Human astrovirus capsid protein releases a membrane lytic peptide upon trypsin maturation.

The human astrovirus (HAstV) is a non-enveloped, single-stranded RNA virus that is a common cause of gastroenteritis. Most non-enveloped viruses use membrane disruption to deliver the viral genome into a host cell after virus uptake. The virus-host factors that allow for HAstV cell entry are currently unknown but thought to be associated with the host-protease-mediated viral maturation. Using in vitro liposome disruption analysis, we identified a trypsin-dependent lipid disruption activity in the capsid protein of HAstV serotype 8. This function was further localized to the P1 domain of the viral capsid core, which was both necessary and sufficient for membrane disruption. Site-directed mutagenesis identified a cluster of four trypsin cleavage sites necessary to retain the lipid disruption activity, which is likely attributed to a short stretch of sequence ending at arginine 313 based on mass spectrometry of liposome-associated peptides. The membrane disruption activity was conserved across several other HAstVs, including the emerging VA2 strain, and effective against a wide range of lipid identities. This work provides key functional insight into the protease maturation process essential to HAstV infectivity and presents a method to investigate membrane penetration by non-enveloped viruses in vitro. IMPORTANCE Human astroviruses (HAstVs) are an understudied family of viruses that cause mild gastroenteritis but have recent cases associated with a more severe neural pathogenesis. Many important elements of the HAstV life cycle are not well understood, and further elucidating them can help understand the various forms of HAstV pathogenesis. In this study, we utilized an in vitro liposome-based assay to describe and characterize a previously unreported lipid disruption activity. This activity is dependent on the protease cleavage of key sites in HAstV capsid core and can be controlled by site-directed mutagenesis. Our group observed this activity in multiple strains of HAstV and in multiple lipid conditions, indicating this may be a conserved activity across the AstV family. The discovery of this function provides insight into HAstV cellular entry, pathogenesis, and a possible target for future therapeutics.

The Capsid Precursor Protein of Astrovirus VA1 Is Proteolytically Processed Intracellularly.

Human astrovirus VA1 has been associated with neurological disease in immunocompromised patients, and its recent propagation in cell culture has opened the possibility to study its biology. Unlike classical human astroviruses, VA1 growth was found to be independent of trypsin during virus replication in vitro. In this work, we show that despite its independence on trypsin activation for cell infection, the VA1 capsid precursor protein, of 86 kDa (VP86), is processed intracellularly, and this proteolytic processing is important for astrovirus VA1 infectivity. Antibodies raised against different regions of the capsid precursor showed that the polyprotein can be processed starting at either its amino- or carboxy-terminal end, and they allowed us to identify those proteins of about 33 (VP33) and 38 (VP38) kDa constitute the core and the spike proteins of the mature infectious virus particles, respectively. The amino-terminal end of the spike protein was found to be Thr-348. Whether the protease involved in intracellular cleavage of the capsid precursor is of viral or cellular origin remains to be determined, but the cleavage is independent of caspases. Also, trypsin is able to degrade the capsid precursor but has no effect on VP33 and VP38 proteins when assembled into virus particles. These studies provide the basis for advancement of the knowledge of astrovirus VA1 cell entry and replication. IMPORTANCE Human astrovirus VA1 has been associated with neurological disease in immunocompromised patients. Its recent propagation in cell culture has facilitated the study of its biology. In this work, we show that despite the ability of this virus to grow in the absence of trypsin, a marked feature of human classical astroviruses, the capsid precursor protein of astrovirus VA1 is cleaved intracellularly to yield the mature infectious particles, formed by two polypeptides, VP33 that constitutes the core domain of the virus particle, and VP38 that forms the spike of the virus. These studies provide a platform to advance our knowledge on astrovirus VA1 cell entry and replication.

Pooling saliva samples as an excellent option to increase the surveillance for SARS-CoV-2 when re-opening community settings.

