Detection of rotavirus and norovirus among children with acute gastroenteritis in Merida and Chihuahua cities, Mexico.

INTRODUCTION: Infantile acute gastroenteritis (AGE) is a leading cause of morbidity and mortality, particularly in developing countries. The most frequent etiological agents of viral gastroenteritis in children are adenovirus, astrovirus, rotavirus, and norovirus, the last two, leading causes. Thus, the aim of this study was to identify the presence of these two viruses in children with AGE, from two cities located in the Southeast and the Northwest regions of México. METHODOLOGY: HuNoVs were detected and characterized by RT-PCR and sequencing, while RVs were detected by RNA electrophoresis. RESULTS: The presence of RV and HuNoV was evaluated in 81 stool samples; 37 were collected between April and July 2013 from patients with acute diarrhea in Merida, and 44 were collected between January and June 2017 in Chihuahua, who attended health services. Despite vaccination, RV resulted in the predominant viruses detected, with 30.8% (25/81) positivity, while HuNoV infection was present in 8.6% (7/81) of the stool samples; GII strains were identified circulating in the Southeast, while GI strains were identified in the Northwest. Moreover, co-infections with both viruses were detected at a prevalence rate of 2.4% (2/81). CONCLUSIONS: The circulation of RV and HuNoV in the country is continuous and should be constantly monitored due to their impact on public health.

Survivin Overexpression Has a Negative Effect on Feline Calicivirus Infection.

It is known that levels of the anti-apoptotic protein survivin are reduced during Murine norovirus MNV-1 and Feline calicivirus (FCV) infection as part of the apoptosis establishment required for virus release and propagation in the host. Recently, our group has reported that overexpression of survivin causes a reduction of FCV protein synthesis and viral progeny production, suggesting that survivin may affect early steps of the replicative cycle. Using immunofluorescence assays, we observed that overexpression of survivin, resulted in the reduction of FCV infection not only in transfected but also in the neighboring nontransfected CrFK cells, thus suggesting autocrine and paracrine protective effects. Cells treated with the supernatants collected from CrFK cells overexpressing survivin showed a reduction in FCV but not MNV-1 protein production and viral yield, suggesting that FCV binding and/or entry were specifically altered. The reduced ability of FCV to bind to the surface of the cells overexpressing survivin, or treated with the supernatants collected from these cells, correlate with the reduction in the cell surface of the FCV receptor, the feline junctional adhesion molecule (fJAM) 1, while no effect was observed in the cells transfected with the pAm-Cyan vector or in cells treated with the corresponding supernatants. Moreover, the overexpression of survivin affects neither Vaccinia virus (VACV) production in CrFK cells nor MNV-1 virus production in RAW 267.4 cells, indicating that the effect is specific for FCV. All of these results taken together indicate that cells that overexpress survivin, or cell treatment with the conditioned medium from these cells, results in the reduction of the fJAM-1 molecule and, therefore, a specific reduction in FCV entry and infection.

The feline calicivirus leader of the capsid protein causes survivin and XIAP downregulation and apoptosis.

Calicivirus infection causes intrinsic apoptosis, leading to viral propagation in the host. During murine norovirus infection, a reduction in the anti-apoptotic protein survivin has been documented. Here we report that in feline calicivirus infection, a downregulation of the anti-apoptotic proteins survivin and XIAP occur, which correlates with the translocation of the pro-apoptotic protein Smac/DIABLO from the mitochondria to the cytoplasm and the activation of caspase-3. Inhibition of survivin degradation by lactacystin treatment caused a delay in apoptosis progression, reducing virus release, without affecting virus production. However, the overexpression of survivin caused a negative effect in viral progeny production. Overexpression of the leader of the capsid protein (LC), but not of the protease-polymerase NS6/7, results in the downregulation of survivin and XIAP, caspase activation and mitochondrial damage. These results indicate that LC is responsible for the induction of apoptosis in transfected cells and most probably in FCV infection.

Annexin A2 associates to feline calicivirus RNA in the replication complexes from infected cells and participates in an efficient viral replication.

Cellular proteins have been identified to participate in calicivirus replication in association with viral proteins and/or viral RNAs. By mass spectrometry from pull-down assays, we identified several cellular proteins bound to the feline calicivirus (FCV) genomic RNA; among them the lipid raft-associated scaffold protein Annexin (Anx) A2. AnxA2 colocalizes with FCV NS6/7 protein and with the dsRNA in infected cells; moreover, it was found associated with the viral RNA in the membrane fraction corresponding to the replication complexes (RCs), suggesting its role during FCV replication. AnxA2-knockdown from CrFK cells prior to infection with FCV caused a delay in the cytopathic effect, a strong reduction of viral non-structural proteins and dsRNA production, and a decrease of FCV yield in both cell-associated and supernatant fractions. Taken together, these results indicate that AnxA2 associates to the genomic RNA of FCV and is required for an efficient FCV replication.

