Virulence Of Different Strains Research Articles

Identification of equine mares as reservoir hosts for pathogenic species of Leptospira.

Equine leptospirosis can result in abortion, stillbirth, neonatal death, placentitis, and uveitis. Horses can also act as subclinical reservoir hosts of infection, which are characterized as asymptomatic carriers that persistently excrete leptospires and transmit disease. In this study, PCR and culture were used to assess urinary shedding of pathogenic Leptospira from 37 asymptomatic mares. Three asymptomatic mares, designated as H2, H8, and H9, were PCR-positive for lipL32, a gene specific for pathogenic species of Leptospira. One asymptomatic mare, H9, was culture-positive, and the recovered isolate was classified as L. kirschneri serogroup Australis serovar Rushan. DNA capture and enrichment of Leptospira genomic DNA from PCR-positive, culture-negative samples determined that asymptomatic mare H8 was also shedding L. kirschneri serogroup Australis, whereas asymptomatic mare H2 was shedding L. interrogans serogroup Icterohaemorrhagiae. Sera from all asymptomatic mares were tested by the microscopic agglutination test (MAT) and 35 of 37 (94.6%) were seropositive with titers ranging from 1:100 to 1:3200. In contrast to asymptomatic mares, mare H44 presented with acute spontaneous abortion and a serum MAT titer of 1:102,400 to L. interrogans serogroup Pomona serovar Pomona. Comparison of L. kirschneri serogroup Australis strain H9 with that of L. interrogans serogroup Pomona strain H44 in the hamster model of leptospirosis corroborated differences in virulence of strains. Since lipopolysaccharide (LPS) is a protective antigen in bacterin vaccines, the LPS of strain H9 (associated with subclinical carriage) was compared with strain H44 (associated with spontaneous abortion). This revealed different LPS profiles and immunoreactivity with reference antisera. It is essential to know what species and serovars of Leptospira are circulating in equine populations to design efficacious vaccines and diagnostic tests. Our results demonstrate that horses in the US can act as reservoir hosts of leptospirosis and shed diverse pathogenic Leptospira species via urine. This report also details the detection of L. kirschneri serogroup Australis serovar Rushan, a species and serotype of Leptospira, not previously reported in the US.

Mass Spectrometry Imaging (MALDI-MSI) as a molecular histology application for characterizing pulmonary lesions caused by PRRSV-1 strains of different virulence

Mass Spectrometry Imaging (MALDI-MSI) as a molecular histology application for characterizing pulmonary lesions caused by PRRSV-1 strains of different virulence

The scene of lung pathology during PRRSV-1 infection.

Porcine reproductive and respiratory syndrome (PRRS) is one of the most economically important infectious diseases for the pig industry worldwide. The disease was firstly reported in 1987 and became endemic in many countries. Since then, outbreaks caused by strains of high virulence have been reported several times in Asia, America and Europe. Interstitial pneumonia, microscopically characterised by thickened alveolar septa, is the hallmark lesion of PRRS. However, suppurative bronchopneumonia and proliferative and necrotising pneumonia are also observed, particularly when a virulent strain is involved. This raises the question of whether the infection by certain strains results in an overstimulation of the proinflammatory response and whether there is some degree of correlation between the strain involved and a particular pattern of lung injury. Thus, it is of interest to know how the inflammatory response is modulated in these cases due to the interplay between virus and host factors. This review provides an overview of the macroscopic, microscopic, and molecular pathology of PRRSV-1 strains in the lung, emphasising the differences between strains of different virulence.