In many countries a second wave of infections caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has occurred, triggering a shortage of reagents needed for diagnosis and compromising the capacity of laboratory testing. There is an urgent need to develop methods to accelerate the diagnostic procedures. Pooling samples represents a strategy to overcome the shortage of reagents, since several samples can be tested using one reaction, significantly increasing the number and speed with which tests can be carried out. We have reported the feasibility to use a direct lysis procedure of saliva as source for RNA to SARS-CoV-2 genome detection by reverse transcription quantitative-PCR (RT-qPCR). Here, we show that the direct lysis of saliva pools, of either five or ten samples, does not compromise the detection of viral RNA. In addition, it is a sensitive, fast, and inexpensive method that can be used for massive screening, especially considering the proximity of the reincorporation of activities in universities, offices, and schools.

Rotavirus cell entry: not so simple after all.

Rotaviruses are important agents of severe gastroenteritis in young children, and show a very selective cell and tissue tropism, as well as significant age and host restriction. In the last few years, these properties have been associated with the initial interaction of the virus with histo-blood group antigens on the cell surface, although post-attachment interactions have also been found to define the susceptibility to infection of human enteroids. These initial interactions seem also to determine the virus entry pathway, as well as the induction of signaling cascades that influence the virus intracellular vesicular traffic and escape from endosomes. Here we review the current knowledge of the different stages of the virus entry journey.

Genómica de rotavirus. Impacto en salud pública / Rotavirus genomics. Public health impact

Resumen: Con la introducción de las vacunas de rotavirus Rotarix (RV1) o RotaTeq (RV5) en programas nacionales de vacunación de diversos países, surgió la preocupación de que la presión inmune generada condujera al aumento en la prevalencia de genotipos virales no incluidos en las vacunas, o bien del surgimiento de nuevas cepas que pudieran escapar a la respuesta inmune protectora inducida por la vacunación. La variación natural de los rotavirus ha hecho que sea muy difícil distinguir si el cambio en las cepas circulantes se debe a la presión selectiva impuesta por las vacunas o bien a la fluctuación natural de las cepas. Si acaso ha habido una presión selectiva, ésta ha sido hasta ahora baja. Sin embargo, es importante mantener la vigilancia epidemiólogica y poner atención al surgimiento de cepas resistentes a la inmunidad, en particular en países en desarrollo en los que se ha descrito una mayor diversidad viral.

Abstract: With the introduction of rotavirus vaccines Rotarix (RV1) or RotaTeq (RV5) in the immunization programs of an increasing number of countries, there is concern that the immune selection pressure induced will cause an increase in the prevalence of virus genotypes not included in the vaccine formulation, or to the appearance of novel rotavirus strains that could evade the protective immune response. The natural fluctuation of rotaviruses makes it difficult to distinguish if the change in the circulating strains is due to the vaccine selective pressure or to the natural diversity fluctuation of viruses. If there has been a selective pressure, it has been low so far. However, it is important to keep an epidemiological surveillance and pay attention to the emergence of strains that are resistant to the vaccine, in particular in those countries where the viral diversity has been shown to be higher.

Nanoscale organization of rotavirus replication machineries.

Rotavirus genome replication and assembly take place in cytoplasmic electron dense inclusions termed viroplasms (VPs). Previous conventional optical microscopy studies observing the intracellular distribution of rotavirus proteins and their organization in VPs have lacked molecular-scale spatial resolution, due to inherent spatial resolution constraints. In this work we employed super-resolution microscopy to reveal the nanometric-scale organization of VPs formed during rotavirus infection, and quantitatively describe the structural organization of seven viral proteins within and around the VPs. The observed viral components are spatially organized as five concentric layers, in which NSP5 localizes at the center of the VPs, surrounded by a layer of NSP2 and NSP4 proteins, followed by an intermediate zone comprised of the VP1, VP2, VP6. In the outermost zone, we observed a ring of VP4 and finally a layer of VP7. These findings show that rotavirus VPs are highly organized organelles.

