Attenuation hotspots in neurotropic human astroviruses.

During the last decade, the detection of neurotropic astroviruses has increased dramatically. The MLB genogroup of astroviruses represents a genetically distinct group of zoonotic astroviruses associated with gastroenteritis and severe neurological complications in young children, the immunocompromised, and the elderly. Using different virus evolution approaches, we identified dispensable regions in the 3' end of the capsid-coding region responsible for attenuation of MLB astroviruses in susceptible cell lines. To create recombinant viruses with identified deletions, MLB reverse genetics (RG) and replicon systems were developed. Recombinant truncated MLB viruses resulted in imbalanced RNA synthesis and strong attenuation in iPSC-derived neuronal cultures confirming the location of neurotropism determinants. This approach can be used for the development of vaccine candidates using attenuated astroviruses that infect humans, livestock animals, and poultry.

Comparison of Nanopore and Synthesis-Based Next-Generation Sequencing Platforms for SARS-CoV-2 Variant Monitoring in Wastewater.

Shortly after the beginning of the SARS-CoV-2 pandemic, many countries implemented sewage sentinel systems to monitor the circulation of the virus in the population. A fundamental part of these surveillance programs is the variant tracking through sequencing approaches to monitor and identify new variants or mutations that may be of importance. Two of the main sequencing platforms are Illumina and Oxford Nanopore Technologies. Here, we compare the performance of MiSeq (Illumina) and MinION (Oxford Nanopore Technologies), as well as two different data processing pipelines, to determine the effect they may have on the results. MiSeq showed higher sequencing coverage, lower error rate, and better capacity to detect and accurately estimate variant abundances than MinION R9.4.1 flow cell data. The use of different variant callers (LoFreq and iVar) and approaches to calculate the variant proportions had a remarkable impact on the results generated from wastewater samples. Freyja, coupled with iVar, may be more sensitive and accurate than LoFreq, especially with MinION data, but it comes at the cost of having a higher error rate. The analysis of MinION R10.4.1 flow cell data using Freyja combined with iVar narrows the gap with MiSeq performance in terms of read quality, accuracy, sensitivity, and number of detected mutations. Although MiSeq should still be considered as the standard method for SARS-CoV-2 variant tracking, MinION's versatility and rapid turnaround time may represent a clear advantage during the ongoing pandemic.

Dynamics of SARS-CoV-2 Alpha (B.1.1.7) variant spread: The wastewater surveillance approach.

Wastewater based epidemiology (WBE) offers an overview of the SARS-CoV-2 variants circulating among the population thereby serving as a proper surveillance method. The variant of concern (VOC) Alpha was first identified in September 2020 in the United Kingdom, and rapidly became dominant across Europe. Our objective was to elucidate the Alpha VOC outcompetition rate and identify mutations in the spike glycoprotein (S) gene, indicative of the circulation of the Alpha VOC and/or other variants in the population through wastewater analysis. In the period covered by this study (November 2020-April 2021), forteen wastewater treatment plants (WWTPs) were weekly sampled. The total number of SARS-CoV-2 genome copies per L (GC/L) was determined with a Real-Time qPCR, targeting the N gene. Surveillance of the Alpha VOC circulation was ascertained using a duplex RT-qPCR, targeting and discriminating the S gene. Our results showed that in a period of 6 weeks the Alpha VOC was present in all the studied WWTPs, and became dominant in 11 weeks on average. The outcompetition rates of the Alpha VOC were estimated, and their relationship with different parameters statistically analyzed. The rapid spread of the Alpha VOC was influenced by its initial input and by the previous circulation of SARS-COV-2 in the population. This latter point could be explained by its higher transmissibility, particularly advantadgeous when a certain degree of herd immunity exists. Moreover, the presence of signature mutations of SARS-COV-2 variants were established by deep-sequencing of the complete S gene. The circulation of the Alpha VOC in the area under study was confirmed, and additionally two combinations of mutations in the S glycoprotein (T73A and D253N, and S477N and A522S) that could affect antibody binding were identified.

Astrovirus replication in human intestinal enteroids reveals multi-cellular tropism and an intricate host innate immune landscape.

