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Introduction: Human respiratory syncytial virus (hRSV) is a main cause of bronchiolitis in infants and its persistence has been described in immunocompromised subjects. However, limited evidence has been reported on the gene expression triggered by the hRSV and the effect of recombinant Taenia solium-derived calreticulin (rTsCRT). Methods: Using a comprehensive microarray approach, we analyzed the transcriptome profile of a macrophage cell line that has supported hRSV persistence for over 150 passages. We compared the gene expression of persistently infected and non-infected macrophages. We also evaluated the effect of rTsCRT on hRSV-infected macrophage gene transcription, as well as on cytokine production and number of copies of the persistent hRSV genome. Results: Our analysis showed that hRSV long-term virus infection significantly alters mRNA expression of antiviral, inflammatory, as well as arginine and lipid metabolism-associated genes, revealing a transcriptional signature that suggests a mixed M1/M2 phenotype. The resulting host-virus equilibrium allows for the regulation of viral replication, while evading the antiviral and proinflammatory responses. Interestingly, rTsCRT stimulus upregulated Tnfα, Il6 and Nos2 mRNA. We found increased levels of both proinflammatory cytokines and nitrite levels in the conditioned media of persistent macrophages treated with rTsCRT. This increase was associated with a significant reduction in viral genome copies. Discussion: hRSV persistently infected macrophages retain responsiveness to external stimuli and demonstrate that the profound changes induced by viral persistence are potentially reversible. Our observations contribute to the understanding of the mechanisms related to hRSV persistence in macrophages and have implications for the development of targeted therapies to eliminate persistent infections or reduce the negative effects related with chronic inflammatory diseases associated with hRSV infection. [ABSTRACT FROM AUTHOR] more...

IntroductionHuman respiratory syncytial virus (hRSV) is a main cause of bronchiolitis in infants and its persistence has been described in immunocompromised subjects. However, limited evidence has been reported on the gene expression triggered by the hRSV and the effect of recombinant Taenia solium-derived calreticulin (rTsCRT).MethodsUsing a comprehensive microarray approach, we analyzed the transcriptome profile of a macrophage cell line that has supported hRSV persistence for over 150 passages. We compared the gene expression of persistently infected and non-infected macrophages. We also evaluated the effect of rTsCRT on hRSV-infected macrophage gene transcription, as well as on cytokine production and number of copies of the persistent hRSV genome.ResultsOur analysis showed that hRSV long-term virus infection significantly alters mRNA expression of antiviral, inflammatory, as well as arginine and lipid metabolism-associated genes, revealing a transcriptional signature that suggests a mixed M1/M2 phenotype. The resulting host-virus equilibrium allows for the regulation of viral replication, while evading the antiviral and proinflammatory responses. Interestingly, rTsCRT stimulus upregulated Tnfα, Il6 and Nos2 mRNA. We found increased levels of both proinflammatory cytokines and nitrite levels in the conditioned media of persistent macrophages treated with rTsCRT. This increase was associated with a significant reduction in viral genome copies.DiscussionhRSV persistently infected macrophages retain responsiveness to external stimuli and demonstrate that the profound changes induced by viral persistence are potentially reversible. Our observations contribute to the understanding of the mechanisms related to hRSV persistence in macrophages and have implications for the development of targeted therapies to eliminate persistent infections or reduce the negative effects related with chronic inflammatory diseases associated with hRSV infection. more...

