Liver immunopathogenesis in fatal cases of dengue in children: detection of viral antigen, cytokine profile and inflammatory mediators.

Introduction: Dengue virus (DENV), the etiologic agent of dengue fever illness, represents a global public health concern, mainly in tropical and subtropical areas across the globe. It is well known that this acute viral disease can progress to severe hemorrhagic stages in some individuals, however, the immunopathogenic basis of the development of more severe forms by these patients is yet to be fully understood. Objective: In this context, we investigated and characterized the histopathological features as well as the cytokine profile and cell subpopulations present in liver tissues from three fatal cases of DENV in children. Methods: Hematoxylin and Eosin, Periodic Acid Schiff and Picro Sirius Red staining were utilized for the histopathological analysis. Immunohistochemistry assay was performed to characterize the inflammatory response and cell expression patterns. Results: Vascular dysfunctions such as hemorrhage, vascular congestion and edema associated with a mononuclear infiltrate were observedin all three cases. Liver tissues exhibited increased presence of CD68+ and TCD8+ cells as well as high expression of MMP-9, TNF-a, RANTES, VEGFR-2 mediators. Viral replication was confirmed by the detection of NS3 protein. Conclusion: Taken together, these results evidenced key factors that may be involved in the development of severe alterations in liver tissues of children in response to DENV infection.

SARS-CoV-2: preliminary study of infected human nasopharyngeal tissue by high resolution microscopy.

BACKGROUND: The novel coronavirus SARS-CoV-2 is the etiological agent of COVID-19. This virus has become one of the most dangerous in recent times with a very high rate of transmission. At present, several publications show the typical crown-shape of the novel coronavirus grown in cell cultures. However, an integral ultramicroscopy study done directly from clinical specimens has not been published. METHODS: Nasopharyngeal swabs were collected from 12 Cuban individuals, six asymptomatic and RT-PCR negative (negative control) and six others from a COVID-19 symptomatic and RT-PCR positive for SARS CoV-2. Samples were treated with an aldehyde solution and processed by scanning electron microscopy (SEM), confocal microscopy (CM) and, atomic force microscopy. Improvement and segmentation of coronavirus images were performed by a novel mathematical image enhancement algorithm. RESULTS: The images of the negative control sample showed the characteristic healthy microvilli morphology at the apical region of the nasal epithelial cells. As expected, they do not display virus-like structures. The images of the positive sample showed characteristic coronavirus-like particles and evident destruction of microvilli. In some regions, virions budding through the cell membrane were observed. Microvilli destruction could explain the anosmia reported by some patients. Virus-particles emerging from the cell-surface with a variable size ranging from 80 to 400 nm were observed by SEM. Viral antigen was identified in the apical cells zone by CM. CONCLUSIONS: The integral microscopy study showed that SARS-CoV-2 has a similar image to SARS-CoV. The application of several high-resolution microscopy techniques to nasopharyngeal samples awaits future use.

Transient Expression in Cytoplasm and Apoplast of Rotavirus VP6 Protein Fused to Anti-DEC205 Antibody in Nicotiana benthamiana and Nicotiana sylvestris.

Rotavirus is the most common cause of severe diarrhea in infants and children worldwide and is responsible for about 215,000 deaths annually. Over 85% of these deaths originate in low-income/developing countries in Asia and Africa. Therefore, it is necessary to explore the development of vaccines that avoid the use of "living" viruses and furthermore, vaccines that have viral antigens capable of generating powerful heterotypic responses. Our strategy is based on the expression of the fusion of the anti-DEC205 single-chain variable fragment (scFv) coupled by an OLLAS tag to a viral protein (VP6) of Rotavirus in Nicotiana plants. It was possible to express transiently in N. benthamiana and N. sylvestris a recombinant protein consisting of the single chain variable fragment linked by an OLLAS tag to the VP6 protein. The presence of the recombinant protein, which had a molecular weight of approximately 75 kDa, was confirmed by immunodetection, in both plant species and in both cellular compartments (cytoplasm and apoplast) where it was expressed. In addition, the recombinant protein was modeled, and it was observed that some epitopes of interest are exposed on the surface, which could favor their immunogenic response.

A novel method for the in vitro assembly of virus-like particles and multimeric proteins.