The actin cytoskeleton is important for rotavirus internalization and RNA genome replication.

Numerous host factors are required for the efficient replication of rotavirus, including the activation and inactivation of several cell signaling pathways. One of the cellular structures that are reorganized during rotavirus infection is the actin cytoskeleton. In this work, we report that the dynamics of the actin microfilaments are important at different stages of the virus life cycle, specifically, during virus internalization and viral RNA synthesis at 6 h post-infection. Our results show that the actin-binding proteins alpha-actinin 4 and Diaph, as well as the Rho-family small GTPase Cdc42 are necessary for an efficient virus entry, while GTPase Rac1 is required for maximal viral RNA synthesis.

Isolation of Neutralizing Monoclonal Antibodies to Human Astrovirus and Characterization of Virus Variants That Escape Neutralization.

Human astroviruses (HAstVs) cause severe diarrhea and represent an important health problem in children under two years of age. Despite their medical importance, the study of these pathogens has been neglected. To better understand the astrovirus antigenic structure and the basis of protective immunity, in this work we produced a panel of neutralizing monoclonal antibodies (Nt-MAbs) to HAstV serotypes 1, 2, and 8 and identified the mutations that allow the viruses to escape neutralization. We first tested the capacity of the recombinant HAstV capsid core and spike domains to elicit Nt-Abs. Hyperimmunization of animals with the two domains showed that although both induced a potent immune response, only the spike was able to elicit antibodies with neutralizing activity. Based on this finding, we used a mixture of the recombinant spike domains belonging to the three HAstV serotypes to immunize mice. Five Nt-MAbs were isolated and characterized; all of them were serotype specific, two were directed to HAstV-1, one was directed to HAstV-2, and two were directed to HAstV-8. These antibodies were used to select single and double neutralization escape variant viruses, and determination of the amino acid changes that allow the viruses to escape neutralization permitted us to define the existence of four potentially independent neutralization epitopes on the HAstV capsid. These studies provide the basis for development of subunit vaccines that induce neutralizing antibodies and tools to explore the possibility of developing a specific antibody therapy for astrovirus disease. Our results also establish a platform to advance our knowledge on HAstV cell binding and entry.IMPORTANCE Human astroviruses (HAstVs) are common etiological agents of acute gastroenteritis in children, the elderly, and immunocompromised patients; some virus strains have also been associated with neurological disease. Despite their medical importance, the study of these pathogens has advanced at a slow pace. In this work, we produced neutralizing antibodies to the virus and mapped the epitopes they recognize on the virus capsid. These studies provide the basis for development of subunit vaccines that induce neutralizing antibodies, as well as tools to explore the development of a specific antibody therapy for astrovirus disease. Our results also establish a platform to advance our knowledge on HAstV cell binding and entry.

Minimal capsid composition of infectious human astrovirus.

Human astrovirus is an important etiological agent of acute gastroenteritis in young children. Despite advances in the characterization of the structure of the virion by cryo-electron microscopy and of capsid proteins by x-ray crystallography, the definition of the minimal polypeptide composition of infectious virus particles has been elusive. In this work we show that mature infectious particles are composed by only two proteins; VP34 that forms the core domain of the virus, and VP27 that constitutes the 30 dimeric spikes present on the virus surface. Our results also indicate that during the transition of immature (90 spikes) to mature (30 spikes) virus particles, that occur during trypsin activation, the viral protein VP25, that most likely forms the 60 spikes that are lost during maturation, detaches from the virus particle. This information is relevant to better understand the biology of virus entry and also for the efficient development of subunit vaccines.

Bats, Primates, and the Evolutionary Origins and Diversification of Mammalian Gammaherpesviruses.

