Orlando Acosta

Amino acid domains 280-297 of VP6 and 531-554 of VP4 are implicated in heat shock cognate protein hsc70-mediated rotavirus infection.

SummaryThe rotavirus infection mechanism seems to be a multi-step process which is still not fully understood. The heat shock cognate protein hsc70 has been proposed as being a co-receptor molecule for rotavirus entry into susceptible cells. In this work, an attempt was made to determine the existence of possible domains for VP4 and VP6 binding to hsc70. We selected amino acid sequences 531–554 from VP4 and 280–297 from VP6 on the basis of already recognized sequences for binding to hsc70. This study determined that DLPs and synthetic peptides from VP6 (aa 280–297) and VP4 (aa 531–554), individually or in combination, inhibited rotavirus RRV, YM and WA entry into MA104 and Caco-2 cells in an additive and dose-dependent manner. Hyperimmune sera against these synthetic peptides blocked infection by infectious TLPs. Capture ELISA results showed that DLPs interact with hsc70, probably through VP6 as the specific interaction between hcs70 and DLPs was disrupted by a VP6 peptide. These results suggest that VP6 takes part during rotavirus cell entry by binding to hsc70. This, as well as previous work, provides insight concerning the function of hsc70 within a multi-step model of rotavirus entry.

Mouse intestinal villi as a model system for studies of rotavirus infection

Rotavirus replicates in the mature enterocytes lining the villi of the small intestine and the availability of an in vitro system for culturing these natural target cells would contribute to substantial advances in the understanding of the pathogenesis of rotavirus. A novel in vitro system was established for culturing isolated small intestinal villi from suckling mice, and the susceptibility of the villus cells to the wild-type murine rotavirus EDIM-Cambridge (ECwt) infection was assessed by immunocytochemistry staining and ELISA. Cell viability of cultured villi infected by rotavirus was estimated to be higher than 70% 16 h post-infection, whereas the accumulated rotavirus structural and non-structural antigen was found to reach a maximum value at 24 h post-infection. Terminal apoptosis was found in about 65% of villus cells 22 h post-infection as detected with either propidium iodide or Hoechst 33342 staining. Mock-infected villus cells exhibited a slight tendency toward more extensive chromatin fragmentation compared to their rotavirus-infected counterpart, mainly when caspase-3 activity was measured. Examination of villus cells by ELISA indicated that the amount of rotavirus structural antigen accumulated at 12 h post-infection was nearly the same regardless of the intestinal section (duodenum, jejunum and ileum) used. The isolation, culture and infection of small intestinal villi from suckling mice has led to the introduction of a useful model for rotavirus studies.

Nerve growth factor and Neurotrophin-3 modulate the rabies infection of adult sensory neurons in primary cultures

With the aim of determining if the proportion of rabies virus (RV)-infected adult neurons from dorsal root ganglion are affected by in vitro treatment with different neurotrophins, experiments using Nerve Growth Factor (NGF), Brain Derived Neurotrophic Factor (BDNF) or Neurotrophin-3 (NT-3) as supplements for cells in culture were performed. Cultures treated with three different concentrations of each of the neurotrophins mentioned were infected with Challenge Virus Standard RV strain. An indirect immunoperoxidase technique was performed for the detection and counting of infected cells. NGF (2 ngml(-1) and 10 ngml(-1)) and NT-3 (1 ngml(-1) and 5 ngml(-1)) induced a significant reduction of infected neurons. None of the cultures treated with BDNF showed changes in the percentage of infected neurons. Likewise, the proportion of infected non-neuronal cells (Schwann cells and fibroblasts) was not altered by the treatment with neurotrophins. In addition, morphometric analysis of total and virus-immunoreactive neurons in culture were carried out, the neurotrophin treatment induced variations in the profile of neurons preferentially infected, since cell diameters in the infected cell population are different in the presence of NGF and NT-3. Data presented here could indicate a putative participation of neurotrophin receptor or biochemical modifications induced by neurotrophin treatment that affect the infection. The primary culture of dorsal root ganglion cells from adult mice is a very useful model for studying the basic phenomena of the RV-neuron interaction.