Human astroviruses (HAstV) are understudied positive-strand RNA viruses that cause gastroenteritis mostly in children and the elderly. Three clades of astroviruses, classic, MLB-type and VA-type have been reported in humans. One limitation towards a better understanding of these viruses has been the lack of a physiologically relevant cell culture model that supports growth of all clades of HAstV. Herein, we demonstrate infection of HAstV strains belonging to all three clades in epithelium-only human intestinal enteroids (HIE) isolated from biopsy-derived intestinal crypts. A detailed investigation of infection of VA1, a member of the non-canonical HAstV-VA/HMO clade, showed robust replication in HIE derived from different patients and from different intestinal regions independent of the cellular differentiation status. Flow cytometry and immunofluorescence analysis revealed that VA1 infects several cell types, including intestinal progenitor cells and mature enterocytes, in HIE cultures. RNA profiling of VA1-infected HIE uncovered that the host response to infection is dominated by interferon (IFN)-mediated innate immune responses. A comparison of the antiviral host response in non-transformed HIE and transformed human colon carcinoma Caco-2 cells highlighted significant differences between these cells, including an increased magnitude of the response in HIE. Additional studies confirmed the sensitivity of VA1 to exogenous IFNs, and indicated that the endogenous IFN response of HIE to curtail the growth of strains from all three clades. Genotypic variation in the permissiveness of different HIE lines to HAstV could be overcome by pharmacologic inhibition of JAK/STAT signaling. Collectively, our data identify HIE as a universal infection model for HAstV and an improved model of the intestinal epithelium to investigate enteric virus-host interactions.

Time Evolution of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in Wastewater during the First Pandemic Wave of COVID-19 in the Metropolitan Area of Barcelona, Spain.

Two large wastewater treatment plants (WWTP), covering around 2.7 million inhabitants, which represents around 85% of the metropolitan area of Barcelona, were sampled before, during, and after the implementation of a complete lockdown. Five one-step reverse transcriptase quantitative PCR (RT-qPCR) assays, targeting the polymerase (IP2 and IP4), the envelope (E), and the nucleoprotein (N1 and N2) genome regions, were employed for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA detection in 24-h composite wastewater samples concentrated by polyethylene glycol (PEG) precipitation. SARS-CoV-2 was detected in a sewage sample collected 41 days ahead of the declaration of the first COVID-19 case. The evolution of SARS-CoV-2 genome copies in wastewater evidenced the validity of water-based epidemiology (WBE) to anticipate COVID-19 outbreaks, to evaluate the impact of control measures, and even to estimate the burden of shedders, including presymptomatic, asymptomatic, symptomatic, and undiagnosed cases. For the latter objective, a model was applied for the estimation of the total number of shedders, evidencing a high proportion of asymptomatic infected individuals. In this way, an infection prevalence of 2.0 to 6.5% was figured. On the other hand, proportions of around 0.12% and 0.09% of the total population were determined to be required for positive detection in the two WWTPs. At the end of the lockdown, SARS-CoV-2 RNA apparently disappeared in the WWTPs but could still be detected in grab samples from four urban sewers. Sewer monitoring allowed for location of specific hot spots of COVID-19, enabling the rapid adoption of appropriate mitigation measures.IMPORTANCE Water-based epidemiology (WBE) is a valuable early warning tool for tracking the circulation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) among the population, including not only symptomatic patients but also asymptomatic, presymptomatic, and misdiagnosed carriers, which represent a high proportion of the infected population. In the specific case of Barcelona, wastewater surveillance anticipated by several weeks not only the original COVID-19 pandemic wave but also the onset of the second wave. In addition, SARS-CoV-2 occurrence in wastewater evidenced the efficacy of the adopted lockdown measures on the circulation of the virus. Health authorities profited from WBE to complement other inputs and adopt rapid and adequate measures to mitigate the effects of the pandemic. For example, sentinel surveillance of specific sewers helped to locate COVID-19 hot spots and to conduct massive numbers of RT-PCR tests among the population.

Monitoring Emergence of the SARS-CoV-2 B.1.1.7 Variant through the Spanish National SARS-CoV-2 Wastewater Surveillance System (VATar COVID-19).