OBJECTIVE: To develop a method for the efficient assembly of viral or multimeric proteins into virus-like particles (VLP) or other macro structures. RESULTS: Protein monomers were assembled by eliminating calcium ions through precipitation. The model protein, rotavirus VP6, assembled into stable, long nanotubes with better quality than the assemblies obtained directly from cell culture. Nanotube length was directly proportional to the initial concentration of VP6 monomers, in accordance with the classic nucleation theory of capsid assembly. The quality of the obtained assemblies was confirmed when the nanotubes were functionalized with metals, yielding unique nanobiomaterials. Assembly efficiency was improved in comparison with other previously proposed methods. CONCLUSIONS: The novel method presented here is simpler and faster than other reported methods for the assembly and disassembly of viral proteins, a step needed for most applications.

Distinctive EBV infection characteristics in children from a developing country.

BACKGROUND: In developing countries, Epstein-Barr virus (EBV) infection is mostly asymptomatic in early childhood. EBV persistence may lead to different malignancies, such as B cell derived lymphomas. In Argentina, most children are seropositive at three years and an increased association between EBV and lymphoma was proved in children under 10 years old by our group. OBJECTIVE: Our aim was to characterize EBV infection at the site of entry and reactivation of viral infection -the tonsils- in order to better understand the mechanism of viral persistence in pediatric patients. METHODS: A cohort of 54 patients was described. We assessed specific antibodies profiles in sera; viral proteins presence by IHC on FFPE samples and EBV type from fresh tissue. RESULTS: EBV type 1 was prevalent, mostly in the youngest patients. Asymptomatic primary infected patients presented higher viral loads and Latency 0/I or II patterns, whereas the Latency III pattern was observed mostly in healthy carriers. There were no differences between groups in the expression of viral lytic antigens. This study discloses new features in patients undergoing primary infection from a developing population. Low viral inoculum and restricted viral antigen expression may be responsible for the lack of symptoms in children from our country.

Ursolic acid: A novel antiviral compound inhibiting rotavirus infection in vitro.

Rotavirus is one of the leading causes of severe acute gastroenteritis in children under 5 years of age, mainly affecting developing countries. Once the disease is acquired, no specific treatment is available; as such, the development of new drugs for effective antirotaviral treatment is critical. Ursolic acid is a pentacyclic triterpenoid with antiviral activity, which has been studied extensively in vitro and in vivo. To study the potential antirotaviral activity of ursolic acid, its toxic potential for viral particles (virucidal effect) and cultured cells (cytotoxicity) was analysed. No effect on virion infectivity was observed with treatments of up to 40 µM ursolic acid, while incipient cytotoxicity started to be evident with 20 µM ursolic acid. The antiviral potential of ursolic acid was evaluated in in-vitro rotavirus infections, demonstrating that 10 µM ursolic acid inhibits rotavirus replication (observed by a decrease in viral titre and the level of the main viral proteins) and affects viral particle maturation (a process associated with the endoplasmic reticulum) 15 h post infection. Interestingly, ursolic acid was also found to hamper the early stages of the viral replication cycle, as a significant reduction in the number and size of viroplasms, consistent with a decrease in VP6 and NSP2 viral proteins, was observed 4 h post infection. As such, these observations demonstrate that ursolic acid exhibits antiviral activity, suggesting that this chemical could be used as a new treatment for rotavirus.

Diagnosing Zika virus infection against a background of other flaviviruses: Studies in high resolution serological analysis.

Zika virus (ZIKV) is a mosquito-borne flavivirus. Homologous proteins of different flaviviruses display high degrees of sequence identity, especially within subgroups. This leads to extensive immunological cross-reactivity and corresponding problems for developing a ZIKV-specific serological assay. In this study, peptide microarrays were employed to identify individual ZIKV antibody targets with promise in differential diagnosis. A total of 1643 overlapping oligopeptides were synthesized and printed onto glass slides. Together, they encompass the full amino acid sequences of ZIKV proteomes of African, Brazilian, USA, and French Polynesian origins. The resulting ZIKV scanning microarray chips were used to screen three pools of sera from recent Zika outbreaks in Senegal and Cape Verde, in Brazil, and from overseas travelers returning to the EU. Together with a mixed pool of well characterized, archived sera of patients suffering from infections by dengue, yellow fever, tick-borne encephalitis, and West Nile viruses, a total of 42 sera went into the study. Sixty-eight antibody target regions were identified. Most of which were hitherto unknown. Alignments and sequence comparisons revealed 13 of which could be classified as bona fide ZIKV-specific. These identified antibody target regions constitute a founding set of analytical tools for serological discrimination of ZIKV from other flaviviruses.