Gammaherpesviruses (γHVs) are generally considered host specific and to have codiverged with their hosts over millions of years. This tenet is challenged here by broad-scale phylogenetic analysis of two viral genes using the largest sample of mammalian γHVs to date, integrating for the first time bat γHV sequences available from public repositories and newly generated viral sequences from two vampire bat species (Desmodus rotundus and Diphylla ecaudata). Bat and primate viruses frequently represented deep branches within the supported phylogenies and clustered among viruses from distantly related mammalian taxa. Following evolutionary scenario testing, we determined the number of host-switching and cospeciation events. Cross-species transmissions have occurred much more frequently than previously estimated, and most of the transmissions were attributable to bats and primates. We conclude that the evolution of the Gammaherpesvirinae subfamily has been driven by both cross-species transmissions and subsequent cospeciation within specific viral lineages and that the bat and primate orders may have potentially acted as superspreaders to other mammalian taxa throughout evolutionary history. IMPORTANCE: It has long been believed that herpesviruses have coevolved with their hosts and are species specific. Nevertheless, a global evolutionary analysis of bat viruses in the context of other mammalian viruses, which could put this widely accepted view to the test, had not been undertaken until now. We present two main findings that may challenge the current view of γHV evolution: multiple host-switching events were observed at a higher rate than previously appreciated, and bats and primates harbor a large diversity of γHVs which may have led to increased cross-species transmissions from these taxa to other mammals.

Polarized rotavirus entry and release from differentiated small intestinal cells.

Rotaviruses infect mature enterocytes from small intestine, however most data about their cellular entry are from studies carried out in non-intestinal polarized or non-polarized cell lines. In this work the entry of porcine rotavirus YM strain into small intestinal cell line IPEC-J2 was studied. It was found that YM and the human rotavirus Wa strain infect preferentially from the basolateral cell surface. Cell infection from the apical and basolateral surfaces was dependent on the presence of cholesterol. The treatment with neuraminidase, sucrose, and bafilomycin suggests that there are differences in the receptor usage and entry mechanism of the virus from the apical and basolateral surface. While cell entry is more efficient from basolateral surface, the viruses egressed mainly from the apical cell side.

Assessment of Epstein-Barr virus nucleic acids in gastric but not in breast cancer by next-generation sequencing of pooled Mexican samples

Gastric (GC) and breast (BrC) cancer are two of the most common and deadly tumours. Different lines of evidence suggest a possible causative role of viral infections for both GC and BrC. Wide genome sequencing (WGS) technologies allow searching for viral agents in tissues of patients with cancer. These technologies have already contributed to establish virus-cancer associations as well as to discovery new tumour viruses. The objective of this study was to document possible associations of viral infection with GC and BrC in Mexican patients. In order to gain idea about cost effective conditions of experimental sequencing, we first carried out an in silico simulation of WGS. The next-generation-platform IlluminaGallx was then used to sequence GC and BrC tumour samples. While we did not find viral sequences in tissues from BrC patients, multiple reads matching Epstein-Barr virus (EBV) sequences were found in GC tissues. An end-point polymerase chain reaction confirmed an enrichment of EBV sequences in one of the GC samples sequenced, validating the next-generation sequencing-bioinformatics pipeline.

Assessment of Epstein-Barr virus nucleic acids in gastric but not in breast cancer by next-generation sequencing of pooled Mexican samples.

Gastric (GC) and breast (BrC) cancer are two of the most common and deadly tumours. Different lines of evidence suggest a possible causative role of viral infections for both GC and BrC. Wide genome sequencing (WGS) technologies allow searching for viral agents in tissues of patients with cancer. These technologies have already contributed to establish virus-cancer associations as well as to discovery new tumour viruses. The objective of this study was to document possible associations of viral infection with GC and BrC in Mexican patients. In order to gain idea about cost effective conditions of experimental sequencing, we first carried out an in silico simulation of WGS. The next-generation-platform IlluminaGallx was then used to sequence GC and BrC tumour samples. While we did not find viral sequences in tissues from BrC patients, multiple reads matching Epstein-Barr virus (EBV) sequences were found in GC tissues. An end-point polymerase chain reaction confirmed an enrichment of EBV sequences in one of the GC samples sequenced, validating the next-generation sequencing-bioinformatics pipeline.