Since its first identification in the United Kingdom in late 2020, the highly transmissible B.1.1.7 variant of SARS-CoV-2 has become dominant in several countries raising great concern. We developed a duplex real-time RT-qPCR assay to detect, discriminate, and quantitate SARS-CoV-2 variants containing one of its mutation signatures, the ΔHV69/70 deletion, and used it to trace the community circulation of the B.1.1.7 variant in Spain through the Spanish National SARS-CoV-2 Wastewater Surveillance System (VATar COVID-19). The B.1.1.7 variant was detected earlier than clinical epidemiological reporting by the local authorities, first in the southern city of Málaga (Andalucía) in week 20_52 (year_week), and multiple introductions during Christmas holidays were inferred in different parts of the country. Wastewater-based B.1.1.7 tracking showed a good correlation with clinical data and provided information at the local level. Data from wastewater treatment plants, which reached B.1.1.7 prevalences higher than 90% for ≥2 consecutive weeks showed that 8.1 ± 2.0 weeks were required for B.1.1.7 to become dominant. The study highlights the applicability of RT-qPCR-based strategies to track specific mutations of variants of concern as soon as they are identified by clinical sequencing and their integration into existing wastewater surveillance programs, as a cost-effective approach to complement clinical testing during the COVID-19 pandemic.

Infectivity of Norovirus GI and GII from Bottled Mineral Water during a Waterborne Outbreak, Spain.

During a waterborne outbreak of norovirus in Spain, we estimated 50% illness doses for a group of exposed (secretor) persons to be 556 (95% CI 319-957) genome copies/day for norovirus GI and 2,934 (95% CI 1,683-5,044) genome copies/day for norovirus GII. Use of a propidium monoazide viability assay reduced these values.

Human Astrovirus MLB Replication In Vitro: Persistence in Extraintestinal Cell Lines.

MLB astroviruses were identified 10 years ago in feces from children with gastroenteritis of unknown etiology and have been unexpectedly detected in severe cases of meningitis/encephalitis, febrile illness of unknown etiology, and respiratory syndromes. The aim of this study was to establish a cell culture system supporting MLB astrovirus replication. We used two clinical strains to infect several cell lines, an MLB1 strain from a gastroenteritis case, and an MLB2 strain associated with a neurologic infection. Efforts to propagate the viruses in the Caco-2 cell line were unsuccessful. In contrast, we identified two human nonintestinal cell lines, Huh-7 and A549, permissive for both genotypes. After serial passages in the Huh-7.5 cell line, the adapted strains were able to establish persistent infections in the Huh-7.5, Huh-7AI, and A549 cell lines, with high viral loads (up to 10 log10 genome copies/ml) detected by quantitative reverse transcription-PCR (RT-qPCR) in the culture supernatant. Immunofluorescence assays demonstrated infection in about 10% of cells in persistently infected cultures. Electron microscopy revealed particles of 32 to 33 nm in diameter after negative staining of cell supernatants and capsid arrays in ultrathin sections with a particularly high production in Huh-7.5 cells. Interferon (IFN) expression by infected cells and effect of exogenous IFN varied depending on the type of infection and the cell line. The availability of a cell culture system to propagate MLB astroviruses represents a key step to better understand their replicative cycle, as well as a source of viruses to conduct a wide variety of basic virologic studies.IMPORTANCE MLB astroviruses are emerging viruses infecting humans. More studies are required to determine their exact epidemiology, but several reports have already identified them as the cause of unexpected clinical diseases, including severe neurologic diseases. Our study provides the first description of a cell culture system for the propagation of MLB astroviruses, enabling the study of their replicative cycle. Moreover, we demonstrated the unknown capacity of MLB astrovirus to establish persistent infections in cell culture. Whether these persistent infections are also established in vivo remains unknown, but the clinical consequences would be of high interest if persistence was confirmed in vivo Finally, our analysis of IFN expression provides some trails to understand the mechanism by which MLB astroviruses can cause persistent infections in the assayed cultures.

Norovirus in Bottled Water Associated with Gastroenteritis Outbreak, Spain, 2016.