Rotavirus VP6 protein mucosally delivered by cell wall-derived particles from Lactococcus lactis induces protection against infection in a murine model.

Rotaviruses are the primary cause of acute gastroenteritis in children worldwide. Although the implementation of live attenuated vaccines has reduced the number of rotavirus-associated deaths, variance in their effectiveness has been reported in different countries. This fact, among other concerns, leads to continuous efforts for the development of new generation of vaccines against rotavirus.In this work, we describe the obtention of cell wall-derived particles from a recombinant Lactococcus lactis expressing a cell wall-anchored version of the rotavirus VP6 protein. After confirming by SDS-PAGE, Western blot, flow cytometry and electronic immunomicroscopy that these particles were carrying the VP6 protein, their immunogenic potential was evaluated in adult BALB/c mice. For that, mucosal immunizations (oral or intranasal), with or without the dmLT [(double mutant Escherichia coli heat labile toxin LT(R192G/L211A)] adjuvant were performed. The results showed that these cell wall-derived particles were able to generate anti-rotavirus IgG and IgA antibodies only when administered intranasally, whether the adjuvant was present or not. However, the presence of dmLT was necessary to confer protection against rotavirus infection, which was evidenced by a 79.5 percent viral shedding reduction.In summary, this work describes the production of cell wall-derived particles which were able to induce a protective immune response after intranasal immunization. Further studies are needed to characterize the immune response elicited by these particles as well as to determine their potential as an alternative to the use of live L. lactis for mucosal antigen delivery.

Detection of the antiviral activity of epicatechin isolated from Salacia crassifolia (Celastraceae) against Mayaro virus based on protein C homology modelling and virtual screening.

Mayaro fever, caused by Mayaro virus (MAYV) is a sub-lethal disease with symptoms that are easily confused with those of dengue fever, except for polyarthralgia, which may culminate in physical incapacitation. Recently, outbreaks of MAYV have been documented in metropolitan areas, and to date, there is no therapy or vaccine available. Moreover, there is no information regarding the three-dimensional structure of the viral proteins of MAYV, which is important in the search for antivirals. In this work, we constructed a three-dimensional model of protein C of MAYV by homology modelling, and this was employed in a manner similar to that of receptors in virtual screening studies to evaluate 590 molecules as prospective antiviral agents. In vitro bioassays were utilized to confirm the potential antiviral activity of the flavonoid epicatechin isolated from Salacia crassifolia (Celastraceae). The virtual screening showed that six flavonoids were promising ligands for protein C. The bioassays showed potent antiviral action of epicatechin, which protected the cells from almost all of the effects of viral infection. An effective concentration (EC50) of 0.247 µmol/mL was observed with a selectivity index (SI) of 7. The cytotoxicity assay showed that epicatechin has low toxicity, with a 50% cytotoxic concentration (CC50) greater than 1.723 µmol/mL. Epicatechin was found to be twice as potent as the reference antiviral ribavirin. Furthermore, a replication kinetics assay showed a strong inhibitory effect of epicatechin on MAYV growth, with a reduction of at least four logs in virus production. Our results indicate that epicatechin is a promising candidate for further testing as an antiviral agent against Mayaro virus and other alphaviruses.

Zoonotic infection of Brazilian primate workers with New World simian foamy virus.