Identification of Host Cell Factors Associated with Astrovirus Replication in Caco-2 Cells.

UNLABELLED: Astroviruses are small, nonenveloped viruses with a single-stranded positive-sense RNA genome causing acute gastroenteritis in children and immunocompromised patients. Since positive-sense RNA viruses have frequently been found to replicate in association with membranous structures, in this work we characterized the replication of the human astrovirus serotype 8 strain Yuc8 in Caco-2 cells, using density gradient centrifugation and free-flow zonal electrophoresis (FFZE) to fractionate cellular membranes. Structural and nonstructural viral proteins, positive- and negative-sense viral RNA, and infectious virus particles were found to be associated with a distinct population of membranes separated by FFZE. The cellular proteins associated with this membrane population in infected and mock-infected cells were identified by tandem mass spectrometry. The results indicated that membranes derived from multiple cell organelles were present in the population. Gene ontology and protein-protein interaction network analysis showed that groups of proteins with roles in fatty acid synthesis and ATP biosynthesis were highly enriched in the fractions of this population in infected cells. Based on this information, we investigated by RNA interference the role that some of the identified proteins might have in the replication cycle of the virus. Silencing of the expression of genes involved in cholesterol (DHCR7, CYP51A1) and fatty acid (FASN) synthesis, phosphatidylinositol (PI4KIIIß) and inositol phosphate (ITPR3) metabolism, and RNA helicase activity (DDX23) significantly decreased the amounts of Yuc8 genomic and antigenomic RNA, synthesis of the structural protein VP90, and virus yield. These results strongly suggest that astrovirus RNA replication and particle assembly take place in association with modified membranes potentially derived from multiple cell organelles. IMPORTANCE: Astroviruses are common etiological agents of acute gastroenteritis in children and immunocompromised patients. More recently, they have been associated with neurological diseases in mammals, including humans, and are also responsible for different pathologies in birds. In this work, we provide evidence that astrovirus RNA replication and virus assembly occur in contact with cell membranes potentially derived from multiple cell organelles and show that membrane-associated cellular proteins involved in lipid metabolism are required for efficient viral replication. Our findings provide information to enhance our knowledge of astrovirus biology and provide information that might be useful for the development of therapeutic interventions to prevent virus replication.

Prevalence of respiratory virus in symptomatic children in private physician office settings in five communities of the state of Veracruz, Mexico.

BACKGROUND: Acute respiratory tract infections are the leading cause of morbidity and mortality in children worldwide. Many studies have described the frequency of viruses in hospitalized patients, but studies describing the prevalence of viruses in the community setting are limited, particularly in developing countries, where most of the deaths from serious respiratory diseases occur. The aim of this study was to evaluate the diversity of respiratory viruses in the community setting using molecular diagnostic tools, as well as the clinical characteristics of respiratory viral infections in the general pediatric practice in Mexico. METHODS: Children with respiratory tract infections attending private pediatric practices during a 10-month period in five cities of the state of Veracruz were included. Nasal swabs were taken and processed by a multiplex detection kit for 15 respiratory viruses. RESULTS: 525 children were included from July 2011 to May 2012; 44% were female, mean age was 45 months. The 3 most frequent clinical diagnosis were: rhinopharyngitis 68%, pharyngitis 18%, and 3.3% influenza-like illness. 71.5% of the samples were positive for virus. The five most frequent pathogens were respiratory syncycitial virus in 18.3% of the children, rhinovirus in 17.5%, influenza A 9.1%, adenovirus 7.2%, and enterovirus 3.4%, although all 15 viruses were detected; there were viral coinfections in 14.1%, and 28.5% of the samples were negative. CONCLUSIONS: A large proportion of respiratory infections in the community setting in Mexico was associated to viruses. Although testing for common respiratory pathogens in children with acute respiratory tract infections may lead to a better understanding of the role of viral pathogens in, and eventually to improvement in the management of, individual patients, additional prospective studies are required to study the need of routinely using such tests in general pediatric practices in resource-limited countries.

