Pavel Isa

A metagenomic analysis of pandemic influenza A (2009 H1N1) infection in patients from North America.

Although metagenomics has been previously employed for pathogen discovery, its cost and complexity have prevented its use as a practical front-line diagnostic for unknown infectious diseases. Here we demonstrate the utility of two metagenomics-based strategies, a pan-viral microarray (Virochip) and deep sequencing, for the identification and characterization of 2009 pandemic H1N1 influenza A virus. Using nasopharyngeal swabs collected during the earliest stages of the pandemic in Mexico, Canada, and the United States (n = 17), the Virochip was able to detect a novel virus most closely related to swine influenza viruses without a priori information. Deep sequencing yielded reads corresponding to 2009 H1N1 influenza in each sample (percentage of aligned sequences corresponding to 2009 H1N1 ranging from 0.0011% to 10.9%), with up to 97% coverage of the influenza genome in one sample. Detection of 2009 H1N1 by deep sequencing was possible even at titers near the limits of detection for specific RT-PCR, and the percentage of sequence reads was linearly correlated with virus titer. Deep sequencing also provided insights into the upper respiratory microbiota and host gene expression in response to 2009 H1N1 infection. An unbiased analysis combining sequence data from all 17 outbreak samples revealed that 90% of the 2009 H1N1 genome could be assembled de novo without the use of any reference sequence, including assembly of several near full-length genomic segments. These results indicate that a streamlined metagenomics detection strategy can potentially replace the multiple conventional diagnostic tests required to investigate an outbreak of a novel pathogen, and provide a blueprint for comprehensive diagnosis of unexplained acute illnesses or outbreaks in clinical and public health settings.

Heat Shock Cognate Protein 70 Is Involved in Rotavirus Cell Entry

ABSTRACT In this work, we have identified the heat shock cognate protein (hsc70) as a receptor candidate for rotaviruses. hsc70 was shown to be present on the surface of MA104 cells, and antibodies to this protein blocked rotavirus infectivity, while not affecting the infectivity of reovirus and poliovirus. Preincubation of the hsc70 protein with the viruses also inhibited their infectivity. Triple-layered particles (mature virions), but not double-layered particles, bound hsc70 in a solid-phase assay, and this interaction was blocked by monoclonal antibodies to the virus surface proteins VP4 and VP7. Rotaviruses were shown to interact with hsc70 at a postattachment step, since antibodies to hsc70 and the protein itself did not inhibit the virus attachment to cells. We propose that the functional rotavirus receptor is a complex of several cell surface molecules that include, among others, hsc70.

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.

Role of sialic acids in rotavirus infection

Rotaviruses are the leading cause of childhood diarrhea. The entry of rotaviruses into the host cell is a complex process that includes several interactions of the outer layer proteins of the virus with different cell surface molecules. The fact that neuraminidase treatment of the cells, or preincubation of the virus with sialic acid-containing compounds decrease the infectivity of some rotavirus strains, suggested that these viruses interact with sialic acid on the cell surface. The infectivity of some other rotavirus strains is not affected by neuraminidase treatment of the cells, and therefore they are considered neuraminidase-resistant. However, the current evidence suggests that even these neuraminidase-resistant strains might interact with sialic acids located in context different from that of the sialic acids used by the neuraminidase-sensitive strains. This review summarizes our current knowledge of the rotavirus-sialic acid interaction, its structural basis, the specificity with which distinct rotavirus isolates interact with sialic acid-containing compounds, and also the potential use of these compounds as therapeutic agents.

Molecular Biology of Rotavirus Cell Entry

Rotaviruses, the leading cause of severe dehydrating diarrhea in infants and young children worldwide, are non-enveloped viruses formed by three concentric layers of protein that enclose a genome of double-stranded RNA. The entry of rotaviruses into epithelial cells appears to be a multistep process during which at least three contacts between the virus and cell receptors occur. Different rotavirus strains display different requirements to infect cells. Some strains depend on the presence of sialic acid on the cell surface; however, interaction with a sialic acid-containing receptor does not seem to be essential, because variants that no longer need sialic acid to infect the cells can be isolated from sialic acid-dependent strains. Comparative characterization of the sialic acid-dependent rotavirus strain RRV, its neuraminidase-resistant variant nar3, and the human rotavirus strain Wa have allowed to show that alpha2beta1 integrin is used by nar3 as its primary cell attachment site, and by RRV in a second interaction subsequent to its initial contact with a sialic acid-containing cell receptor. These first two interactions are mediated by the virus spike protein VP4. After attaching to the cell, all three strains interact with integrin alphaVbeta3 and protein hsc70, interactions perhaps important for the virus to penetrate into the cells interior. The cell molecules proposed to serve as rotavirus receptors have been found associated with cholesterol and glycosphingolipid-enriched lipid microdomains, and disorganization of these domains greatly inhibits rotavirus infectivity. We propose that the functional rotavirus receptor is a complex of several cell molecules most likely immersed in plasma membrane lipid microdomains.

Different Rotavirus Strains Enter MA104 Cells through Different Endocytic Pathways: the Role of Clathrin-Mediated Endocytosis

ABSTRACT Rotaviruses, the single most important agents of acute severe gastroenteritis in children, are nonenveloped viruses formed by a three-layered capsid that encloses a genome formed by 11 segments of double-stranded RNA. The mechanism of entry of these viruses into the host cell is not well understood. The best-studied strain, RRV, which is sensitive to neuraminidase (NA) treatment of the cells, uses integrins α2β1 and αvβ3 and the heat shock protein hsc70 as receptors and enters MA104 cells through a non-clathrin-, non-caveolin-mediated pathway that depends on a functional dynamin and on the presence of cholesterol on the cell surface. In this work, using a combination of pharmacological, biochemical, and genetic approaches, we compared the entry characteristics of four rotavirus strains known to have different receptor requirements. We chose four rotavirus strains that represent all phenotypic combinations of NA resistance or sensitivity and integrin dependence or independence. We found that even though all the strains share their requirements for hsc70, dynamin, and cholesterol, three of them differ from the simian strain RRV in the endocytic pathway used. The human strain Wa, porcine strain TFR-41, and bovine strain UK seem to enter the cell through clathrin-mediated endocytosis, since treatments that inhibit this pathway block their infectivity; consistent with this entry route, these strains were sensitive to changes in the endosomal pH. The inhibition of other endocytic mechanisms, such as macropinocytosis or caveola-mediated uptake, had no effect on the internalization of the rotavirus strains tested here.