Norovirus (NoV) is the main non-bacterial pathogen causing outbreaks of gastroenteritis and is considered to be the leading cause of foodborne illness. This study aims to determine whether lettuce-encapsulated bacteria can express histo-blood group antigen (HBGA)–like substances to bind to NoV and, if so, to explore its role in protecting NoV from disinfection practices. Fifteen bacterial strains (HBGA-SEBs) were isolated from the lettuce microbiome and studied as they were proved to have the ability to express HBGA-like substances through indirect ELISA detection. By using attachment assay, HBGA-SEBs showed great abilities in carrying NoVs regarding the evaluation of binding capacity, especially for the top four strains from genera Wautersiella, Sphingobacterium, and Brachybacterium, which could absorb more than 60% of free-flowing NoVs. Meanwhile, the direct viral–bacterial binding between HBGA-like substance-expressing bacteria (HBGA-SEB) and NoVs was observed by TEM. Subsequently, results of simulated environmental experiments showed that the binding of NoVs with HBGA-SEBs did have detrimental effects on NoV reduction, which were evident in short-time high-temperature treatment (90°C) and UV exposure. Finally, by considering the relative abundance of homologous microorganisms of HBGA-SEBs in the lettuce microbiome (ca. 36.49%) and the reduction of NoVs in the simulated environments, we suggested putting extra attention on the daily disinfection of foodborne-pathogen carriers to overcome the detrimental effects of direct viral–bacterial interactions on the reduction of NoVs. more...

Norovirus (NoV) is the main non-bacterial pathogen causing outbreaks of gastroenteritis and is considered to be the leading cause of foodborne illness. This study aims to determine whether lettuce-encapsulated bacteria can express histo-blood group antigen (HBGA)–like substances to bind to NoV and, if so, to explore its role in protecting NoV from disinfection practices. Fifteen bacterial strains (HBGA-SEBs) were isolated from the lettuce microbiome and studied as they were proved to have the ability to express HBGA-like substances through indirect ELISA detection. By using attachment assay, HBGA-SEBs showed great abilities in carrying NoVs regarding the evaluation of binding capacity, especially for the top four strains from genera Wautersiella , Sphingobacterium , and Brachybacterium , which could absorb more than 60% of free-flowing NoVs. Meanwhile, the direct viral–bacterial binding between HBGA-like substance-expressing bacteria (HBGA-SEB) and NoVs was observed by TEM. Subsequently, results of simulated environmental experiments showed that the binding of NoVs with HBGA-SEBs did have detrimental effects on NoV reduction, which were evident in short-time high-temperature treatment (90°C) and UV exposure. Finally, by considering the relative abundance of homologous microorganisms of HBGA-SEBs in the lettuce microbiome (ca. 36.49%) and the reduction of NoVs in the simulated environments, we suggested putting extra attention on the daily disinfection of foodborne-pathogen carriers to overcome the detrimental effects of direct viral–bacterial interactions on the reduction of NoVs. [ABSTRACT FROM AUTHOR] more...

The detection of Zika virus (ZIKV) in immunoprivileged anatomical sites, potential sites for viral persistence, may guide the confirmation of undefined cases of ZIKV infection and also bring to light unknown pathways of viral transmission. Thus, this study aimed to characterize ZIKV infection in stratified, standardized placental samples in women with exanthematic febrile manifestations during pregnancy and compare findings to the standard investigation protocol of official health agencies. To this end, a case series of placental findings within a prospective cohort study was conducted over a period of 24 months. Serum/urine were obtained at the time of clinical case identification. Placental sampling was performed following standard investigation protocol (samples of 1.0 cm sent to a reference laboratory) and in a systematic way at various regions, such as chorionic plate, chorionic villi, basal plate, amniotic membrane, and umbilical cord, for subsequent ZIKV identification and quantification. Clinical information was obtained and histological preparation with hematoxylin–eosin staining for morphological evaluation was performed. This case series included 17 placentas systematically collected. Of these, 14 were positive by qRT-PCR for ZIKV, 5 in the umbilical cord, 7 in the amniotic membrane, 7 in the chorionic plate, 13 in the chorionic villi, and 7 in the basal plate, whereas none were reported by the reference laboratory. The most common morphological and anatomopathological findings were increased stromal cellularity, villitis, calcification, maternal vascular malperfusion, placental hypoplasia, and maternal–fetal hemorrhage (intervillous thrombi). Seven women presented positive testing for ZIKV in serological and/or molecular tests during gestation in urine. While viral quantification in urine ranged from 101 to 103 FFU eq/ml, that in different placental regions ranged from 103 to 108 FFU eq/g. Thus, ZIKV can infect different regions of the placenta and umbilical cord of pregnant women, showing that the systematic collection and adequate storage of the placenta is fundamental for the detection of ZIKV in this organ. The detection of ZIKV in the placenta after several months of initial symptoms suggests that this tissue may be a site for viral persistence during pregnancy. more...