In April 2016, an outbreak of gastrointestinal illness (4,136 cases) occurred in Catalonia, Spain. We detected high levels of norovirus genotypes I and II in office water coolers associated with the outbreak. Infectious viral titer estimates were 33-49 genome copies/L for genotype I and 327-660 genome copies/L for genotype II.

Replication of Human Norovirus RNA in Mammalian Cells Reveals Lack of Interferon Response.

UNLABELLED: Human noroviruses (HuNoVs), named after the prototype strain Norwalk virus (NV), are a leading cause of acute gastroenteritis outbreaks worldwide. Studies on the related murine norovirus (MNV) have demonstrated the importance of an interferon (IFN) response in host control of virus replication, but this remains unclear for HuNoVs. Despite the lack of an efficient cell culture infection system, transfection of stool-isolated NV RNA into mammalian cells leads to viral RNA replication and virus production. Using this system, we show here that NV RNA replication is sensitive to type I (α/ß) and III (interleukin-29 [IL-29]) IFN treatment. However, in cells capable of a strong IFN response to Sendai virus (SeV) and poly(I·C), NV RNA replicates efficiently and generates double-stranded RNA without inducing a detectable IFN response. Replication of HuNoV genogroup GII.3 strain U201 RNA, generated from a reverse genetics system, also does not induce an IFN response. Consistent with a lack of IFN induction, NV RNA replication is enhanced neither by neutralization of type I/III IFNs through neutralizing antibodies or the soluble IFN decoy receptor B18R nor by short hairpin RNA (shRNA) knockdown of mitochondrial antiviral signaling protein (MAVS) or interferon regulatory factor 3 (IRF3) in the IFN induction pathways. In contrast to other positive-strand RNA viruses that block IFN induction by targeting MAVS for degradation, MAVS is not degraded in NV RNA-replicating cells, and an SeV-induced IFN response is not blocked. Together, these results indicate that HuNoV RNA replication in mammalian cells does not induce an IFN response, suggesting that the epithelial IFN response may play a limited role in host restriction of HuNoV replication. IMPORTANCE: Human noroviruses (HuNoVs) are a leading cause of epidemic gastroenteritis worldwide. Due to lack of an efficient cell culture system and robust small-animal model, little is known about the innate host defense to these viruses. Studies on murine norovirus (MNV) have shown the importance of an interferon (IFN) response in host control of MNV replication, but this remains unclear for HuNoVs. Here, we investigated the IFN response to HuNoV RNA replication in mammalian cells using Norwalk virus stool RNA transfection, a reverse genetics system, IFN neutralization reagents, and shRNA knockdown methods. Our results show that HuNoV RNA replication in mammalian epithelial cells does not induce an IFN response, nor can it be enhanced by blocking the IFN response. These results suggest a limited role of the epithelial IFN response in host control of HuNoV RNA replication, providing important insights into our understanding of the host defense to HuNoVs that differs from that to MNV.

Plasmid-based human norovirus reverse genetics system produces reporter-tagged progeny virus containing infectious genomic RNA.

Human norovirus (HuNoV) is the leading cause of gastroenteritis worldwide. HuNoV replication studies have been hampered by the inability to grow the virus in cultured cells. The HuNoV genome is a positive-sense single-stranded RNA (ssRNA) molecule with three open reading frames (ORFs). We established a reverse genetics system driven by a mammalian promoter that functions without helper virus. The complete genome of the HuNoV genogroup II.3 U201 strain was cloned downstream of an elongation factor-1α (EF-1α) mammalian promoter. Cells transfected with plasmid containing the full-length genome (pHuNoVU201F) expressed the ORF1 polyprotein, which was cleaved by the viral protease to produce the mature nonstructural viral proteins, and the capsid proteins. Progeny virus produced from the transfected cells contained the complete NoV genomic RNA (VP1, VP2, and VPg) and exhibited the same density in isopycnic cesium chloride gradients as native infectious NoV particles from a patient's stool. This system also was applied to drive murine NoV RNA replication and produced infectious progeny virions. A GFP reporter construct containing the GFP gene in ORF1 produced complete virions that contain VPg-linked RNA. RNA from virions containing the encapsidated GFP-genomic RNA was successfully transfected back into cells producing fluorescent puncta, indicating that the encapsidated RNA is replication-competent. The EF-1α mammalian promoter expression system provides the first reverse genetics system, to our knowledge, generalizable for human and animal NoVs that does not require a helper virus. Establishing a complete reverse genetics system expressed from cDNA for HuNoVs now allows the manipulation of the viral genome and production of reporter virions.

