Susana Guix

Molecular Epidemiology of Caliciviruses Causing Outbreaks and Sporadic Cases of Acute Gastroenteritis in Spain

ABSTRACT The molecular epidemiology of human caliciviruses (HuCVs) causing sporadic cases and outbreaks of acute gastroenteritis around eastern Spain (Catalonia and the Valencian Community) was studied by reverse transcription-PCR (RT-PCR) and by sequencing part of the RNA polymerase gene in open reading frame 1. HuCVs were detected in 44 of 310 stool specimens (14.19%) negative for other enteric pathogens obtained from children with acute gastroenteritis. Norwalk-like viruses (NLVs) were the most common cause of the gastroenteritis outbreaks investigated here. They were detected in 14 out of 25 (56%) outbreaks with an identified pathogen. Genotypes producing both sporadic cases and outbreaks were diverse, with a predominance of GGII strains related to genotypes Melksham and Lordsdale. Five strains clustered with a “new variant” designated GGIIb, which was detected circulating throughout quite a few European countries in the years 2000 and 2001. The emergence mechanism of these strains might be the occurrence of intertypic recombinations between different viruses. The nucleotide sequence of part of the capsid gene (ORF2) from three of these strains demonstrated their relationship with Mexico virus.

Molecular Epidemiology of Astrovirus Infection in Barcelona, Spain

ABSTRACT A 3-year study involving 2,347 gastroenteritis samples was conducted to determine the prevalence, time distribution, and medical significance of human astrovirus infection in Barcelona, Spain. The overall incidence of astrovirus was found to be 4.9%. Mixed infections with other enteric agents were detected in 17.2% of all astrovirus-positive samples. During the 3-year period, the highest astrovirus incidence was reported in the winter months, although infections also occurred in summer. The peak detection rate was observed in children between 2 and 4 years of age. Overall, HAstV-1 was the most prevalent type, followed by HAstV-4, HAstV-3, HAstV-8, and HAstV-2. HAstV-5, HAstV-6, and HAstV-7 were not detected during these 3 years. From our serotype data for each age group, we observed that HAstV-1, HAstV-2, and HAstV-3 affected mostly children younger than 3 years of age, while HAstV-4 and HAstV-8 had a greater impact in older children. Genetic variability was analyzed between astroviruses isolated in Barcelona and strains isolated in other parts of the world. A fourth lineage was described for HAstV-1, most likely due to the large number of assayed samples, which may also explain the high level of genetic variability observed in the astrovirus isolates.

New tools for the study and direct surveillance of viral pathogens in water.

Half a century ago scientists attempted the detection of poliovirus in water. Since then other enteric viruses responsible for gastroenteritis and hepatitis have replaced enteroviruses as the main target for detection. However, most viral outbreaks are restricted to norovirus and hepatitis A virus, making them the main targets in water. The inclusion of virus analysis in regulatory standards for viruses in water samples must overcome several shortcomings such as the technical difficulties and high costs of virus monitoring, the lack of harmonised and standardised assays and the challenge posed by the ever-changing nature of viruses. However, new tools are nowadays available for the study and direct surveillance of viral pathogens in water that may contribute to fulfil these requirements.

Norwalk Virus RNA Is Infectious in Mammalian Cells

ABSTRACT Human noroviruses are positive-sense RNA viruses and are the leading cause of epidemic acute viral gastroenteritis in developed countries. The absence of an in vitro cell culture model for human norovirus infection has limited the development of effective antivirals and vaccines. Human histo-blood group antigens have been regarded as receptors for norovirus infection, and expression of the α(1,2) fucosyltransferase gene (FUT2) responsible for the secretor phenotype is required for susceptibility to Norwalk virus (NV) infection. We report for the first time that transfection of NV RNA, isolated from stool samples from human volunteers, into human hepatoma Huh-7 cells leads to viral replication, with expression of viral antigens, RNA replication, and release of viral particles into the medium. Prior treatment of the RNA with proteinase K completely abolishes RNA infectivity, suggesting a key role of an RNA-protein complex. Although overexpression of the human FUT2 gene enhances virus binding to cells, it is not sufficient to allow a complete viral infection, and viral spread from NV-transfected cells to naïve cells does not occur. Finally, no differences in NV RNA replication are observed between Huh-7 and Huh-7.5.1 cells, which contain an inactivating mutation in retinoic acid-inducible gene I (RIG-I), suggesting that the RIG-I pathway does not play a role in limiting NV replication. Our results strongly suggest that the block(s) to NV replication in vitro is at the stage of receptor and/or coreceptor binding and/or uncoating, either because cells lack some specific factor or activation of cellular antiviral responses independent of RIG-I inhibits virus replication.

Fine-Tuning Translation Kinetics Selection as the Driving Force of Codon Usage Bias in the Hepatitis A Virus Capsid

Hepatitis A virus (HAV), the prototype of genus Hepatovirus, has several unique biological characteristics that distinguish it from other members of the Picornaviridae family. Among these, the need for an intact eIF4G factor for the initiation of translation results in an inability to shut down host protein synthesis by a mechanism similar to that of other picornaviruses. Consequently, HAV must inefficiently compete for the cellular translational machinery and this may explain its poor growth in cell culture. In this context of virus/cell competition, HAV has strategically adopted a naturally highly deoptimized codon usage with respect to that of its cellular host. With the aim to optimize its codon usage the virus was adapted to propagate in cells with impaired protein synthesis, in order to make tRNA pools more available for the virus. A significant loss of fitness was the immediate response to the adaptation process that was, however, later on recovered and more associated to a re-deoptimization rather than to an optimization of the codon usage specifically in the capsid coding region. These results exclude translation selection and instead suggest fine-tuning translation kinetics selection as the underlying mechanism of the codon usage bias in this specific genome region. Additionally, the results provide clear evidence of the Red Queen dynamics of evolution since the virus has very much evolved to re-adapt its codon usage to the environmental cellular changing conditions in order to recover the original fitness.

