Norovirus evolves as one or more distinct clonal populations in immunocompromised hosts.

Noroviruses are a major cause of acute gastroenteritis worldwide and can establish chronic infection in immunocompromised individuals. To investigate the mechanisms of norovirus evolution during chronic infection, we selected seven representative patients from a National Institutes of Health study cohort who sustained norovirus infection for periods ranging from 73 to 1,492 days. Six patients shed viruses belonging to a single genotype (GII.2[PNA], GII.4 New Orleans[P4], GII.4 Den Haag[P4], GII.3[P21], GII.6[P7], or GII.14[P7]) over the period examined, while one patient sequentially shed two genotypes (GII.6[P7] followed by GII.4 Sydney[P31]). Norovirus genomes from consecutive stool samples were sequenced at high resolution (>3,300 reads/nucleotide position) using the Illumina platform and subjected to bioinformatics analysis. Norovirus sequences could be resolved into one or more discrete clonal RNA genomes that persisted within these patients over time. Phylogenetic analyses inferred that clonal populations originated from a single founder virus and not by reinfection with community strains. Estimated evolutionary rates of clonal populations during persistent infection were similar to those of noroviruses from acute infection in the global database, suggesting that inherently higher RNA-dependent polymerase error rates were not associated with the ability to persist. The high-resolution analysis of norovirus diversity and evolution at the population level described here should allow a better understanding of adaptive mutations sustained during chronic infection. IMPORTANCE Noroviruses are an important cause of chronic diarrhea in patients with compromised immune systems. Presently, there are no effective therapies to clear the virus, which can persist for years in the intestinal tract. The goal of our study was to develop a better understanding of the norovirus strains that are associated with these long-term infections. With the remarkable diversity of norovirus strains detected in the immunocompromised patient cohort we studied, it appears that most, if not all, noroviruses circulating in nature may have the capacity to establish a chronic infection when a person is unable to mount an effective immune response. Our work is the most comprehensive genetic data set generated to date in which near full-length genomes from noroviruses associated with chronic infection were analyzed by high-resolution next-generation sequencing. Analysis of this data set led to our discovery that certain patients in our cohort were shedding noroviruses that could be subdivided into distinct haplotypes or populations of viruses that were co-evolving independently. The ability to track haplotypes of noroviruses during chronic infection will allow us to fine-tune our understanding of how the virus adapts and maintains itself in the human host, and how selective pressures such as antiviral drugs can affect these distinct populations.

Widespread and dynamic expression of granzyme C by skin-resident antiviral T cells.

After recognition of cognate antigen (Ag), effector CD8+ T cells secrete serine proteases called granzymes in conjunction with perforin, allowing granzymes to enter and kill target cells. While the roles for some granzymes during antiviral immune responses are well characterized, the function of others, such as granzyme C and its human ortholog granzyme H, is still unclear. Granzyme C is constitutively expressed by mature, cytolytic innate lymphoid 1 cells (ILC1s). Whether other antiviral effector cells also produce granzyme C and whether it is continually expressed or responsive to the environment is unknown. To explore this, we analyzed granzyme C expression in different murine skin-resident antiviral lymphocytes. At steady-state, dendritic epidermal T cells (DETCs) expressed granzyme C while dermal γδ T cells did not. CD8+ tissue-resident memory T cells (TRM) generated in response to cutaneous viral infection with the poxvirus vaccinia virus (VACV) also expressed granzyme C. Both DETCs and virus-specific CD8+ TRM upregulated granzyme C upon local VACV infection. Continual Ag exposure was not required for maintained TRM expression of granzyme C, although re-encounter with cognate Ag boosted expression. Additionally, IL-15 treatment increased granzyme C expression in both DETCs and TRM. Together, our data demonstrate that granzyme C is widely expressed by antiviral T cells in the skin and that expression is responsive to both environmental stimuli and TCR engagement. These data suggest that granzyme C may have functions other than killing in tissue-resident lymphocytes.

Identification of a new polymorphism on the wild-type canine distemper virus genome: could this contribute to vaccine failures?

The canine distemper virus (CDV) is responsible for a multisystem infectious disease with high prevalence in dogs and wild carnivores and has vaccination as the main control measure. However, recent studies show an increase in cases including vaccinated dogs in different parts of the world. There are several reasons for vaccine failures, including differences between vaccine strains and wild-type strains. In this study, a phylogenetic analysis of CDV strains from samples of naturally infected, vaccinated, and symptomatic dogs in Goiânia, Goiás, Brazil was performed with partial sequencing of the hemagglutinin (H) gene of CDV. Different sites of amino acid substitutions were found, and one strain had the Y549H mutation, typically present in samples from wild animals. Substitutions in epitopes (residues 367, 376, 379, 381, 386, and 388) that may interfere with the vaccine's ability to provide adequate protection against infection for CDV were observed. The identified strains were grouped in the South America 1/Europe lineage, with a significant difference from other lineages and vaccine strains. Twelve subgenotypes were characterized, considering a nucleotide identity of at least 98% among the strains. These findings highlight the relevance of canine distemper infection and support the need better monitoring of the circulating strains that contribute to elucidate if there is a need for vaccine update.