A new killing assay with the Caenorhabditis elegans PX627 mutant to assess the virulence of uropathogenic Escherichia coli strains

The nematode Caenorhabditis elegans (C. elegans) is a prime invertebrate host model for studying uropathogenic Escherichia coli (UPEC) pathogenesis. The aim of this work was to develop a new C. elegans killing assay based on feeding bacteria by the nematode throughout its life from the egg. With this model, the lifespan of C. elegans rrf-3, temperature-sterile, mutant, and PX627, auxin-inducible infertile, mutant fed UPEC strains, was compared. The behavior of three clinical UPEC strains and the non-pathogenic Escherichia coli OP50 strain was analyzed. Survival curves were generated by the Kaplan–Meier method and compared by the log-rank test over 10 days of follow-up. There was no significant difference between the survival curves obtained with each of the two C. elegans mutants (PX627 and rrf-3) fed with each of the strains of E. coli (OP50, G1722, G1473 or ER41). The UPEC strains were classified according to their virulence in vivo in the C. elegans PX627 mutant. The most virulent strain was ER41 which harbored the virulence genes fimA, papC and hlyA, expressed hemolysis in vitro and showed no antibiotic resistance. The least virulent strain was G1722 which only harbored the two adhesion factor genes, was not hemolytic and was resistant to multiple antibiotics. The C. elegans PX627 mutant fed with UPEC bacteria from the egg stage is a simple and inexpensive invertebrate animal model for assessing the in vivo virulence of different strains. The early exposure of C. elegans to pathogenic bacteria at the egg stage, without the need to change the incubation temperature, is an advantage over previously described C. elegans killing assays.

PRRSV-1 induced lung lesion is associated with an imbalance between costimulatory and coinhibitory immune checkpoints.

Porcine reproductive and respiratory syndrome virus (PRRSV) induces a dysregulation on the innate and adaptive immune responses. T-cell activation requires a proper interaction and precise balance between costimulatory and coinhibitory molecules, commonly known as immune checkpoints. This study aims to evaluate the expression of immune checkpoints in lung and tracheobronchial lymph node from piglets infected with two PRRSV-1 strains of different virulence during the early stage of infection. Seventy 4-week-old piglets were grouped into three experimental groups: (i) control, (ii) 3249-infected group (low virulent strain), and (iii) Lena-infected group (virulent strain) and were euthanized at 1, 3, 6, 8, and 13 days post-infection (dpi). Lung and tracheobronchial lymph node were collected to evaluate histopathological findings, PRRSV viral load and mRNA expression of costimulatory (CD28, CD226, TNFRSF9, SELL, ICOS, and CD40) and coinhibitory (CTLA4, TIGIT, PD1/PDL1, TIM3, LAG3, and IDO1) molecules through RT-qPCR. Our findings highlight a mild increase of costimulatory molecules together with an earlier and stronger up-regulation of coinhibitory molecules in both organs from PRRSV-1-infected animals, especially in the lung from virulent Lena-infected animals. The simultaneous expression of coinhibitory immune checkpoints could work in synergy to control and limit the inflammation-induced tissue damage. Further studies should be addressed to determine the role of these molecules in later stages of PRRSV infection.

Interaction between Porcine Alveolar Macrophage-Tang Cells and Streptococcus suis Strains of Different Virulence: Phagocytosis and Apoptosis

Streptococcus suis is an important swine bacterial pathogen that activates macrophages to secrete inflammatory cytokines. Primary porcine alveolar macrophages (PAMs) are inconvenient to obtain, but it is unknown whether immortalized PAM-Tang cells can replace them as a better cell model for the study of the interaction between S. suis and macrophages. In this study, the phagocytic integrity, polarization, and pro-inflammatory cytokine secretion of PAM-Tang cells were confirmed by live-cell imaging, electron microscopy, confocal microscopy, and ELISA. Interestingly, the S. suis serotype 9 avirulent strain W7119 induced higher levels of adhesion and pro-inflammatory cytokines in PAM-Tang cells than the S. suis serotype 2 virulent strain 700794. Prolonged incubation with S. suis caused more cytotoxic cell damage, and the virulent strain induced higher levels of cytotoxicity to PAM-Tang cells. The virulent strain also induced higher levels of apoptosis in PAM-Tang cells, as shown by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL) assay. In addition, it is the first report of virulent and avirulent S. suis inducing PAM-Tang polarization towards pro-inflammatory M1 macrophages and p53- and caspase-dependent apoptosis in PAMs. Taken together, this study contributes to a better understand of interactions between macrophages and S. suis isolates of different virulence, and confirms that PAM-Tang cells provide a long-term, renewable resource for investigating macrophage infections with bacteria.