Inhibiting rotavirus infection by membrane-impermeant thiol/disulfide exchange blockers and antibodies against protein disulfide isomerase.

Objectives: Determining the effect of membrane-impermeant thiol/disulfide exchange inhibitors on rhesus rotavirus infectivity in MA104 cells and investigating protein disulfide isomerase (PDI) as a potential target for these inhibitors. Methods: Cells were treated with DTNB [5,5-dithio-bis-(2-nitrobenzoic acid)], bacitracin or anti-PDI antibodies and then infected with virus. Triple-layered particles (TLPs) were also pretreated with inhibitors before inoculation. The effects of these inhibitors on α-sarcin co-entry, virus binding to cells and PDI-TLP interaction were also examined. FACS analysis, cell-surface protein biotin-labeling, lipid-raft isolation and ELISA were performed to determine cell-surface PDI expression. Results: Infectivity became reduced by 50% when cells or TLPs were treated with 1 or 6 mM DTNB, respectively; infectivity became reduced by 50% by 20 mM bacitracin treatment of cells whereas TLPs were insensitive to bacitracin treatment; anti-PDI antibodies decreased viral infectivity by about 45%. The presence of DTNB (2.5 mM) or bacitracin (20 mM) was unable to prevent virus binding to cells and rotavirus-induced α-sarcin co-entry. Conclusions: It was concluded that thiol/disulfide exchange was involved in rotavirus entry process and that cell-surface PDI was at least a potential target for DTNB and bacitracin-induced infectivity inhibition.

Inflammatory and oxidative stress in rotavirus infection

Rotaviruses are the single leading cause of life-threatening diarrhea affecting children under 5 years of age. Rotavirus entry into the host cell seems to occur by sequential interactions between virion proteins and various cell surface molecules. The entry mechanisms seem to involve the contribution of cellular molecules having binding, chaperoning and oxido-reducing activities. It appears to be that the receptor usage and tropism of rotaviruses is determined by the species, cell line and rotavirus strain. Rotaviruses have evolved functions which can antagonize the host innate immune response, whereas are able to induce endoplasmic reticulum (ER) stress, oxidative stress and inflammatory signaling. A networking between ER stress, inflammation and oxidative stress is suggested, in which release of calcium from the ER increases the generation of mitochondrial reactive oxygen species (ROS) leading to toxic accumulation of ROS within ER and mitochondria. Sustained ER stress potentially stimulates inflammatory response through unfolded protein response pathways. However, the detailed characterization of the molecular mechanisms underpinning these rotavirus-induced stressful conditions is still lacking. The signaling events triggered by host recognition of virus-associated molecular patterns offers an opportunity for the development of novel therapeutic strategies aimed at interfering with rotavirus infection. The use of N-acetylcysteine, non-steroidal anti-inflammatory drugs and PPARγ agonists to inhibit rotavirus infection opens a new way for treating the rotavirus-induced diarrhea and complementing vaccines.

Inhibition of rotavirus infection in cultured cells by N-acetyl-cysteine, PPARγ agonists and NSAIDs.

Although the current rotavirus vaccines have shown good tolerance and significant efficacy, it would be useful to develop alternative or complementary strategies aimed at preventing or treating acute diarrhoeal disease caused by this viral agent. A variety of antiviral strategies other than vaccines have been assayed for rotavirus infection management. The recently demonstrated sensitivity of rotavirus infectivity to thiol/disulfide reagents prompted assays for screening drugs that potentially affect cellular redox reactions. MA104 or Caco-2 cells were inoculated with the rotavirus strains RRV, Wa, Wi or M69 and then incubated with different concentrations of drugs belonging to a selected group of 60 drugs that are currently used in humans for purposes other than rotavirus infection treatment. Eighteen of these drugs were able to inhibit rotavirus infectivity to different extents. A more systematic evaluation was performed with drugs that could be used in children such as N-acetylcysteine and ascorbic acid, in addition to ibuprofen, pioglitazone and rosiglitazone, all of which affecting cellular pathways potentially needed by the rotavirus infection process. Evidence is provided here that rotavirus infectivity is significantly inhibited by NAC in different cell-culture systems. These findings suggest that NAC has the potential to be used as a therapeutic tool for treatment and prevention of rotavirus disease in children.

