 days prior to VV or IAV infections. Means and standard errors of the means are shown in panels A, C, and D. Means are shown as lines in panel B. A two-way analysis of variance and Bonferroni statistical analysis were completed for experiments shown in panels A, C, and D, and t tests were completed for the experiment shown in panel B. Four to five mice/group were used for each experiment. O.D., optical density; HAI, hemagglutination inhibition.T-cell and Ab responses, together with innate immune mechanisms, collaborate to control viral replication and limit pathogenesis. To examine the effect of chronic MNV infection on VV-induced or IAV-induced pathogenesis, we infected C57BL/6 mice i.n. with a lethal or sublethal dose of VV or IAV and monitored body weight over a 16-day period. MNV-CR6p2 infection had no significant effect on morbidity or mortality from either virus (Fig. ​(Fig.33 and ​and4).4). Since MNV isolates are highly diverse, we decided to examine the effects of a second strain of MNV (MNV-CW3) which is fully cleared in immunocompetent mice. Mice that cleared MNV-CW3 (19 days post-MNV infection) were infected i.n. with VV or IAV. Once again, this strain of MNV had no effect on VV-induced or IAV-induced morbidity or mortality (Fig. ​(Fig.33 and ​and4).4). Future studies should address the extent to which other MNV strains affect the generation of adaptive immune responses to secondary viral infections.Open in a separate windowFIG. 3.MNV does not increase morbidity following subsequent i.n. infection with VV or IAV. MNV-infected and naïve C57BL/6 mice were infected i.n. with a sublethal dose of VV (∼1 × 10³ PFU) (A) or IAV (∼50 50% tissue culture infective dose units) (B), and weight loss was recorded for 16 days postinfection. MNV infections were completed 19 days prior to VV or IAV infections. A two-way analysis of variance and Bonferroni statistical analysis were completed. Four to five mice/group were used for each experiment.Open in a separate windowFIG. 4.MNV does not increase mortality following subsequent i.n. infection with VV or IAV. MNV-infected and naïve C57BL/6 mice were infected i.n. with VV (∼1 × 10⁴ PFU) (A) or IAV (∼500 50% tissue culture infective dose units) (B), and survival was monitored for 16 days postinfection. MNV infections were completed 19 days prior to VV or IAV infections. Eight to 10 mice/group were used for each experiment.Taken together, these data demonstrate that MNV infection has no significant effects on the measured immune response to VV or IAV. Our results cannot, however, be simply extrapolated to other viruses or microorganisms. Rather, the effect of MNV infection on host immunity in mouse model disease systems needs to be established on a system-by-system basis. Without this knowledge, the possible confounding effects of MNV infection will continue to undermine the confidence in results obtained using mice in colonies in which MNV infections are endemic.     

两种核酸提取方法对小鼠诺如病毒RNA提取效能的比较

目的比较两种核酸提取方法对小鼠诺如病毒RNA的提取效能。方法用Trizol提取法和QIAamp Vira lRNA Min iKit提取法分别提取感染小鼠诺如病毒（Murine Norovirus,MNV）的小鼠小肠组织样品RNA和细胞培养物RNA,测定RNA浓度;用MNV特异的引物对分离的核酸样品进行一步法RT—PCR扩增。结果Trizol提取法提取小肠组织的RNA浓度高于QIAamp Viral RNA Mini Kit提取法;QIAamp Viral RNA Mini Kit提取得到的细胞培养物RNA浓度高于Trizol提取法。经QIAamp Viral RNA Mini Kit提取的两种核酸样品均能扩增出特异条带,而Trizol提取的核酸样品未见特异条带。结论在MNV的检测中,QIAampViralRNAKit更适合组织样品中MNV病毒核酸的提取。     

Cleavage map and proteolytic processing of the murine norovirus nonstructural polyprotein in infected cells