Negative effect of heat shock on feline calicivirus release from infected cells is associated with the control of apoptosis.

FCV infection causes rapid cytopathic effects, and its replication results in the induction of apoptosis changes in cultured cells. It is well established that the survival of apoptotic cells can be enhanced by the expression of heat-shock proteins (Hsp) to prevent damage or facilitate recovery. Hsps can act as molecular chaperones, but they can also have anti-apoptotic roles by binding to apoptotic proteins and inhibiting the activation of caspases, the primary mediators of apoptosis. Because apoptosis occurs during FCV infection and heat shock (HS) treatment has a cytoprotective role due to the expression of Hsps, we studied the effect of the HS response to hyperthermia during FCV infection in cultured cells. We found that FCV infection does not inhibit the expression of Hsp70 induced by HS and that non-structural and structural protein synthesis was not modified during HS treatment. However, HS caused a delay in the appearance of a cytopathic effect in infected cells, as well as a reduction in the extracellular but not in the cell-associated viral yield. This antiviral effect of HS correlates with the inhibition of caspase-3 activation. Thus, the HS-induced reduction in virus production appeared to be associated with the control of apoptosis, supporting previous data that indicate that apoptosis is necessary for FCV release.

Norovirus genome circularization and efficient replication are facilitated by binding of PCBP2 and hnRNP A1.

Sequences and structures within the terminal genomic regions of plus-strand RNA viruses are targets for the binding of host proteins that modulate functions such as translation, RNA replication, and encapsidation. Using murine norovirus 1 (MNV-1), we describe the presence of long-range RNA-RNA interactions that were stabilized by cellular proteins. The proteins potentially responsible for the stabilization were selected based on their ability to bind the MNV-1 genome and/or having been reported to be involved in the stabilization of RNA-RNA interactions. Cell extracts were preincubated with antibodies against the selected proteins and used for coprecipitation reactions. Extracts treated with antibodies to poly(C) binding protein 2 (PCBP2) and heterogeneous nuclear ribonucleoprotein (hnRNP) A1 significantly reduced the 5'-3' interaction. Both PCBP2 and hnRNP A1 recombinant proteins stabilized the 5'-3' interactions and formed ribonucleoprotein complexes with the 5' and 3' ends of the MNV-1 genomic RNA. Mutations within the 3' complementary sequences (CS) that disrupt the 5'-3'-end interactions resulted in a significant reduction of the viral titer, suggesting that the integrity of the 3'-end sequence and/or the lack of complementarity with the 5' end is important for efficient virus replication. Small interfering RNA-mediated knockdown of PCBP2 or hnRNP A1 resulted in a reduction in virus yield, confirming a role for the observed interactions in efficient viral replication. PCBP2 and hnRNP A1 induced the circularization of MNV-1 RNA, as revealed by electron microscopy. This study provides evidence that PCBP2 and hnRNP A1 bind to the 5' and 3' ends of the MNV-1 viral RNA and contribute to RNA circularization, playing a role in the virus life cycle.

Nucleolin interacts with the feline calicivirus 3' untranslated region and the protease-polymerase NS6 and NS7 proteins, playing a role in virus replication.

Cellular proteins play many important roles during the life cycle of all viruses. Specifically, host cell nucleic acid-binding proteins interact with viral components of positive-stranded RNA viruses and regulate viral translation, as well as RNA replication. Here, we report that nucleolin, a ubiquitous multifunctional nucleolar shuttling phosphoprotein, interacts with the Norwalk virus and feline calicivirus (FCV) genomic 3' untranslated regions (UTRs). Nucleolin can also form a complex in vitro with recombinant Norwalk virus NS6 and -7 (NS6/7) and can be copurified with the analogous protein from feline calicivirus (p76 or NS6/7) from infected feline kidney cells. Nucleolin RNA levels or protein were not modified during FCV infection; however, as a consequence of the infection, nucleolin was seen to relocalize from the nucleoli to the nucleoplasm, as well as to the perinuclear area where it colocalizes with the feline calicivirus NS6/7 protein. In addition, antibodies to nucleolin were able to precipitate viral RNA from feline calicivirus-infected cells, indicating a direct or indirect association of nucleolin with the viral RNA during virus replication. Small interfering RNA (siRNA)-mediated knockdown of nucleolin resulted in a reduction of the cytopathic effect and virus yield in CrFK cells. Taken together, these results demonstrate that nucleolin is a nucleolar component that interacts with viral RNA and NS6/7 and is required for feline calicivirus replication.