Clinical surveillance for human astrovirus in Monastir region, Tunisia.

BACKGROUND/AIMS: Astroviruses (AstVs) are enteric viruses that can cause gastroenteritis in children. This study is part of monitoring the circulation of astroviruses in children hospitalized and/or outpatients for acute gastroenteritis at the primary care center of Ouerdanine or at the Pediatric Department of the University Hospital Fattouma-Bourguiba (Monastir, Tunisia). The aims of our study were to know the prevalence of human astrovirus in clinical samples of children, characterize the strains and evaluate the infectivity of isolated strains on cell culture. METHODS: Fifty stool samples were collected from children under five years old in the region of Monastir (Tunisia) from October 2010 to June 2011. All specimens were subjected to RT-PCR amplification followed by sequencing and phylogenetic analysis. RESULTS: The study shows a low prevalence of astrovirus (4 %) in children. The two positive samples obtained were HAstV type 3. Samples that were RT-PCR positive were cultured in CaCO-2 cells and the presence of infectious viral particles was confirmed. The phylogenetic analysis shows that the different HAstV-3 strains isolated in Tunisia are grouped into two clusters. The first cluster includes strains obtained in 2004, which belong to lineage HAstV-3a, while strains isolated in 2010 belong to lineage HAstV-3c. CONCLUSIONS: This study is part of monitoring the circulation of astroviruses in children younger than five years old from Monastir region, Tunisia. The results show low prevalence (4 %). All genotyped samples belonged to lineage HAstV-3c, which could be presently emerging. Two different lineages have been isolated in Tunisia: HAstV-3a in 2004 and HAstV-3c in 2010.

Human astroviruses.

Human astroviruses (HAtVs) are positive-sense single-stranded RNA viruses that were discovered in 1975. Astroviruses infecting other species, particularly mammalian and avian, were identified and classified into the genera Mamastrovirus and Avastrovirus. Through next-generation sequencing, many new astroviruses infecting different species, including humans, have been described, and the Astroviridae family shows a high diversity and zoonotic potential. Three divergent groups of HAstVs are recognized: the classic (MAstV 1), HAstV-MLB (MAstV 6), and HAstV-VA/HMO (MAstV 8 and MAstV 9) groups. Classic HAstVs contain 8 serotypes and account for 2 to 9% of all acute nonbacterial gastroenteritis in children worldwide. Infections are usually self-limiting but can also spread systemically and cause severe infections in immunocompromised patients. The other groups have also been identified in children with gastroenteritis, but extraintestinal pathologies have been suggested for them as well. Classic HAstVs may be grown in cells, allowing the study of their cell cycle, which is similar to that of caliciviruses. The continuous emergence of new astroviruses with a potential zoonotic transmission highlights the need to gain insights on their biology in order to prevent future health threats. This review focuses on the basic virology, pathogenesis, host response, epidemiology, diagnostic assays, and prevention strategies for HAstVs.

Hepatitis A virus adaptation to cellular shutoff is driven by dynamic adjustments of codon usage and results in the selection of populations with altered capsids.