Murine norovirus (MNV) is presently the only member of the genus Norovirus in the Caliciviridae that can be propagated in cell culture. The goal of this study was to elucidate the proteolytic processing strategy of MNV during an authentic replication cycle in cells. A proteolytic cleavage map of the ORF1 polyprotein was generated, and the virus-encoded 3C-like (3CL) proteinase (Pro) mediated cleavage at five dipeptide cleavage sites, 341E/G342, Q705/N706, 870E/G871, 994E/A995, and 1177Q/G1178, that defined the borders of six proteins with the gene order p38.3 (Nterm)-p39.6 (NTPase)-p18.6-p14.3 (VPg)-p19.2 (Pro)-p57.5 (Pol). Bacterially expressed MNV 3CL Pro was sufficient to mediate trans cleavage of the ORF1 polyprotein containing the mutagenized Pro sequence into products identical to those observed during cotranslational processing of the authentic ORF1 polyprotein in vitro and to those observed in MNV-infected cells. Immunoprecipitation and Western blot analysis of proteins produced in virus-infected cells demonstrated efficient cleavage of the proteinase-polymerase precursor. Evidence for additional processing of the Nterm protein in MNV-infected cells by caspase 3 was obtained, and Nterm sequences 118DRPD121 and 128DAMD131 were mapped as caspase 3 cleavage sites by site-directed mutagenesis. The availability of the MNV nonstructural polyprotein cleavage map in concert with a permissive cell culture system should facilitate studies of norovirus replication.     

A Comparison of the ATP Generating Pathways Used by S. Typhimurium to Fuel Replication within Human and Murine Macrophage and Epithelial Cell Lines

The metabolism of S. Typhimurium within infected host cells plays a fundamental role in virulence since it enables intracellular proliferation and dissemination and affects the innate immune response. An essential requirement for the intracellular replication of S. Typhimurium is the need to regenerate ATP. The metabolic route used to fulfil this requirement is the subject of the present study. For infection models we used human and murine epithelial and macrophage cell lines. The epithelial cell lines were mIC_c12, a transimmortalised murine colon enterocyte cell line that shows many of the characteristics of a primary epithelial cell line, and HeLa cells. The model macrophage cell lines were THP-1A human monocyte/macrophages and RAW 264.7 murine macrophages. Using a mutational approach combined with an exometabolomic analysis, we showed that neither fermentative metabolism nor anaerobic respiration play major roles in energy generation in any of the cell lines studied. Rather, we identified overflow metabolism to acetate and lactate as the foremost route by which S. Typhimurium fulfils its energy requirements.     

Persistent Salmonellosis Causes Pancreatitis in a Murine Model of Infection

Pancreatitis, a known risk factor for the development of pancreatic ductal adenocarcinoma, is a serious, widespread medical condition usually caused by alcohol abuse or gallstone-mediated ductal obstruction. However, many cases of pancreatitis are of an unknown etiology. Pancreatitis has been linked to bacterial infection, but causality has yet to be established. Here, we found that persistent infection of mice with the bacterial pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium) was sufficient to induce pancreatitis reminiscent of the human disease. Specifically, we found that pancreatitis induced by persistent S. Typhimurium infection was characterized by a loss of pancreatic acinar cells, acinar-to-ductal metaplasia, fibrosis and accumulation of inflammatory cells, including CD11b⁺ F4/80⁺, CD11b⁺ Ly6C^int Ly6G⁺ and CD11b⁺ Ly6C^hi Ly6G⁻ cells. Furthermore, we found that S. Typhimurium colonized and persisted in the pancreas, associated with pancreatic acinar cells in vivo, and could invade cultured pancreatic acinar cells in vitro. Thus, persistent infection of mice with S. Typhimurium may serve as a useful model for the study of pancreatitis as it relates to bacterial infection. Increased knowledge of how pathogenic bacteria can cause pancreatitis will provide a more integrated picture of the etiology of the disease and could lead to the development of new therapeutic approaches for treatment and prevention of pancreatitis and pancreatic ductal adenocarcinoma.     