A novel endogenous betaretrovirus in the common vampire bat (Desmodus rotundus) suggests multiple independent infection and cross-species transmission events.

The Desmodus rotundus endogenous betaretrovirus (DrERV) is fixed in the vampire bat D. rotundus population and in other phyllostomid bats but is not present in all species from this family. DrERV is not phylogenetically related to Old World bat betaretroviruses but to betaretroviruses from rodents and New World primates, suggesting recent cross-species transmission. A recent integration age estimation of the provirus in some taxa indicates that an exogenous counterpart might have been in recent circulation.

DNA microarray for detection of gastrointestinal viruses.

Gastroenteritis is a clinical illness of humans and other animals that is characterized by vomiting and diarrhea and caused by a variety of pathogens, including viruses. An increasing number of viral species have been associated with gastroenteritis or have been found in stool samples as new molecular tools have been developed. In this work, a DNA microarray capable in theory of parallel detection of more than 100 viral species was developed and tested. Initial validation was done with 10 different virus species, and an additional 5 species were validated using clinical samples. Detection limits of 1 × 10(3) virus particles of Human adenovirus C (HAdV), Human astrovirus (HAstV), and group A Rotavirus (RV-A) were established. Furthermore, when exogenous RNA was added, the limit for RV-A detection decreased by one log. In a small group of clinical samples from children with gastroenteritis (n = 76), the microarray detected at least one viral species in 92% of the samples. Single infection was identified in 63 samples (83%), and coinfection with more than one virus was identified in 7 samples (9%). The most abundant virus species were RV-A (58%), followed by Anellovirus (15.8%), HAstV (6.6%), HAdV (5.3%), Norwalk virus (6.6%), Human enterovirus (HEV) (9.2%), Human parechovirus (1.3%), Sapporo virus (1.3%), and Human bocavirus (1.3%). To further test the specificity and sensitivity of the microarray, the results were verified by reverse transcription-PCR (RT-PCR) detection of 5 gastrointestinal viruses. The RT-PCR assay detected a virus in 59 samples (78%). The microarray showed good performance for detection of RV-A, HAstV, and calicivirus, while the sensitivity for HAdV and HEV was low. Furthermore, some discrepancies in detection of mixed infections were observed and were addressed by reverse transcription-quantitative PCR (RT-qPCR) of the viruses involved. It was observed that differences in the amount of genetic material favored the detection of the most abundant virus. The microarray described in this work should help in understanding the etiology of gastroenteritis in humans and animals.

Rotaviruses reach late endosomes and require the cation-dependent mannose-6-phosphate receptor and the activity of cathepsin proteases to enter the cell.