Molecular Anatomy of 2009 Influenza Virus A (H1N1)

Influenza A viruses are a major cause of morbidity and mortality worldwide and affect large segments of the population every year. The nature of their genome, formed by eight segments of single-stranded RNA, favors the constant evolution of the virus by two main mechanisms: the accumulation of single nucleotide mutations in the viral genes introduced by an error-prone viral RNA polymerase and the reassortment of genes between two strains of different origin. The viral genome encodes 11 proteins. Most have been shown to play a role in shaping the virulence scenario of influenza A viruses, including the adaptation of infection and transmission into new host species, the ability to modulate the host immune response, and the capacity to replicate efficiently at low temperature. On the surface of the virus particles there are two principal polypeptides, the hemagglutinin (HA) and the neuraminidase (NA), which are the target for the neutralizing antibodies immune response. There are 16 HA and 9 NA different subtypes in the influenza A virus that circulate in humans and animals. When a virus strain with a new HA or NA subtype appears in the human population by genetic reassortment, it usually causes a pandemic because there is no preexisting immunity against the new virus. This was the case for the three pandemics that occurred during the last century (1918, 1957, and 1968) and also for the first pandemic of the 21(st) century, caused by the currently circulating A (H1N1) 2009 virus, which was generated by gene reassortment between a virus present in pigs of North America and a virus that circulates in the swine population of Euroasia.

Survey of equine rotaviruses shows conservation of one P genotype in background of two G genotypes

SummaryDIG-labelled ssRNA probes were prepared from variable regions of VP4 and VP7 cognate genes, and used in hybridization assays for P and G genotyping of group A cell culture-adapted equine rotaviruses and fecal samples collected from foals with and without diarrhea. The probes confirmed known P and G serotypes of sixteen cell culture-adapted strains. From one-hundred and twenty-one rotavirus-positive samples, 83 reacted when tested for their P and G genotype specific probes. From these, 71 were found to contain G3 P12 genotypes, and 11 G14 P12 genotypes. No sample reacted with H1 or L338 P and G genotype probes. This suggests that the equine rotavirus population is conservative, containing predominantly one P genotype and two G genotypes. One isolate (26/94) whose dsRNA was visualized in an agarose gel did not react with any of the equine probes, and was found to belong to G8 and P1 genotypes. This is the fourth example of a single unique equine isolate (after H1, L338, and R-22). The remaining thirty-eight untypable field isolates had no detectable dsRNA after storage for 1 to 3 years.

RNA silencing of rotavirus gene expression.

Abstract RNA interference (RNAi) is a double-stranded RNA (dsRNA)-triggered mechanism for suppressing gene expression, which is conserved in evolution and has emerged as a powerful tool to study gene function. Rotaviruses, the leading cause of severe diarrhea in young children, are formed by three concentric layers of protein, and a genome composed of 11 segments of dsRNA. Here, we show that the RNAi machinery can be triggered to silence rotavirus gene expression by sequence-specific short interfering RNAs (siRNAs). RNAi is also useful for the study of the virus-cell interactions, through the silencing of cellular genes that are potentially important for the replication of the virus. Interestingly, while the translation of mRNAs is readily stopped by the RNAi machinery, the viral transcripts involved in virus genome replication do not seem to be susceptible to RNAi. Since gene silencing by RNAi is very efficient and specific, this system could become a novel therapeutic approach for rotavirus and other virus infections, once efficient methods for in vivo delivery of siRNAs are developed. Although the use of RNAi as an antiviral therapeutic tool remains to be demonstrated, there is no doubt that this technology will influence drastically the way postgenomic virus research is conducted.

Gangliosides have a functional role during rotavirus cell entry.

ABSTRACT Cell entry of rotaviruses is a complex process, which involves sequential interactions with several cell surface molecules. Among the molecules implicated are gangliosides, glycosphingolipids with one or more sialic acid (SA) residues. The role of gangliosides in rotavirus cell entry was studied by silencing the expression of two key enzymes involved in their biosynthesis—the UDP-glucose:ceramide glucosyltransferase (UGCG), which transfers a glucose molecule to ceramide to produce glucosylceramide GlcCer, and the lactosyl ceramide-α-2,3–sialyl transferase 5 (GM3-s), which adds the first SA to lactoceramide-producing ganglioside GM3. Silencing the expression of both enzymes resulted in decreased ganglioside levels (as judged by GM1a detection). Four rotavirus strains tested (human Wa, simian RRV, porcine TFR-41, and bovine UK) showed a decreased infectivity in cells with impaired ganglioside synthesis; however, their replication after bypassing the entry step was not affected, confirming the importance of gangliosides for cell entry of the viruses. Interestingly, viral binding to the cell surface was not affected in cells with inhibited ganglioside synthesis, but the infectivity of all strains tested was inhibited by preincubation of gangliosides with virus prior to infection. These data suggest that rotaviruses can attach to cell surface in the absence of gangliosides but require them for productive cell entry, confirming their functional role during rotavirus cell entry.