Comparative pathogenesis of peste des petits ruminants virus strains of difference virulence

Peste des petits ruminants (PPR) is an acute disease of small ruminants caused by a morbillivirus. Clinical observation of the disease in the field revealed that several species of small ruminants are affected to varying degrees. This difference in disease-related effects could depend either on the host or on the virulence of the virus strain. A previous study highlighted the difference in virulence between two strains of PPRV used to infect Saanen goats. For this breed, PPRV Morocco 2008 strain (MA08) was highly virulent while PPRV Côte d’Ivoire 1989 (IC89) strain induced mild disease. Experimental studies generally based on healthy and young animals do not permit exploration of the natural variability of the host susceptibility to PPRV. Therefore, building on the previous study on Saanen goats, the current study focussed on this breed of goat and used commercially available animals with an unknown history of infection with other pathogens. Results confirmed the previous disease pattern for PPRV IC89 and MA08 strains. Viral RNA detection, macroscopic and histological lesions were stronger for the highly virulent MA08 strain. We show here for the first time that viral RNA can be detected in the tissues of vaccinated animals. Viral RNA was also detected for the first time in serum samples, which is in agreement with the role of circulating immune cells in transporting the virus into host target organs. Thus, this study provides insight into the pathogenesis of strains of different virulence of PPRV and will help to better understand the onset of the disease.

Global profiling of protein lysine malonylation in Toxoplasma gondii strains of different virulence and genetic backgrounds.

Lysine malonylation is a post-translational modification (PTM), which regulates many cellular processes. Limited information is available about the level of lysine malonylation variations between Toxoplasma gondii strains of distinct genetic lineages. Yet, insights into such variations are needed to understand the extent to which lysine malonylation contributes to the differences in the virulence and repertoire of virulence factors between T. gondii genotypes. In this study, we profiled lysine malonylation in T. gondii using quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) and immuno-affinity purification. This analysis was performed on three T. gondii strains with distinctive pathogenicity in mice, including RH strain (type I), PRU strain (type II), and VEG strain (type III). In total, 111 differentially malonylated proteins and 152 sites were upregulated, and 17 proteins and 17 sites were downregulated in RH strain versus PRU strain; 50 proteins and 59 sites were upregulated, 50 proteins and 53 sites were downregulated in RH strain versus VEG strain; and 72 proteins and 90 sites were upregulated, and 7 proteins and 8 sites were downregulated in VEG strain versus PRU strain. Differentially malonylated proteins were involved in key processes, such as those mediating the regulation of protein metabolism, stress response, glycolysis, and actin cytoskeleton. These results reveal an association between lysine malonylation and intra-species virulence differences in T. gondii and offer a new resource for elucidating the contribution of lysine malonylation to energy metabolism and virulence in T. gondii.

Construction of a telomerase-immortalized porcine tracheal epithelial cell model for swine-origin mycoplasma infection