Studying neurotrophin antiviral effect on rabies-infected dorsal root ganglia cultures

Neurotrophin (NT)-induced modulation of rabies virus adsorption, transcription, and replication were analyzed in adult mouse dorsal root ganglia cultures. Different types of nerve growth factor and NT-3 treatment were tested before infection (pretreatment), during infection (transtreatment) and after withdrawing the viral inoculum (post-treatment). NT pretreatment for 4 days prior to infection produced a significant increase in the quantity of virus adsorbed into cultures and a concomitant increase in genomic viral RNA as measured by real time polymerase chain reaction (PCR). NT pretreatment triggered increased expression of two rabies virus receptors (NCAM and p75NTR); however, no increase in rabies virus transcription and expression could be observed. By contrast, NT treatment during and after infection (trans- and post-treatment) induced a strong decrease in the quantity of viral nucleoprotein genomic and messenger nucleoprotein RNAs. These findings suggested that NT had an intrinsic inhibitory effect on rabies virus infection, which was not counterbalanced by NTs’ rabies virus receptor—enhancing property and viral uptake. Adult mouse dorsal root ganglion cultures can be regarded as being a useful model for detecting therapeutic targets and evaluating experimental antiviral drugs.

N‐Acetylcysteine Treatment of Rotavirus‐Associated Diarrhea in Children

Rotaviruses are the leading cause of severe, acute, and dehydrating diarrhea affecting children under 5 years of age worldwide. Despite an important reduction in rotavirus‐caused deaths as a consequence of the rotavirus vaccine, alternative or complementary strategies for preventing or treating rotavirus‐associated diarrhea are needed mainly in the poorest countries. We describe the cases of four rotavirus‐unvaccinated 12–13‐month‐old girls and a 5‐year‐old boy who developed rotavirus‐associated diarrhea confirmed by enzyme‐linked immunosorbent assay, Western blotting, and immunochemistry analyses. After the first day of diarrheal episodes, three of the five patients were immediately administered oral N‐acetylcysteine (NAC) 60 mg/kg daily, divided into three equal doses every 8 hours. The other two patients did not receive NAC and served as controls. Administration of NAC resulted in a decreased number of diarrheal episodes, excretion of fecal rotavirus antigen, and resolution of symptoms after 2 days of treatment. Our results suggest that NAC treatment after the first diarrheal episode could be an efficient strategy for treating rotavirus‐affected children and preventing the associated severe life‐threatening accompanying dehydration.

CPm gene diversity in field isolates of Citrus tristeza virus from Colombia

The nucleotide sequence diversity of the CPm gene from 28 field isolates of Citrus tristeza virus (CTV) was assessed by SSCP and sequence analyses. These isolates showed two major shared haplotypes, which differed in distribution: A1 was the major haplotype in 23 isolates from different geographic regions, whereas R1 was found in isolates from a discrete region. Phylogenetic reconstruction clustered A1 within an independent group, while R1 was grouped with mild isolates T30 from Florida and T385 from Spain. Some isolates contained several minor haplotypes, which were very similar to, and associated with, the major haplotype.

Unusual Electrophoretic Properties of the Coat Protein of Raspberry Ringspot Nepovirus

Coat protein of raspberry ringspot nepovirus (RRV) migrated during electrophoresis in a discontinuous buffer system as two components which were capable of interchanging during this procedure. However, only one component was formed during electrophoresis in a continuous buffer system. Treatment of the protein before electrophoresis with iodoacetamide, which alkylates sulfhydryl groups, prevented the formation of the faster migrating electrophoretic component. Thus the faster migrating component is probably an artifact caused by S-S bond formation. Although the coat proteins of two other nepoviruses did not behave in this way, the results obtained with RRV emphasize the need for caution in interpreting electrophoretic heterogeneity in virus proteins. The estimated molecular weight of RRV coat protein was 57,000.