UNLABELLED: Hepatitis A virus (HAV) has a highly biased and deoptimized codon usage compared to the host cell and fails to inhibit host protein synthesis. It has been proposed that an optimal combination of abundant and rare codons controls the translation speed required for the correct capsid folding. The artificial shutoff host protein synthesis results in the selection of variants containing mutations in the HAV capsid coding region critical for folding, stability, and function. Here, we show that these capsid mutations resulted in changes in their antigenicity; in a reduced stability to high temperature, low pH, and biliary salts; and in an increased efficacy of cell entry. In conclusion, the adaptation to cellular shutoff resulted in the selection of large-plaque-producing virus populations. IMPORTANCE: HAV has a naturally deoptimized codon usage with respect to that of its cell host and is unable to shut down the cellular translation. This fact contributes to the low replication rate of the virus, in addition to other factors such as the highly inefficient internal ribosome entry site (IRES), and explains the outstanding physical stability of this pathogen in the environment mediated by a folding-dependent highly cohesive capsid. Adaptation to artificially induced cellular transcription shutoff resulted in a redeoptimization of its capsid codon usage, instead of an optimization. These genomic changes are related to an overall change of capsid folding, which in turn induces changes in the cell entry process. Remarkably, the adaptation to cellular shutoff allowed the virus to significantly increase its RNA uncoating efficiency, resulting in the selection of large-plaque-producing populations. However, these populations produced much-debilitated virions.

Hepatitis A virus genotype distribution during a decade of universal vaccination of preadolescents.

A universal vaccination program among preadolescents was implemented in Catalonia, Spain, during the period of 1999-2013 and its effectiveness has been clearly demonstrated by an overall significant attack rate reduction. However, reductions were not constant over time, and increases were again observed in 2002-2009 due to the occurrence of huge outbreaks. In the following years, in the absence of large outbreaks, the attack rate decreased again to very low levels. However, an increase of symptomatic cases in the <5 age group has recently been observed. This is an unexpected observation since children younger than 6 are mostly asymptomatic. Such a long vaccination campaign offers the opportunity to analyze not only the effectiveness of vaccination, but also the influence of the circulating genotypes on the incidence of hepatitis A among the different age groups. This study has revealed the emergence of genotype IC during a foodborne outbreak, the short-lived circulation of vaccine-escape variants isolated during an outbreak among the men-having-sex-with-men group, and the association of genotype IIIA with the increase of symptomatic cases among the very young. From a public health perspective, two conclusions may be drawn: vaccination is better at an early age, and the vaccination schedule must be complete and include all recommended vaccine doses.

Molecular basis of the behavior of hepatitis a virus exposed to high hydrostatic pressure.

Food-borne hepatitis A outbreaks may be prevented by subjecting foods at risk of virus contamination to moderate treatments of high hydrostatic pressure (HHP). A pretreatment promoting hepatitis A virus (HAV) capsid-folding changes enhances the virucidal effect of HHP, indicating that its efficacy depends on capsid conformation. HAV populations enriched in immature capsids (125S provirions) are more resistant to HHP, suggesting that mature capsids (150S virions) are more susceptible to this treatment. In addition, the monoclonal antibody (MAb) K24F2 epitope contained in the immunodominant site is a key factor for the resistance to HHP. Changes in capsid folding inducing a loss of recognition by MAb K24F2 render more susceptible conformations independently of the origin of such changes. Accordingly, codon usage-associated folding changes and changes stimulated by pH-dependent breathings, provided they confer a loss of recognition by MAb K24F2, induce a higher susceptibility to HHP. In conclusion, the resistance of HAV to HHP treatments may be explained by a low proportion of 150S particles combined with a good accessibility of the epitope contained in the immunodominant site close to the 5-fold axis.

Standardized multiplex one-step qRT-PCR for hepatitis A virus, norovirus GI and GII quantification in bivalve mollusks and water.

A quadruplex Real-Time RT-PCR assay for the simultaneous quantitative detection of hepatitis A virus (HAV), norovirus (NoV) GI and GII, and mengovirus (used as process control for determination of the virus/nucleic acid extraction efficiency) has been developed. This multiplex assay has been comparatively evaluated with the individual monoplex assays and showed to be slightly less sensitive, with average ΔCq values of 0.90, 0.28 and 0.44 for HAV, NoV GI and NoV GII, respectively, in standard curves of viral RNA, or 0.32, 0.37 and 0.51 for the same viruses respectively, in naturally-contaminated samples. These ΔCq values were mostly negligible since it represented, in the worst case scenario, a loss of 0.43 log in genome copy numbers. The quadruplex assay shows similar theoretical detection limits than the monoplex assay for NoV GII, and 10 times higher for HAV and NoV GI. However, when naturally-contaminated food and water samples were tested, these theoretical detection thresholds were often exceeded and very low genome copy numbers (below the limit of detection) could be quantified. The quadruplex assay fulfills the requirements of the method developed by the European Committee on Standardization (CEN) for virus detection in selected foodstuffs with significant advantages in labor and reagent costs.