Primary porcine tracheal epithelial cells (PTECs) are an appropriate model for studying the molecular mechanism of various porcine respiratory diseases, including swine-origin mycoplasmas, which are isolated from respiratory tract of pigs and mainly found on the mucosal surface surrounding swine trachea. However, the short proliferation ability of primary PTECs greatly limits their lifespan. In this study, primary PTECs were carefully isolated and cultured, and immortal PTECs were constructed by transfecting primary PTECs with the recombinant constructed plasmid pEGFP-hTERT containing human telomerase reverse transcriptase (hTERT). Immortal PTECs (hTERT-PTECs) maintained both the morphological and functional characteristics of primary PTECs, as indicated by the expression of cytokeratin 18, cell-cycle analysis, proliferation assay, Western blotting, telomerase activity assay, karyotype analysis and quantitative RT-PCR. Compared to primary PTECs, hTERT-PTECs had an extended replicative lifespan, higher telomerase activity, and enhanced proliferative activity. In addition, this cell line resulted in a lack of transformed and grown tumors in nude mice, suggesting that it could be safely applied in further studies. Moreover, hTERT-PTECs were vulnerable to all swine-origin mycoplasmas through quantitative analysis as indicated by 50% color changing unit (CCU50) calculation, and no significant differences of adhesion ability between primary and immortal PTECs were observed. For the representative swine mycoplasma Mycoplasma hyopneumoniae (Mhp), except for DNA copies quantitative real-time PCR assay, indirect immunofluorescence assay and Western blotting analysis also depicted that hTERT-PTECs was able to adhere to different Mhp strains of different virulence. In summary, like primary PTECs, hTERT-PTECs could be widely used as an adhesion cell model for swine-origin mycoplasmas and in infection studies of various porcine respiratory pathogens.

Integrative RNA profiling of TBEV-infected neurons and astrocytes reveals potential pathogenic effectors

Tick-borne encephalitis virus (TBEV), the most medically relevant tick-transmitted flavivirus in Eurasia, targets the host central nervous system and frequently causes severe encephalitis. The severity of TBEV-induced neuropathogenesis is highly cell-type specific and the exact mechanism responsible for such differences has not been fully described yet. Thus, we performed a comprehensive analysis of alterations in host poly-(A)/miRNA/lncRNA expression upon TBEV infection in vitro in human primary neurons (high cytopathic effect) and astrocytes (low cytopathic effect). Infection with severe but not mild TBEV strain resulted in a high neuronal death rate. In comparison, infection with either of TBEV strains in human astrocytes did not. Differential expression and splicing analyses with an in silico prediction of miRNA/mRNA/lncRNA/vd-sRNA networks found significant changes in inflammatory and immune response pathways, nervous system development and regulation of mitosis in TBEV Hypr-infected neurons. Candidate mechanisms responsible for the aforementioned phenomena include specific regulation of host mRNA levels via differentially expressed miRNAs/lncRNAs or vd-sRNAs mimicking endogenous miRNAs and virus-driven modulation of host pre-mRNA splicing. We suggest that these factors are responsible for the observed differences in the virulence manifestation of both TBEV strains in different cell lines. This work brings the first complex overview of alterations in the transcriptome of human astrocytes and neurons during the infection by two TBEV strains of different virulence. The resulting data could serve as a starting point for further studies dealing with the mechanism of TBEV-host interactions and the related processes of TBEV pathogenesis.

Immunobiological properties of inactivated anti-highly pathogenic avian influenza vaccines based on antigens of А/Н5М avian influenza virus strains of different virulence

Immunobiological properties of inactivated anti-highly pathogenic avian influenza vaccines based on antigens of А/Н5М avian influenza virus strains of different virulence

Activation of T-bet, FOXP3, and EOMES in Target Organs From Piglets Infected With the Virulent PRRSV-1 Lena Strain.

Transcription factors (TFs) modulate genes involved in cell-type-specific proliferative and migratory properties, metabolic features, and effector functions. Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important pathogen agents in the porcine industry; however, TFs have been poorly studied during the course of this disease. Therefore, we aimed to evaluate the expressions of the TFs T-bet, GATA3, FOXP3, and Eomesodermin (EOMES) in target organs (the lung, tracheobronchial lymph node, and thymus) and those of different effector cytokines (IFNG, TNFA, and IL10) and the Fas ligand (FASL) during the early phase of infection with PRRSV-1 strains of different virulence. Target organs from mock-, virulent Lena-, and low virulent 3249-infected animals humanely euthanized at 1, 3, 6, 8, and 13 days post-infection (dpi) were collected to analyze the PRRSV viral load, histopathological lesions, and relative quantification through reverse transcription quantitative PCR (RT-qPCR) of the TFs and cytokines. Animals belonging to both infected groups, but mainly those infected with the virulent Lena strain, showed upregulation of the TFs T-bet, EOMES, and FOXP3, together with an increase of the cytokine IFN-γ in target organs at the end of the study (approximately 2 weeks post-infection). These results are suggestive of a stronger polarization to Th1 cells and regulatory T cells (Tregs), but also CD4+ cytotoxic T lymphocytes (CTLs), effector CD8+ T cells, and γδT cells in virulent PRRSV-1-infected animals; however, their biological functionality should be the object of further studies.