Spread of Gamma (P.1) Sub-Lineages Carrying Spike Mutations Close to the Furin Cleavage Site and Deletions in the N-Terminal Domain Drives Ongoing Transmission of SARS-CoV-2 in Amazonas, Brazil.

The Amazonas was one of the most heavily affected Brazilian states by the COVID-19 epidemic. Despite a large number of infected people, particularly during the second wave associated with the spread of the Variant of Concern (VOC) Gamma (lineage P.1), SARS-CoV-2 continues to circulate in the Amazonas. To understand how SARS-CoV-2 persisted in a human population with a high immunity barrier, we generated 1,188 SARS-CoV-2 whole-genome sequences from individuals diagnosed in the Amazonas state from 1st January to 6th July 2021, of which 38 were vaccine breakthrough infections. Our study reveals a sharp increase in the relative prevalence of Gamma plus (P.1+) variants, designated Pango Lineages P.1.3 to P.1.6, harboring two types of additional Spike changes: deletions in the N-terminal (NTD) domain (particularly Δ144 or Δ141-144) associated with resistance to anti-NTD neutralizing antibodies or mutations at the S1/S2 junction (N679K or P681H) that probably enhance the binding affinity to the furin cleavage site, as suggested by our molecular dynamics simulations. As lineages P.1.4 (S:N679K) and P.1.6 (S:P681H) expanded (Re > 1) from March to July 2021, the lineage P.1 declined (Re < 1) and the median Ct value of SARS-CoV-2 positive cases in Amazonas significantly decreases. Still, we did not find an increased incidence of P.1+ variants among breakthrough cases of fully vaccinated patients (71%) in comparison to unvaccinated individuals (93%). This evidence supports that the ongoing endemic transmission of SARS-CoV-2 in the Amazonas is driven by the spread of new local Gamma/P.1 sublineages that are more transmissible, although not more efficient to evade vaccine-elicited immunity than the parental VOC. Finally, as SARS-CoV-2 continues to spread in human populations with a declining density of susceptible hosts, the risk of selecting more infectious variants or antibody evasion mutations is expected to increase. IMPORTANCE The continuous evolution of SARS-CoV-2 is an expected phenomenon that will continue to happen due to the high number of cases worldwide. The present study analyzed how a Variant of Concern (VOC) could still circulate in a population hardly affected by two COVID-19 waves and with vaccination in progress. Our results showed that the answer behind that was a new generation of Gamma-like viruses, which emerged locally carrying mutations that made it more transmissible and more capable of spreading, partially evading prior immunity triggered by natural infections or vaccines. With thousands of new cases daily, the current pandemics scenario suggests that SARS-CoV-2 will continue to evolve and efforts to reduce the number of infected subjects, including global equitable access to COVID-19 vaccines, are mandatory. Thus, until the end of pandemics, the SARS-CoV-2 genomic surveillance will be an essential tool to better understand the drivers of the viral evolutionary process.

Human bocavirus detection and quantification in fecal and serum specimens from recipients of allogeneic hematopoietic stem cell transplantation: A longitudinal study.

OBJECTIVES: The aim of this study was to evaluate the occurrence of human bocavirus (HBoV) and to determine viral loads in samples of patients admitted for allogeneic hematopoietic stem cell transplantation (allo-HSCT). METHODS: Fecal and serum samples were collected from 19 patients, during a 24-month period. Samples were screened by quantitative polymerase chain reaction TaqMan assay, with specific probe and primers targeting the NP1 gene of all HBoVs genotypes (HBoV-1 to - 4), and viral loads were determined using serial dilutions of a recombinant plasmid. RESULTS: HBoV DNA was detected in 42.1% (8 of 19) of the patients in at least one type of sample (feces and/or serum) during the study period, with 75% (6 of 8) of the patients being positive in both types of sample. Viral shedding in feces had a median of 26 days (range, 5 to 121) and viremia was detected in 87.5% (7 of 8) of the patients. The HBoV loads in fecal samples were higher than in sera and, in most cases, HBoV was detected earlier in fecal than in sera samples. In six HBoV-positive patients (6 of 8) diarrhea was observed concomitantly to viral detection in fecal samples. CONCLUSIONS: A high frequency and loads of HBoV in allo-HSCT recipients was observed, especially in fecal samples. Positivity in fecal samples was an early predictor of HBoV presence.