Identification of human astrovirus genome-linked protein (VPg) essential for virus infectivity.

Viral genome-linked proteins (VPgs) have been identified in several single-stranded positive-sense RNA virus families. The presence of such protein in the family Astroviridae has not been fully elucidated, although a putative VPg coding region in open reading frame 1a (ORF1a) of astrovirus with high amino acid sequence similarity to the VPg coding region of Caliciviridae has been previously identified. In this work we present several experimental findings that show that human astrovirus (HAstV) RNA encodes a VPg essential for viral infectivity: (i) RNase treatment of RNA purified from astrovirus-infected cells results in a single protein of 13 to 15 kDa, compatible with the predicted astrovirus VPg size; (ii) the antibody used to detect this 13- to 15-kDa protein is specifically directed against a region that includes the putative VPg coding region; (iii) the 13- to 15-kDa protein detected has been partially sequenced and the sequence obtained is contained in the computationally predicted VPg; (iv) the protein resulting from this putative VPg coding region is a highly disordered protein, resembling the VPg of sobemo-, calici- and potyviruses; (v) proteolytic treatment of the genomic RNA leads to loss of infectivity; and (vi) mutagenesis of Tyr-693 included in the putative VPg protein is lethal for HAstV replication, which strongly supports its functional role in the covalent link with the viral RNA.

Analysis of SARS-CoV-2 in wastewater for prevalence estimation and investigating clinical diagnostic test biases.

Here we analyze SARS-CoV-2 genome copies in Catalonia's wastewater during the Omicron peak and develop a mathematical model to estimate the number of infections and the temporal relationship between reported and unreported cases. 1-liter samples from 16 wastewater treatment plants were collected and used in a compartmental epidemiological model. The average correlation between genome copies and reported cases was 0.85, with an average delay of 8.8 days. The model estimated that 53% of the population was infected, compared to the 19% reported cases. The under-reporting was highest in November and December 2021. The maximum genome copies shed in feces by an infected individual was estimated to range from 1.4×108 gc/g to 4.4×108 gc/g. Our framework demonstrates the potential of wastewater data as a leading indicator for daily new infections, particularly in contexts with low detection rates. It also serves as a complementary tool for prevalence estimation and offers a general approach for integrating wastewater data into compartmental models.

Epidemiology of GII.4 and GII.2 norovirus outbreaks in closed and semi-closed institutions in 2017 and 2018.

Norovirus infections are a leading cause of acute gastroenteritis outbreaks worldwide, with genotypes GII.2 and GII.4 being the most prevalent. The aim of this study was to compare the characteristics of GII.2 and GII.4 norovirus outbreaks reported in Catalonia in closed or semi-closed institutions in 2017 and 2018. The epidemiological and clinical characteristics of GII.2 and GII.4 outbreaks were compared using the chi-square test or Fisher's exact test for categorical variables and the Mann-Whitney U test for continuous variables. Odds ratios and their 95% confidence intervals were estimated. 61 outbreaks were reported: GII.4 was the causative agent in 12 outbreaks (30%) and GII.2 in 9 outbreaks (22.5%). GII.2 outbreaks were detected more frequently in schools or summer camps (66.7%) and GII.4 outbreaks in nursing homes (91.7%) (p = 0.01). Ninety-three people were affected in GII.2 outbreaks and 94 in GII.4 outbreaks. The median age was 15 years (range: 1-95 years) in GII.2 outbreaks and 86 years (range: 0-100 years) in GII.4 outbreaks (p < 0.001). Nausea, abdominal pain, and headache were observed more frequently in persons affected by GII.2 outbreaks (p < 0.05). Symptomatic cases presented a higher viral load suggestive of greater transmission capacity, although asymptomatic patients presented relevant loads indicative of transmission capacity.