Time Series Transcriptomic Analysis of Bronchoalveolar Lavage Cells from Piglets Infected with Virulent or Low-Virulent Porcine Reproductive and Respiratory Syndrome Virus 1.

ABSTRACTPorcine reproductive and respiratory syndrome virus (PRRSV) has evolved to escape the immune surveillance for a survival advantage leading to a strong modulation of host’s immune responses and favoring secondary bacterial infections. However, limited data are available on how the immunological and transcriptional responses elicited by virulent and low-virulent PRRSV-1 strains are comparable and how they are conserved during the infection. To explore the kinetic transcriptional signature associated with the modulation of host immune response at lung level, a time-series transcriptomic analysis was performed in bronchoalveolar lavage cells upon experimental in vivo infection with two PRRSV-1 strains of different virulence, virulent subtype 3 Lena strain or the low-virulent subtype 1 3249 strain. The time-series analysis revealed overlapping patterns of dysregulated genes enriched in T-cell signaling pathways among both virulent and low-virulent strains, highlighting an upregulation of co-stimulatory and co-inhibitory immune checkpoints that were disclosed as Hub genes. On the other hand, virulent Lena infection induced an early and more marked “negative regulation of immune system process” with an overexpression of co-inhibitory receptors genes related to T-cell and NK cell functions, in association with more severe lung lesion, lung viral load, and BAL cell kinetics. These results underline a complex network of molecular mechanisms governing PRRSV-1 immunopathogenesis at lung level, revealing a pivotal role of co-inhibitory and co-stimulatory immune checkpoints in the pulmonary disease, which may have an impact on T-cell activation and related pathways. These immune checkpoints, together with the regulation of cytokine-signaling pathways, modulated in a virulence-dependent fashion, orchestrate an interplay among pro- and anti-inflammatory responses.IMPORTANCE Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the major threats to swine health and global production, causing substantial economic losses. We explore the mechanisms involved in the modulation of host immune response at lung level performing a time-series transcriptomic analysis upon experimental infection with two PRRSV-1 strains of different virulence. A complex network of molecular mechanisms was revealed to control the immunopathogenesis of PRRSV-1 infection, highlighting an interplay among pro- and anti-inflammatory responses as a potential mechanism to restrict inflammation-induced lung injury. Moreover, a pivotal role of co-inhibitory and co-stimulatory immune checkpoints was evidenced, which may lead to progressive dysfunction of T cells, impairing viral clearance and leading to persistent infection, favoring as well secondary bacterial infections or viral rebound. However, further studies should be conducted to evaluate the functional role of immune checkpoints in advanced stages of PRRSV infection and explore a possible T-cell exhaustion state.

Identification of Differential Responses of Goat PBMCs to PPRV Virulence Using a Multi-Omics Approach.

Peste des petits ruminants (PPR) is an acute transboundary infectious viral disease of small ruminants, mainly sheep and goats. Host susceptibility varies considerably depending on the PPR virus (PPRV) strain, the host species and breed. The effect of strains with different levels of virulence on the modulation of the immune system has not been thoroughly compared in an experimental setting so far. In this study, we used a multi-omics approach to investigate the host cellular factors involved in different infection phenotypes. Peripheral blood mononuclear cells (PBMCs) from Saanen goats were activated with a T-cell mitogen and infected with PPRV strains of different virulence: Morocco 2008 (high virulence), Ivory Coast 1989 (low virulence) and Nigeria 75/1 (live attenuated vaccine strain). Our results showed that the highly virulent strain replicated better than the other two in PBMCs and rapidly induced cell death and a stronger inhibition of lymphocyte proliferation. However, all the strains affected lymphocyte proliferation and induced upregulation of key antiviral genes and proteins, meaning a classical antiviral response is orchestrated regardless of the virulence of the PPRV strain. On the other hand, the highly virulent strain induced stronger inflammatory responses and activated more genes related to lymphocyte migration and recruitment, and inflammatory processes. Both transcriptomic and proteomic approaches were successful in detecting viral and antiviral effectors under all conditions. The present work identified key immunological factors related to PPRV virulence in vitro.