In Macrophages,Caspase-1 Activation by SopE and the Type III Secretion System-1 of S. Typhimurium Can Proceed in the Absence of Flagellin

The innate immune system is of vital importance for protection against infectious pathogens. Inflammasome mediated caspase-1 activation and subsequent release of pro-inflammatory cytokines like IL-1β and IL-18 is an important arm of the innate immune system. Salmonella enterica subspecies 1 serovar Typhimurium (S. Typhimurium, SL1344) is an enteropathogenic bacterium causing diarrheal diseases. Different reports have shown that in macrophages, S. Typhimurium may activate caspase-1 by at least three different types of stimuli: flagellin, the type III secretion system 1 (T1) and the T1 effector protein SopE. However, the relative importance and interdependence of the different factors in caspase-1 activation is still a matter of debate. Here, we have analyzed their relative contributions to caspase-1 activation in LPS-pretreated RAW264.7 macrophages. Using flagellar mutants (fliGHI, flgK) and centrifugation to mediate pathogen-host cell contact, we show that flagellins account for a small part of the caspase-1 activation in RAW264.7 cells. In addition, functional flagella are of key importance for motility and host cell attachment which is a prerequisite for mediating caspase-1 activation via these three stimuli. Using site directed mutants lacking several T1 effector proteins and flagellin expression, we found that SopE elicits caspase-1 activation even when flagellins are absent. In contrast, disruption of essential genes of the T1 protein injection system (invG, sipB) completely abolished caspase-1 activation. However, a robust level of caspase-1 activation is retained by the T1 system (or unidentified T1 effectors) in the absence of flagellin and SopE. T1-mediated inflammasome activation is in line with recent work by others and suggests that the T1 system itself may represent the basic caspase-1 activating stimulus in RAW264.7 macrophages which is further enhanced independently by SopE and/or flagellin.     

Chemical Derivatives of a Small Molecule Deubiquitinase Inhibitor Have Antiviral Activity against Several RNA Viruses

Most antiviral treatment options target the invading pathogen and unavoidably encounter loss of efficacy as the pathogen mutates to overcome replication restrictions. A good strategy for circumventing drug resistance, or for pathogens without treatment options, is to target host cell proteins that are utilized by viruses during infection. The small molecule WP1130 is a selective deubiquitinase inhibitor shown previously to successfully reduce replication of noroviruses and some other RNA viruses. In this study, we screened a library of 31 small molecule derivatives of WP1130 to identify compounds that retained the broad-spectrum antiviral activity of the parent compound in vitro but exhibited improved drug-like properties, particularly increased aqueous solubility. Seventeen compounds significantly reduced murine norovirus infection in murine macrophage RAW 264.7 cells, with four causing decreases in viral titers that were similar or slightly better than WP1130 (1.9 to 2.6 log scale). Antiviral activity was observed following pre-treatment and up to 1 hour postinfection in RAW 264.7 cells as well as in primary bone marrow-derived macrophages. Treatment of the human norovirus replicon system cell line with the same four compounds also decreased levels of Norwalk virus RNA. No significant cytotoxicity was observed at the working concentration of 5 µM for all compounds tested. In addition, the WP1130 derivatives maintained their broad-spectrum antiviral activity against other RNA viruses, Sindbis virus, LaCrosse virus, encephalomyocarditis virus, and Tulane virus. Thus, altering structural characteristics of WP1130 can maintain effective broad-spectrum antiviral activity while increasing aqueous solubility.     

Streptococcus pneumoniae Enhances Human Respiratory Syncytial Virus Infection In Vitro and In Vivo

Human respiratory syncytial virus (HRSV) and Streptococcus pneumoniae are important causative agents of respiratory tract infections. Both pathogens are associated with seasonal disease outbreaks in the pediatric population, and can often be detected simultaneously in infants hospitalized with bronchiolitis or pneumonia. It has been described that respiratory virus infections may predispose for bacterial superinfections, resulting in severe disease. However, studies on the influence of bacterial colonization of the upper respiratory tract on the pathogenesis of subsequent respiratory virus infections are scarce. Here, we have investigated whether pneumococcal colonization enhances subsequent HRSV infection. We used a newly generated recombinant subgroup B HRSV strain that expresses enhanced green fluorescent protein and pneumococcal isolates obtained from healthy children in disease-relevant in vitro and in vivo model systems. Three pneumococcal strains specifically enhanced in vitro HRSV infection of primary well-differentiated normal human bronchial epithelial cells grown at air-liquid interface, whereas two other strains did not. Since previous studies reported that bacterial neuraminidase enhanced HRSV infection in vitro, we measured pneumococcal neuraminidase activity in these cultures but found no correlation with the observed infection enhancement in our model. Subsequently, a selection of pneumococcal strains was used to induce nasal colonization of cotton rats, the best available small animal model for HRSV. Intranasal HRSV infection three days later resulted in strain-specific enhancement of HRSV replication in vivo. One S. pneumoniae strain enhanced HRSV both in vitro and in vivo, and was also associated with enhanced syncytium formation in vivo. However, neither pneumococci nor HRSV were found to spread from the upper to the lower respiratory tract, and neither pathogen was transmitted to naive cage mates by direct contact. These results demonstrate that pneumococcal colonization can enhance subsequent HRSV infection, and provide tools for additional mechanistic and intervention studies.     