HIV reservoirs persist in infected individuals despite combination antiretroviral therapy and can be identified in secondary lymphoid tissues, in intestinal tissues, in the central nervous system as well as in blood. Clinical trials have begun to explore effects of small molecule interventions to perturb the latent viral infection, but only limited information is available regarding the impacts of HIV cure-related clinical interventions on viral reservoirs found in tissues. Of the 14 HIV cure-related clinical trials since 2012 that have evaluated the effects of small molecule interventions in vivo, four trials have examined the impacts of the interventions in peripheral blood as well as other tissues that harbor persistent HIV. The additional tissues examined include cerebral spinal fluid, intestines and lymph nodes. We provide a comparison contrast analyses of the data across anatomical compartments tested in these studies to reveal where peripheral blood analyses reflect outcomes in other tissues as well as where the data reveal differences between tissue outcomes. We also summarize the current knowledge on these topics and highlight key open questions that need to be addressed experimentally to move the HIV cure research field closer to the development of an intervention strategy capable of eliciting long-term antiretroviral free remission of HIV disease. more...

The detection of Zika virus (ZIKV) in immunoprivileged anatomical sites, potential sites for viral persistence, may guide the confirmation of undefined cases of ZIKV infection and also bring to light unknown pathways of viral transmission. Thus, this study aimed to characterize ZIKV infection in stratified, standardized placental samples in women with exanthematic febrile manifestations during pregnancy and compare findings to the standard investigation protocol of official health agencies. To this end, a case series of placental findings within a prospective cohort study was conducted over a period of 24 months. Serum/urine were obtained at the time of clinical case identification. Placental sampling was performed following standard investigation protocol (samples of 1.0 cm sent to a reference laboratory) and in a systematic way at various regions, such as chorionic plate, chorionic villi, basal plate, amniotic membrane, and umbilical cord, for subsequent ZIKV identification and quantification. Clinical information was obtained and histological preparation with hematoxylin–eosin staining for morphological evaluation was performed. This case series included 17 placentas systematically collected. Of these, 14 were positive by qRT-PCR for ZIKV, 5 in the umbilical cord, 7 in the amniotic membrane, 7 in the chorionic plate, 13 in the chorionic villi, and 7 in the basal plate, whereas none were reported by the reference laboratory. The most common morphological and anatomopathological findings were increased stromal cellularity, villitis, calcification, maternal vascular malperfusion, placental hypoplasia, and maternal–fetal hemorrhage (intervillous thrombi). Seven women presented positive testing for ZIKV in serological and/or molecular tests during gestation in urine. While viral quantification in urine ranged from 101 to 103 FFU eq/ml, that in different placental regions ranged from 103 to 108 FFU eq/g. Thus, ZIKV can infect different regions of the placenta and umbilical cord of pregnant women, showing that the systematic collection and adequate storage of the placenta is fundamental for the detection of ZIKV in this organ. The detection of ZIKV in the placenta after several months of initial symptoms suggests that this tissue may be a site for viral persistence during pregnancy. [ABSTRACT FROM AUTHOR] more...

HIV reservoirs persist in infected individuals despite combination antiretroviral therapy and can be identified in secondary lymphoid tissues, in intestinal tissues, in the central nervous system as well as in blood. Clinical trials have begun to explore effects of small molecule interventions to perturb the latent viral infection, but only limited information is available regarding the impacts of HIV cure-related clinical interventions on viral reservoirs found in tissues. Of the 14 HIV cure-related clinical trials since 2012 that have evaluated the effects of small molecule interventions in vivo , four trials have examined the impacts of the interventions in peripheral blood as well as other tissues that harbor persistent HIV. The additional tissues examined include cerebral spinal fluid, intestines and lymph nodes. We provide a comparison contrast analyses of the data across anatomical compartments tested in these studies to reveal where peripheral blood analyses reflect outcomes in other tissues as well as where the data reveal differences between tissue outcomes. We also summarize the current knowledge on these topics and highlight key open questions that need to be addressed experimentally to move the HIV cure research field closer to the development of an intervention strategy capable of eliciting long-term antiretroviral free remission of HIV disease. [ABSTRACT FROM AUTHOR] more...