Indole decreases the virulence of pathogenic vibrios belonging to the Harveyi clade.

Indole is a signaling molecule secreted by over 85species of bacteria, including several Vibrio species, and it has been reported to affect different bacterial phenotypes such as biofilm formation, motility, and virulence. In this study, we aimed at investigating the inter-strain variability of the effect of indole in 12 different strains belonging to the Harveyi clade of vibrios. Indole reduced the virulence of all strains towards gnotobiotic brine shrimp larvae. The survival rate of brine shrimp larvae challenged with vibrios pretreated with indole was increased by 1.3-fold to 1.8-fold. Additionally, indole significantly decreased the biofilm formation in all of the strains, decreased the swimming motility in eight of the strains, and decreased swarming motility in five of the strains. When cultured in the presence of exogenous indole, the mRNA level of the pirA and pirB toxin genes were down-regulated to 65% and 46%, and to 62% and 55% in the AHPND-causing strains Vibrio parahaemolyticus M0904 and Vibrio campbellii S01, respectively. These data indicate that indole has a significant impact on the virulence of different strains belonging to the Harveyi clade of vibrios. Our results suggest that indole signaling is a valid target for the development of novel therapeutics in order to control infections caused by Harveyi clade vibrios in aquaculture.

Dynamics of African swine fever virus (ASFV) infection in domestic pigs infected with virulent, moderate virulent and attenuated genotype II ASFV European isolates.

This study aimed to compare the infection dynamics of three genotype II African swine fever viruses (ASFV) circulating in Europe. Eighteen domestic pigs divided into three groups were infected intramuscularly or by direct contact with two haemadsorbent ASFVs (HAD) from Poland (Pol16/DP/ OUT21) and Estonia (Est16/WB/Viru8), and with the Latvian non-HAD ASFV (Lv17/WB/Rie1). Parameters, such as symptoms, pathogenicity, and distribution of the virus in tissues, humoral immune response, and dissemination of the virus by blood, oropharyngeal and rectal routes, were investigated. The Polish ASFV caused a case of rapidly developing fatal acute disease, while the Estonian ASFV caused acute to sub-acute infections and two animals survived. In contrast, animals infected with the ASFV from Latvia developed a more subtle, mild, or even subclinical disease. Oral excretion was sporadic or even absent in the attenuated group, whereas in animals that developed an acute or sub-acute form of ASF, oral excretion began at the same time the ASFV was detected in the blood, or even 3 days earlier, and persisted up to 22 days. Regardless of virulence, blood was the main route of transmission of ASFV and infectious virus was isolated from persistently infected animals for at least 19 days in the attenuated group and up to 44 days in the group of moderate virulence. Rectal excretion was limited to the acute phase of infection. In terms of diagnostics, the ASFV genome was detected in contact pigs from oropharyngeal samples earlier than in blood, independently of virulence. Together with blood, both samples could allow to detect ASFV infection for a longer period. The results presented here provide quantitative data on the spread and excretion of ASFV strains of different virulence among domestic pigs that can help to better focus surveillance activities and, thus, increase the ability to detect ASF introductions earlier.

Porcine reproductive and respiratory syndrome virus impacts on gut microbiome in a strain virulence-dependent fashion.