Potential role of fomites in the vehicular transmission of human astroviruses.

ABSTRACT The persistence of human astroviruses dried on representative porous (paper) and nonporous (china) surfaces was investigated. Long-term astrovirus survival on fomites was monitored by an integrated cell culture-reverse transcription-PCR procedure. Viruses were applied to inanimate surfaces in the presence and absence of fecal material, and their survival was assayed at 4 and 20°C with high relative humidity. Astroviruses exhibited a notable persistence when dried on porous and nonporous materials, particularly at low temperature. Short-term survival of astroviruses on fomites was compared to that of other enteric viruses significant for health, such as rotavirus, adenovirus, poliovirus, and hepatitis A virus. Overall, astroviruses persisted better than poliovirus and adenovirus, although they exhibited a shorter survival than rotavirus and hepatitis A virus. Astroviruses show a high level of persistence at the desiccation step, which is of major significance in determining the chance of subsequent virus survival dried on fomites. Astroviruses are able to survive on inert surfaces long enough to suggest that fomites may play a relevant role in the secondary transmission of astrovirus diarrhea.

Norwalk virus does not replicate in human macrophages or dendritic cells derived from the peripheral blood of susceptible humans

Human noroviruses are difficult to study due to the lack of an efficient in vitro cell culture system or small animal model. Murine norovirus replicates in murine macrophages (MPhi) and dendritic cells (DCs), raising the possibility that human NoVs might replicate in such human cell types. To test this hypothesis, we evaluated DCs and MPhi derived from monocyte subsets and CD11c(+) DCs isolated from peripheral blood mononuclear cells of individuals susceptible to Norwalk virus (NV) infection. These cells were exposed to NV and replication was evaluated by immunofluorescence and by quantitative RT-PCR. A few PBMC-derived DCs expressed NV proteins. However, NV RNA did not increase in any of the cells tested. These results demonstrate that NV does not replicate in human CD11c(+) DCs, monocyte-derived DCs and MPhi, but abortive infection may occur in a few DCs. These results suggest that NV tropism is distinct from that of murine noroviruses.

Human norovirus occurrence and diversity in the Llobregat river catchment, Spain

Human noroviruses (NoV) were quantified and characterized in an 18 month survey conducted along the Llobregat river catchment in Spain. Sample types included freshwater, untreated and treated wastewater and drinking water. High NoV genome copy numbers were reported, reaching up to 10(6)  l(-1) and 10(9)  l(-1) in freshwater and raw sewage respectively. In both types of samples, GII NoV genome copies outnumbered those of GI, although without significance. All samples of semi-treated and treated drinking water were negative for NoV. A clear seasonality of NoV occurrence was observed both in river water and sewage samples, with significantly higher genome copy numbers in the cold than in the warm months period. Mean NoV log reduction rates after biological treatment of sewage were 2.2 and 3.1 for GI and GII respectively. A total of 77 NoV strains isolated in the Llobregat river catchment could be phylogenetically characterized, 44 belonging to GI and 33 to GII. The most prevalent genotype was GI.4, followed by GII.4 and GII.21. Several variants of the pandemic GII.4 strain were detected in the environment, corroborating their circulation among the population.

Quantification and genotyping of human sapoviruses in the Llobregat river catchment, Spain.

ABSTRACT Human sapoviruses (SaVs) were quantified and characterized in an 18-month survey conducted along the Llobregat river catchment area in Spain. Sample types included freshwater, untreated and treated wastewater, and drinking water. All genogroups were recovered, and a seasonal distribution was observed. This is the first report of SaV quantification and genotyping in the environment outside Japan.

Inhibition of cellular protein secretion by norwalk virus nonstructural protein p22 requires a mimic of an endoplasmic reticulum export signal.

Protein trafficking between the endoplasmic reticulum (ER) and Golgi apparatus is central to cellular homeostasis. ER export signals are utilized by a subset of proteins to rapidly exit the ER by direct uptake into COPII vesicles for transport to the Golgi. Norwalk virus nonstructural protein p22 contains a YXΦESDG motif that mimics a di-acidic ER export signal in both sequence and function. However, unlike normal ER export signals, the ER export signal mimic of p22 is necessary for apparent inhibition of normal COPII vesicle trafficking, which leads to Golgi disassembly and antagonism of Golgi-dependent cellular protein secretion. This is the first reported function for p22. Disassembly of the Golgi apparatus was also observed in cells replicating Norwalk virus, which may contribute to pathogenesis by interfering with cellular processes that are dependent on an intact secretory pathway. These results indicate that the ER export signal mimic is critical to the antagonistic function of p22, shown herein to be a novel antagonist of ER/Golgi trafficking. This unique and well-conserved human norovirus motif is therefore an appealing target for antiviral drug development.