Defective interfering (DI) RNAs have been detected in several human viruses. HCV in-frame deletions mutants (IFDMs), missing mainly the envelope proteins, have been found in patient sera and liver tissues. IFDMs replicate independently and can be trans-packaged into infectious virions in the presence of full length viral genome. So far, their biological role is unclear. In this study, we have isolated and cloned IFDMs from sera samples and liver tissues of patients infected with HCV genotypes 1b, 2a, and 3a. IFDMs were present in up to 26% of samples tested. Using the in vitro HCV cell culture system, co-expression of the wild type (wt) HCV replicon with HCV IFDMs RNA resulted in increased HCV replication. Additionally, co-transfection of the HCV full length genome RNA and a defective mutant missing the envelope region led to increased viral release, collectively suggesting an important biological role for IFDMs in the virus life cycle. Recently, exosomes, masters of intercellular communication, have been implicated in the transport of HCV viral genomes. We report for the first time that exosomal RNA isolated from HCV sera samples contains HCV defective genomes. We also demonstrate that inhibition of exosomal biogenesis and release influences HCV viral replication. Overall, we provide evidence that the presence of HCV IFDMs affects both viral replication and release. IFDMs exploit exosomes as means of transport, a way to evade the immune system, to spread more efficiently and possibly maintain persistent infection. more...

Kaposi’s sarcoma-associated herpesvirus (KSHV) has an etiologic role in Kaposi’s sarcoma, primary effusion lymphoma and multicentric Castleman’s disease. These diseases are most common in immunocompromised individuals, especially those with AIDS. Similar to all herpesviruses, KSHV infection is lifelong. KSHV infection in tumor cells is primarily latent, with only a small subset of cells undergoing lytic infection. During latency, the KSHV genome persists as a multiple copy, extrachromosomal episome in the nucleus. In order to persist in proliferating tumor cells, the viral genome replicates once per cell cycle and then segregates to daughter cell nuclei. KSHV only expresses several genes during latent infection. Prominent among these genes, is the latency-associated nuclear antigen (LANA). LANA is responsible for KSHV genome persistence and also exerts transcriptional regulatory effects. LANA mediates KSHV DNA replication and in addition, is responsible for segregation of replicated genomes to daughter nuclei. LANA serves as a molecular tether, bridging the viral genome to mitotic chromosomes to ensure that KSHV DNA reaches progeny nuclei. N-terminal LANA attaches to mitotic chromosomes by binding histones H2A/H2B at the surface of the nucleosome. C-terminal LANA binds specific KSHV DNA sequence and also has a role in chromosome attachment. In addition to the essential roles of N- and C-terminal LANA in genome persistence, internal LANA sequence is also critical for efficient episome maintenance. LANA’s role as an essential mediator of virus persistence makes it an attractive target for inhibition in order to prevent or treat KSHV infection and disease. more...

Kaposi's sarcoma-associated herpesvirus (KSHV) has an etiologic role in Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease. These diseases are most common in immunocompromised individuals, especially those with AIDS. Similar to all herpesviruses, KSHV infection is lifelong. KSHV infection in tumor cells is primarily latent, with only a small subset of cells undergoing lytic infection. During latency, the KSHV genome persists as a multiple copy, extrachromosomal episome in the nucleus. In order to persist in proliferating tumor cells, the viral genome replicates once per cell cycle and then segregates to daughter cell nuclei. KSHV only expresses several genes during latent infection. Prominent among these genes, is the latency-associated nuclear antigen (LANA). LANA is responsible for KSHV genome persistence and also exerts transcriptional regulatory effects. LANA mediates KSHV DNA replication and in addition, is responsible for segregation of replicated genomes to daughter nuclei. LANA serves as a molecular tether, bridging the viral genome to mitotic chromosomes to ensure that KSHV DNA reaches progeny nuclei. N-terminal LANA attaches to mitotic chromosomes by binding histones H2A/H2B at the surface of the nucleosome. C-terminal LANA binds specific KSHV DNA sequence and also has a role in chromosome attachment. In addition to the essential roles of N- and C-terminal LANA in genome persistence, internal LANA sequence is also critical for efficient episome maintenance. LANA's role as an essential mediator of virus persistence makes it an attractive target for inhibition in order to prevent or treat KSHV infection and disease. [ABSTRACT FROM AUTHOR] more...