Simian foamy viruses (SFVs) are retroviruses present in nearly all nonhuman primates (NHPs), including Old World primates (OWP) and New World primates (NWP). While all confirmed human infections with SFV are from zoonotic transmissions originating from OWP, little is known about the zoonotic transmission potential of NWP SFV. We conducted a longitudinal, prospective study of 56 workers occupationally exposed to NWP in Brazil. Plasma from these workers was tested using Western blot (WB) assays containing NWP SFV antigens. Genomic DNA from blood and buccal swabs was analyzed for the presence of proviral SFV sequences by three nested PCR tests and a new quantitative PCR assay. Exposure histories were obtained and analyzed for associations with possible SFV infection. Ten persons (18%) tested seropositive and two persons were seroindeterminate (3.6%) for NWP SFV. Six persons had seroreactivity over 2-3 years suggestive of persistent infection. All SFV NWP WB-positive workers reported at least one incident involving NWP, including six reporting NWP bites. NWP SFV viral DNA was not detected in the blood or buccal swabs from all 12 NWP SFV seroreactive workers. We also found evidence of SFV seroreversion in three workers suggestive of possible clearance of infection. Our findings suggest that NWP SFV can be transmitted to occupationally-exposed humans and can elicit specific humoral immune responses but infection remains well-controlled resulting in latent infection and may occasionally clear.

Herpes Simplex Virus Type 1 Neuronal Infection Perturbs Golgi Apparatus Integrity through Activation of Src Tyrosine Kinase and Dyn-2 GTPase.

Herpes simplex virus type 1 (HSV-1) is a ubiquitous pathogen that establishes a latent persistent neuronal infection in humans. The pathogenic effects of repeated viral reactivation in infected neurons are still unknown. Several studies have reported that during HSV-1 epithelial infection, the virus could modulate diverse cell signaling pathways remodeling the Golgi apparatus (GA) membranes, but the molecular mechanisms implicated, and the functional consequences to neurons is currently unknown. Here we report that infection of primary neuronal cultures with HSV-1 triggers Src tyrosine kinase activation and subsequent phosphorylation of Dynamin 2 GTPase, two players with a role in GA integrity maintenance. Immunofluorescence analyses showed that HSV-1 productive neuronal infection caused a scattered and fragmented distribution of the GA through the cytoplasm, contrasting with the uniform perinuclear distribution pattern observed in control cells. In addition, transmission electron microscopy revealed swollen cisternae and disorganized stacks in HSV-1 infected neurons compared to control cells. Interestingly, PP2, a selective inhibitor for Src-family kinases markedly reduced these morphological alterations of the GA induced by HSV-1 infection strongly supporting the possible involvement of Src tyrosine kinase. Finally, we showed that HSV-1 tegument protein VP11/12 is necessary but not sufficient to induce Dyn2 phosphorylation. Altogether, these results show that HSV-1 neuronal infection triggers activation of Src tyrosine kinase, phosphorylation of Dynamin 2 GTPase, and perturbation of GA integrity. These findings suggest a possible neuropathogenic mechanism triggered by HSV-1 infection, which could involve dysfunction of the secretory system in neurons and central nervous system.

Detection of the VP6 gene of group F and G rotaviruses in broiler chicken fecal samples from the Amazon region of Brazil.

The aim of this study was to detect rotavirus F (RVF) and rotavirus G (RVG) in fecal specimens of broiler chickens in Brazil. During 2008 and 2011, a total of 85 fecal samples were collected. The viral genome was extracted, followed by polyacrylamide gel electrophoresis (PAGE), reverse transcription polymerase chain reaction (RT-PCR), and nucleotide sequencing. Samples were screened for rotaviruses by PAGE, and RVF and RVG genome banding patterns were not seen. Using RT-PCR, it was found that 9.4 % (8/85) of the pools contained RVF, whereas 10.6 % (9/85) contained RVG. The predicted amino acid sequences of RVF and RVG from Brazilian samples were 94.4-95.7 % and 96.8-96.9 % identical, respectively, to those of prototypes from Germany. The detection of RVF and RVG in this study provides important epidemiological data about the simultaneous circulation of rotaviruses affecting broiler flocks in the Amazon region of Brazil.

Seroprevalence in Household Raised Pigs Indicate High Exposure to GII Noroviruses in Rural Nicaragua.