SummaryPorcine reproductive and respiratory syndrome (PRRS) is a viral disease defined by reproductive problems, respiratory distress and a negative impact on growth rate and general condition. Virulent PRRS virus (PRRSV) strains have emerged in the last years with evident knowledge gaps in their impact on the host immune response. Thus, the present study examines the impact of acute PRRS virus (PRRSV) infection, with two strains of different virulence, on selected immune parameters and on the gut microbiota composition of infected pigs using 16S rRNA compositional sequencing. Pigs were infected with a low virulent (PRRS_3249) or a virulent (Lena) PRRSV‐1 strain and euthanized at 1, 3, 6, 8 or 13 days post‐inoculation (dpi). Faeces were collected from each animal at the necropsy time‐point. Alpha and beta diversity analyses demonstrated that infection, particularly with the Lena strain, impacted the microbiome composition from 6 dpi onwards. Taxonomic differences revealed that infected pigs had higher abundance of Treponema and Methanobrevibacter (FDR < 0.05). Differences were more considerable for Lena‐ than for PRRS_3249‐infected pigs, showing the impact of strain virulence in the intestinal changes. Lena‐infected pigs had reduced abundancies of anaerobic commensals such as Roseburia, Anaerostipes, Butyricicoccus and Prevotella (P < 0.05). The depletion of these desirable commensals was significantly correlated to infection severity measured by viraemia, clinical signs, lung lesions and immune parameters (IL‐6, IFN‐γ and Hp serum levels). Altogether, the results from this study demonstrate the indirect impact of PRRSV infection on gut microbiome composition in a strain virulence‐dependent fashion and its association with selected immune markers.

Inhibition of nitric oxide production of activated mice peritoneal macrophages is independent of the Toxoplasma gondii strain.

BACKGROUND Toxoplasma gondii causes toxoplasmosis and is controlled by activated macrophages. However, infection of macrophages by tachyzoites induces TGF-β signaling (TGF-s) inhibiting nitric oxide (NO) production. NO inhibition may be a general escape mechanism of distinct T. gondii strains.OBJECTIVES To evaluate in activated macrophages the capacity of T. gondii strains of different virulence and genetics (RH, type I; ME-49, type II; VEG, type III; P-Br, recombinant) to evade the NO microbicidal defense system and determine LC3 loading to the parasitophorous vacuole.METHODS Activated peritoneal macrophages were infected with the different T. gondii strains, NO-production was evaluated by the Griess reagent, and inducible nitric oxide synthase expression, TGF-s, and LC3 localisation assayed by immunofluorescence.FINDINGS Only RH persisted in macrophages, while VEG was more resistant than P-Br and ME-49. All strains induced TGF-s, degradation of inducible nitric oxide synthase, and NO-production inhibition from 2 to 24 h of infection, but only RH sustained these alterations for 48 h. By 24 h of infection, TGF-s lowered in macrophages infected by ME-49, and P-Br, and NO-production recovered, while VEG sustained TGF-s and NO-production inhibition longer. LC3 loading to parasitophorous vacuole was strain-dependent: higher for ME-49, P-Br and VEG, lower for RH. All strains inhibited NO-production, but only RH sustained this effect probably because it persisted in macrophages due to additional evasive mechanisms as lower LC3 loading to parasitophorous vacuole.MAIN CONCLUSIONS These results support that T. gondii can escape the NO microbicidal defense system at the initial phase of the infection, but only the virulent strain sustain this evasion mechanism.

Analysis of Virus Population Profiles within Pigs Infected with Virulent Classical Swine Fever Viruses: Evidence for Bottlenecks in Transmission but Absence of Tissue-Specific Virus Variants.