Having co-evolved with humans, herpesviruses have adapted to exploit the host molecular machinery to ensure viral persistence. The cellular protein Signal Transducer and Activator of Transcription 3 (STAT3) is a leading example. STAT3 is a prominent transcription factor that functions in a variety of physiologic processes including embryonic development, inflammation, immunity, and wound healing. Generally activated via growth factor and cytokine signaling, STAT3 can transcriptionally drive oncoproteins, pro-survival and pro-proliferative proteins as well as angiogenic factors, thereby contributing to cancer. As in most non-viral cancers, STAT3 is constitutively active in EBV-related B and epithelial cell cancers and in animal models of KSHV-cancers. Again, similar to non-viral cancers, STAT3 contributes to gammaherpesvirus (EBV and KSHV)-mediated cancers by driving cell proliferation, invasion and angiogenesis. Being herpesviruses, EBV and KSHV establish latency in humans with episodic lytic activation. Importantly, both viruses activate STAT3 almost immediately upon infection of primary cells. In the setting of infection of primary B cells by EBV, this rapidly activated STAT3 plays a key role in suppressing the DNA damage response (DDR) to EBV-oncogene triggered replication stress, thereby facilitating B cell proliferation and ultimately establishment of latency. STAT3 also contributes to maintenance of latency by curbing lytic activation of EBV and KSHV in latent cells that express high levels of STAT3. In this way, gammaherpesviruses exploit STAT3 to overcome cellular anti-proliferative and anti-lytic barriers to promote viral persistence. These investigations into gammaherpesviruses and STAT3 have simultaneously revealed a novel function for STAT3 in suppression of the DDR, a process fundamental to physiologic cell proliferation as well as development of cancer. [ABSTRACT FROM AUTHOR] more...

Having co-evolved with humans, herpesviruses have adapted to exploit the host molecular machinery to ensure viral persistence. The cellular protein Signal Transducer and Activator of Transcription 3 (STAT3) is a leading example. STAT3 is a prominent transcription factor that functions in a variety of physiologic processes including embryonic development, inflammation, immunity, and wound healing. Generally activated via growth factor and cytokine signaling, STAT3 can transcriptionally drive oncoproteins, pro-survival and pro-proliferative proteins as well as angiogenic factors, thereby contributing to cancer. As in most non-viral cancers, STAT3 is constitutively active in EBV-related B and epithelial cell cancers and in animal models of KSHV-cancers. Again, similar to non-viral cancers, STAT3 contributes to gammaherpesvirus (EBV and KSHV)-mediated cancers by driving cell proliferation, invasion and angiogenesis. Being herpesviruses, EBV and KSHV establish latency in humans with episodic lytic activation. Importantly, both viruses activate STAT3 almost immediately upon infection of primary cells. In the setting of infection of primary B cells by EBV, this rapidly activated STAT3 plays a key role in suppressing the DNA damage response (DDR) to EBV-oncogene triggered replication stress, thereby facilitating B cell proliferation and ultimately establishment of latency. STAT3 also contributes to maintenance of latency by curbing lytic activation of EBV and KSHV in latent cells that express high levels of STAT3. In this way, gammaherpesviruses exploit STAT3 to overcome cellular anti-proliferative and anti-lytic barriers to promote viral persistence. These investigations into gammaherpesviruses and STAT3 have simultaneously revealed a novel function for STAT3 in suppression of the DDR, a process fundamental to physiologic cell proliferation as well as development of cancer. more...