Information about porcine norovirus (PoNoV), genetically similar to human NoV (HuNoV), is limited from rural areas where household-raised pigs are heavily exposed to faecal material which could facilitate transmission. Histo-blood group antigens (HBGAs) are known susceptibility factors to NoV in humans and in a germfree piglet model but their role in susceptibility in the porcine population remains unknown. This study reports: (i) the seroprevalence and antibody titres to human norovirus (NoV) VLPs in household raised pigs; (ii) the distribution of HBGAs in relation to NoV IgG antibody titres and further characterization by blocking of GII.4 VLP binding to pig gastric mucins (PGM). The majority of pigs were seropositive to all three VLPs tested (58-70%) with seropositivity and cross-reactivity increasing significantly with age. However, pig sera could not block the binding of NoV GII.4 VLPs (Dijon) to PGM suggesting no previous infection with this genotype. The majority of the pigs were H-positive (84%), a susceptibility factor for human infections. IgG antibody titres were however higher in H-negative (GMT = 247) as compared with H-positive (GMT = 57) pigs, but after age stratification, this difference in antibody titres was only observed in pigs ≤1 month of age. In conclusion, serological data show that the porcine population of Nicaragua is highly exposed to NoV infections, and the association to HBGAs warrants further investigation.

Activation-dependent mitochondrial translocation of Foxp3 in human hepatocytes.

Foxp3 is considered to be the master regulator for the development and function of regulatory T cells (Treg). Recently Foxp3, has been detected in extra lymphoid tissue, and in hepatocytes and has been associated with hepatocellular carcinoma (HCC), although its role has not been defined. Since it is expected that there is a relationship between protein localization, activity and cellular function, the aim of this study was to explore the subcellular localization of Foxp3 in resting and stimulated human hepatocytes. Foxp3 expression was measured by flow cytometry, subcellular fractioning, and immunofluorescence, and this data was used to track the shuttling of Foxp3 in different subcellular compartments in hepatocytes (HepG2 cell line), stimulated by using the PKC activators (PMA), core and preS1/2 antigen from hepatitis B virus (HBV). Our data shows that besides the nuclear location, mitochondrial translocation was detected after stimulation with PMA and at to a lesser extent, with preS1/2. In addition, Foxp3 is localizes at outer mitochondrial membrane. These results suggest a non-canonical role of Foxp3 in the mitochondrial compartment in human hepatocytes, and opens a new field about their role in liver damages during HBV infection.

Human herpesvirus 8 establishes a latent infection in prostates of Tobago men resulting in increased macrophage infiltration.

BACKGROUND: The Caribbean island of Tobago, which is 97% African ancestry, has one of the highest rates of prostate cancer in the world. We have previously reported that human herpesvirus 8 (HHV-8) infection is significantly associated with prostate cancer in Tobago. In this study, we extend those results testing the hypothesis that HHV-8 seropositive Tobagonian men have a chronic HHV-8 infection in their prostates that is associated with increased inflammation. METHODS: Prostate sections were screened by immunohistochemistry for the expression of HHV-8 proteins K8.1 and LANA-1 and for presence of B cells (CD20) and macrophages (CD68). RESULTS: HHV-8 antigen expression representing lytic and latent infections was seen in 73.9% of prostates from HHV-8 seropositive subjects. Latent infections were seen predominantly in glandular epithelia whereas lytic gene expression was seen mainly in macrophages in prostate stroma. Macrophage infiltrates were significantly increased in sections expressing HHV-8 proteins. CONCLUSION: HHV-8 establishes a chronic latent infection in the prostate, which is associated with an increased macrophage infiltrate.

Genetic linkage of capsid protein-encoding RNA segments in group A equine rotaviruses.

Rotavirus virions are formed by three concentric protein layers that enclose the 11 dsRNA genome segments and the viral proteins VP1 and VP3. Interactions amongst the capsid proteins (VP2, VP6, VP7 and VP4) have been described to play a major role in viral fitness, whilst restricting the reassortment of the genomic segments during co-infection with different rotavirus strains. In this work we describe and characterize the linkage between VP6 and VP7 proteins based on structural and genomic analyses of group A rotavirus strains circulating in Argentinean horses. Strains with the VP7 genotype G3 showed a strong association with the VP6 genotype I6, whilst strains with G14 were associated with the I2 genotype. Most of the differences on the VP6 and VP7 proteins were observed in interactive regions between the two proteins, suggesting that VP6 : VP7 interactions may drive the co-evolution and co-segregation of their respective gene segments.

Genetic heterogeneity of the VP6 gene and predominance of G6P[5] genotypes of Brazilian porcine rotavirus C field strains.