UNLABELLED: Rotaviruses (RVs) enter cells through different endocytic pathways. Bovine rotavirus (BRV) UK uses clathrin-mediated endocytosis, while rhesus rotavirus (RRV) employs an endocytic process independent of clathrin and caveolin. Given the differences in the cell internalization pathway used by these viruses, we tested if the intracellular trafficking of BRV UK was the same as that of RRV, which is known to reach maturing endosomes (MEs) to infect the cell. We found that BRV UK also reaches MEs, since its infectivity depends on the function of Rab5, the endosomal sorting complex required for transport (ESCRT), and the formation of endosomal intraluminal vesicles (ILVs). However, unlike RRV, the infectivity of BRV UK was inhibited by knocking down the expression of Rab7, indicating that it has to traffic to late endosomes (LEs) to infect the cell. The requirement for Rab7 was also shared by other RV strains of human and porcine origin. Of interest, most RV strains that reach LEs were also found to depend on the activities of Rab9, the cation-dependent mannose-6-phosphate receptor (CD-M6PR), and cathepsins B, L, and S, suggesting that cellular factors from the trans-Golgi network (TGN) need to be transported by the CD-M6PR to LEs to facilitate RV cell infection. Furthermore, using a collection of UK × RRV reassortant viruses, we found that the dependence of BRV UK on Rab7, Rab9, and CD-M6PR is associated with the spike protein VP4. These findings illustrate the elaborate pathway of RV entry and reveal a new process (Rab9/CD-M6PR/cathepsins) that could be targeted for drug intervention. IMPORTANCE: Rotavirus is an important etiological agent of severe gastroenteritis in children. In most instances, viruses enter cells through an endocytic pathway that delivers the viral particle to vesicular organelles known as early endosomes (EEs). Some viruses reach the cytoplasm from EEs, where they start to replicate their genome. However, other viruses go deeper into the cell, trafficking from EEs to late endosomes (LEs) to disassemble and reach the cytoplasm. In this work, we show that most RV strains have to traffic to LEs, and the transport of endolysosomal proteases from the Golgi complex to LEs, mediated by the mannose-6-phosphate receptor, is necessary for the virus to exit the vesicular compartment and efficiently start viral replication. We also show that this deep journey into the cell is associated with the virus spike protein VP4. These findings illustrate the elaborate pathway of RV entry that could be used for drug intervention.

Discovery of a novel polyomavirus in acute diarrheal samples from children.

Polyomaviruses are small circular DNA viruses associated with chronic infections and tumors in both human and animal hosts. Using an unbiased deep sequencing approach, we identified a novel, highly divergent polyomavirus, provisionally named MX polyomavirus (MXPyV), in stool samples from children. The ∼5.0 kB viral genome exhibits little overall homology (<46% amino acid identity) to known polyomaviruses, and, due to phylogenetic variation among its individual proteins, cannot be placed in any existing taxonomic group. PCR-based screening detected MXPyV in 28 of 834 (3.4%) fecal samples collected from California, Mexico, and Chile, and 1 of 136 (0.74%) of respiratory samples from Mexico, but not in blood or urine samples from immunocompromised patients. By quantitative PCR, the measured titers of MXPyV in human stool at 10% (weight/volume) were as high as 15,075 copies. No association was found between the presence of MXPyV and diarrhea, although girls were more likely to shed MXPyV in the stool than boys (p=0.012). In one child, viral shedding was observed in two stools obtained 91 days apart, raising the possibility of chronic infection by MXPyV. A multiple sequence alignment revealed that MXPyV is a closely related variant of the recently reported MWPyV and HPyV10 polyomaviruses. Further studies will be important to determine the association, if any, of MXPyV with disease in humans.

Rotavirus infection induces the unfolded protein response of the cell and controls it through the nonstructural protein NSP3.

The unfolded protein response (UPR) is a cellular mechanism that is triggered in order to cope with the stress caused by the accumulation of misfolded proteins in the endoplasmic reticulum (ER). This response is initiated by the endoribonuclease inositol-requiring enzyme 1 (IRE1), activating transcription factor 6 (ATF6), and PKR-like ER kinase, which increase the expression of the genes involved in the folding and degradation processes and decrease the protein input into the ER by inhibiting translation. It has been shown that viruses both induce and manipulate the UPR in order to protect the host cells from an ER stress-mediated death, thus permitting the translation of viral proteins and the efficient replication of the virus. To understand the cellular events that occur during the rotavirus replication cycle, we examined the activation of the three UPR arms following infection, using luciferase reporters driven by promoters of the ER stress-responsive genes and real-time reverse transcription-PCR to determine the levels of the stress-induced mRNAs. Our findings indicated that during rotavirus infection two of the three arms of the UPR (IRE1 and ATF6) become activated; however, these pathways are interrupted at the translational level by the general inhibition of protein synthesis caused by NSP3. This response seems to be triggered by more than one viral protein synthesized during the replication of the virus, but not by the viral double-stranded RNA (dsRNA), since cells transfected with psoralen-inactivated virions, or with naked viral dsRNA, did not induce UPR.