Mycobacterium kansasii-induced death of murine macrophages involves endoplasmic reticulum stress responses mediated by reactive oxygen species generation or calpain activation

Although pathogenic mechanisms of tuberculosis have been extensively studied, little is known about the pathogenic mechanisms of Mycobacterium kansasii. In this work the influence of virulence and ER-stress mediated apoptosis of macrophages during two different strains of M. kansasii infection was investigated. We show that M. kansasii infection is associated with ER stress-mediated apoptosis in the murine macrophage cell line RAW 264.7. Infection of RAW 264.7 cells in vitro with apoptosis-inducing a clinical isolate of M. kansasii SM-1 (SM-1) resulted in strong induction of ER stress responses compared with M. kansasii type strain (ATCC 12478)-infected RAW 264.7 cells. Interestingly, inhibition of calpain prevented the induction of CHOP and Bip in ATCC 12478-infected RAW 264.7 cells but not in RAW 264.7 cells infected with SM-1. In contrast, reactive oxygen species (ROS) were significantly increased only in RAW 264.7 cells infected with SM-1. We propose that ROS generation is important for triggering ER stress-mediated apoptosis during SM-1 infection, whereas ATCC 12478-induced, ER stress-mediated apoptosis is associated with calpain activation. Our results demonstrate that the ER stress pathway plays important roles in the pathogenesis of M. kansasii infections, and that different strains of M. kansasii induce different patterns of ER stress-mediated apoptosis.     

Actinobacillus pleuropneumoniae Possesses an Antiviral Activity against Porcine Reproductive and Respiratory Syndrome Virus

Pigs are often colonized by more than one bacterial and/or viral species during respiratory tract infections. This phenomenon is known as the porcine respiratory disease complex (PRDC). Actinobacillus pleuropneumoniae (App) and porcine reproductive and respiratory syndrome virus (PRRSV) are pathogens that are frequently involved in PRDC. The main objective of this project was to study the in vitro interactions between these two pathogens and the host cells in the context of mixed infections. To fulfill this objective, PRRSV permissive cell lines such as MARC-145, SJPL, and porcine alveolar macrophages (PAM) were used. A pre-infection with PRRSV was performed at 0.5 multiplicity of infection (MOI) followed by an infection with App at 10 MOI. Bacterial adherence and cell death were compared. Results showed that PRRSV pre-infection did not affect bacterial adherence to the cells. PRRSV and App co-infection produced an additive cytotoxicity effect. Interestingly, a pre-infection of SJPL and PAM cells with App blocked completely PRRSV infection. Incubation of SJPL and PAM cells with an App cell-free culture supernatant is also sufficient to significantly block PRRSV infection. This antiviral activity is not due to LPS but rather by small molecular weight, heat-resistant App metabolites (<1 kDa). The antiviral activity was also observed in SJPL cells infected with swine influenza virus but to a much lower extent compared to PRRSV. More importantly, the PRRSV antiviral activity of App was also seen with PAM, the cells targeted by the virus in vivo during infection in pigs. The antiviral activity might be due, at least in part, to the production of interferon γ. The use of in vitro experimental models to study viral and bacterial co-infections will lead to a better understanding of the interactions between pathogens and their host cells, and could allow the development of novel prophylactic and therapeutic tools.