Classical swine fever virus (CSFV) contains a specific motif within the E2 glycoprotein that differs between strains of different virulence. In the highly virulent CSFV strain Koslov, this motif comprises residues S763/L764 in the polyprotein. However, L763/P764 represent the predominant alleles in published CSFV genomes. In this study, changes were introduced into the CSFV strain Koslov (here called vKos_SL) to generate modified CSFVs with substitutions at residues 763 and/or 764 (vKos_LL, vKos_SP, and vKos_LP). The properties of these mutant viruses, in comparison to those of vKos_SL, were determined in pigs. Each of the viruses was virulent and induced typical clinical signs of CSF, but the vKos_LP strain produced them significantly earlier. Full-length CSFV cDNA amplicons (12.3 kb) derived from sera of infected pigs were deep sequenced and cloned to reveal the individual haplotypes that contributed to the single-nucleotide polymorphism (SNP) profiles observed in the virus population. The SNP profiles for vKos_SL and vKos_LL displayed low-level heterogeneity across the entire genome, whereas vKos_SP and vKos_LP displayed limited diversity with a few high-frequency SNPs. This indicated that vKos_SL and vKos_LL exhibited a higher level of fitness in the host and more stability at the consensus level, whereas several consensus changes were observed in the vKos_SP and vKos_LP sequences, pointing to adaptation. For each virus, only a subset of the variants present within the virus inoculums were maintained in the infected pigs. No clear tissue-dependent quasispecies differentiation occurred within inoculated pigs; however, clear evidence for transmission bottlenecks to contact animals was observed, with subsequent loss of sequence diversity.IMPORTANCE The surface-exposed E2 protein of classical swine fever virus is required for its interaction with host cells. A short motif within this protein varies between strains of different virulence. The importance of two particular amino acid residues in determining the properties of a highly virulent strain of the virus has been analyzed. Each of the different viruses tested proved highly virulent, but one of them produced earlier, but not more severe, disease. By analyzing the virus genomes present within infected pigs, it was found that the viruses which replicated within inoculated animals were only a subset of those within the virus inoculum. Furthermore, following contact transmission, it was shown that a very restricted set of viruses had transferred between animals. There were no significant differences in the virus populations present in various tissues of the infected animals. These results indicate mechanisms of virus population change during transmission between animals.

Rabies viruses of different virulence regulates inflammatory responses both in vivo and in vitro via MAPK and NF-κB pathway

Immune responses and central nervous system dysfunction are two main factors to be considered during rabies virus (RABV) infection. However, the mechanisms by which RABV strains of different virulence regulate with chemokine expression and the signaling pathways responsible for the immune responses in the terminal stage of infection both in vivo and in vitro have not been fully elucidated. In this study, we found low expression levels of proinflammatory chemokines in the mouse brain upon infection with street RABV strains (CXZ17 and HN10) at the late stage of infection. We also examined the difference in inflammatory response upon infection with RABV strains of different virulence in a mouse model. We found that the expression of proinflammatory chemokines increased to a varying degree upon infection with street RABV (CXZ17 and HN10) or laboratory-fixed RABV (CVS-11, aG, and CTN); CXCL10, CCL5, and CCL2 were the most significantly upregulated chemokines in brain tissue and microglial BV-2 cells in response to infection with RABV strains of different virulence. Our data also demonstrate significant activation of the MAPK and NF-κB pathways in mouse brain tissue at the late stage of RABV infection. We also found (i) low phosphorylation signals of MAPK and NF-κB p65 in neuronal cells upon infection with CXZ17 and HN10 in the mouse brain and (ii) strong phosphorylation signals in cerebrovascular endothelial cells and neuronal cells upon CTN or aG infection. Moreover, we quantified the nuclear localization status of MAPK signals and NF-κB p65 upon infection with CVS-11, aG, and CTN in BV-2 cells in vitro. We also found (i) that the activation of the p38, ERK1/2, and NF-κB p65 pathway, which stimulates CXCL10, CCL5, and CCL2 expression upon infection with RABV strains of different virulence (aG, CTN, and CVS-11), is triggered after virus entry into BV-2 cells and (ii) that the expression of CXCL10, CCL5, and CCL2 is required for the activation of NF-κB, p38, and ERK1/2, but not JNK. Overall, our study provides insight into the regulation of inflammatory responses mediated by MAPK and NF-κB in the mouse brain and in microglial cells upon RABV infection of different virulence.