The aim of this study was to investigate the genetic diversity of the VP6, VP7, and VP4 genes of 15 Brazilian wild-type porcine RVC strains identified in diarrheic fecal samples. The VP6 gene analysis demonstrated heterogeneity between the 15 RVC strains, which clustered in three distinct genotypes (I1, I5, and I6). In the VP7 and VP4 gene analysis, the genotype combination G6P[4] was detected in only one strain (UEL-77), while G6P[5] was the most commonly (n = 14) detected in RVC strains identified in the Brazilian pig herds evaluated, indicating its probable predominance in this country, mainly in 2014.

Equine arteritis virus gP5 protein induces apoptosis in cultured insect cells.

Equine Arteritis Virus (EAV) has been shown to induce apoptosis in vitro but the induction of this mechanism has not been previously associated with any viral gene product. In this work, we found a cytotoxicity effect of the EAV gP5 protein on baculovirus-insect cells and a low yield of protein recovery. Besides, different morphological features by electron transmission microscopy, DNA fragmentation in agarose gel, TUNEL analysis and caspase 3 activity were found. All these findings indicate that the EAV gP5 protein induces apoptosis in insect cells.

The equine arteritis virus isolate from the 2010 Argentinian outbreak.

A semen sample from a stallion infected during the 2010 equine arteritis virus (EAV) outbreak was received for viral isolation prior to castration of the animal. The virus was identified using a polyclonal antibody immunofluorescence test. Reverse-transcription polymerase chain reaction (RT-PCR) was used to amplify a region of the GP5 gene with primers GL105F and GL673R. The PCR products were purified and sequences of both strands were determined in a MegaBACE™1000 with inner primers CR2 and EAV32. A phylogenetic dataset was built with the previously reported sequences of five strains isolated in Argentina, together with a group of selected sequences obtained from GenBank. The unrooted neighbour-joining tree was constructed using molecular evolutionary genetic analysis (MEGA) and bootstrap analyses were conducted using 1,000 replicate datasets. Evolutionary distances were computed using the maximum composite likelihood method. A NetNGlyc server analysis at the Technical University of Denmark (www.cbs.dtu.dk/services/NetNGlyc/) was used to predict N-glycosylation in GP5 sequences. The phylogenetic analysis revealed that the new strain GLD-LP-ARG), together with other strains previously isolated, belongs to the European group EU-1 but in a different branch. The new strain shows 99% nucleotide identity with strain Al1and 98.1% with the Belgian strain 08P178. Persistently infected stallions and their cryopreserved semen constitute a reservoir of EAV, which ensures its persistence in the horse population around the world. These findings reinforce the importance of careful monitoring of persistently infected stallions, as well as semen straws, by RT-PCR or test mating, in accordance with national regulations.

Effect of Bovine Viral Diarrhea Virus on the ovarian functionality and in vitro reproductive performance of persistently infected heifers.

The aim of this study was to study the effect of Bovine Viral Diarrhea Virus on the reproductive female tract by means of analyzing the ovarian follicular population of persistently infected (PI) heifers, and evaluating the performance of oocytes procured form those heifers in in vitro fertilization procedures. Seven BVDV PI Aberdeen Angus and British crossbred heifers ranging from 18 to 36 months of age were spayed and their ovaries used for viral isolation, microscopic examination, and in vitro fertilization procedures. Bovine Viral Diarrhea Virus was detected from the follicular fluid and sera of all PI heifers. Microscopic examination of the ovaries from PI heifers showed a significant drop in the number of follicles cortical regions, compared with controls. A comparative analysis of the stages of follicular development showed a significant decrease in the number of primordial and tertiary follicles in the cortical regions of ovaries from PI heifers. Viral antigen was detected by immunohistochemistry, and was widely distributed throughout the ovarian tissues. There were differences in the rate of cleavage and embryo development between oocytes obtained from the ovaries of control animals and PI heifers. Furthermore, two developed embryos obtained from oocytes from one of the PI heifers were positive to BVDV, as well as two media from in vitro fertilization (IVF) procedures. The results of this study demonstrate that BVDV PI heifers exhibit alterations in follicular population through of the early interaction between the virus and germ cell line affecting directly the mechanisms involved in the ontogenesis of the ovary.