Sapovirus detection and quantification in fecal samples from allogeneic hematopoietic stem cell transplant recipients.

Sapovirus are important agents of acute gastroenteritis (AGE) and they are associated with outbreaks and sporadic cases worldwide. They infect people of all ages, but mainly children, the elderly and immunocompromised individuals are affected. The aim of this study was investigate sapovirus and to determine viral loads in fecal samples from patient undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT). Fecal samples were submitted to extraction of the genetic material using a commercial kit, and RT-qPCR TaqMan was used for sapovirus screening and determination of viral loads, using a standard curve with serial dilutions of a recombinant plasmid. Positive samples were sequence by Sanger method. Sapovirus was detected in one patient, 5.3% (1/19). Viral excretion lasted for 16 days. Viral load varied from 1.73 × 106 to 8.97 × 106 GC/g. One of the positive samples was characterized as GI.1 genotype. This is the first study to determine sapovirus loads in samples from allo-HSCT and to identify GI.1 genotype in immunocompromised patients.

Immunoinformatic construction of an adenovirus-based modular vaccine platform and its application in the design of a SARS-CoV-2 vaccine.

The current SARS-CoV-2 pandemic has imposed new challenges and demands for health systems, especially in the development of new vaccine strategies. Vaccines for many pathogens were developed based on the display of foreign epitopes in the variable regions of the human adenovirus (HAdV) major capsid proteins (hexon, penton and fiber). The humoral immune response against the HAdV major capsid proteins was demonstrated to play a role in the development of an immune response against the epitopes in display. Through the immunoinformatic profiling of the major capsid proteins of HAdVs from different species, we developed a modular concept that can be used in the development of vaccines based on HAdV vectors. Our data suggests that different immunomodulatory potentials can be observed in the conserved regions, present in the hexon and penton proteins, from different species. Using this modular approach, we developed a HAdV-5 based vaccine strategy for SARS-CoV-2, constructed through the display of SARS-CoV-2 epitopes indicated by our prediction analysis as immunologically relevant. The sequences of the HAdV vector major capsid proteins were also edited to enhance the IFN-gamma induction and antigen presenting cells activation. This is the first study proposing a modular HAdV platform developed to aid the design of new vaccines by inducing an immune response more suited for the epitopes in display.

Classical human astroviruses in symptomatic and asymptomatic children of Goiás, Brazil: Positivity rates, viral loads, and molecular characterization.

Classical human astroviruses (HAstV) are agents of nonbacterial acute gastroenteritis (AGE), being predominant among children. There are only a few studies reporting HAstV loads in samples from patients with AGE, data are even scarcer regarding asymptomatic patients. The aim of this study was to evaluate the occurrence and estimate the viral load of HAstV and to perform molecular characterization of positive samples obtained from children, up to 6 years old, with and without AGE. One fecal sample was obtained from each of the 250 children enrolled in the study, from May 2014 to April 2015. Real-time reverse transcription-polymerase chain reaction (RT-qPCR TaqMan) was performed, followed by a conventional RT-PCR directed to ORF2, region C, of the positive samples. Then, these amplicons were sequenced and a phylogenetic analysis was performed to determine the HAstV-1 lineages. A global positivity index of 3.2% (8 of 250) was observed for HAstV with a similar frequency (50%) in both symptomatic and asymptomatic group. Viral loads ranged from 2.8 × 105 to 1.6 × 1011 genome copy/mL Four samples were characterized as HAstV-1, lineage 1a and two as HAstV-4, lineage 4c. Our findings show similar HAstV positivity rates for children with and without AGE, providing evidence of HAstV-1a and HAstV-4c lineage cocirculation in the Central West region of Brazil. Data contributes to the molecular epidemiology of these agents in the region.

Recombinant noroviruses detected in Mid-West region of Brazil in two different periods 2009-2011 and 2014-2015: Atypical breakpoints of recombination and detection of distinct GII.P7-GII.6 lineages.

Noroviruses are an important cause of acute gastroenteritis. The high incidence of norovirus is a reflection of its great genomic and antigenic variability resultant of evolutionary mechanisms, such as recombination. Herein, the main objective of this study was to characterize partially two regions of norovirus genome (RdRp and VP1) from fecal samples, collected in two different time periods (2009-2011 and 2014-2015) in the Mid-West region of Brazil. Twenty samples were sequenced and characterized (GI.P5-GI.5, GII.P16-GII.3, GI.P7-GI.7, GII.Pe-GII.4 and GII.P7-GII.6). Sequences of GII.Pe-GII.4 genotype were also characterized as Sydney 2012 variant. Genotypes GII.P7-GII.6, GII.P16-GII.3 and GII.Pe-GII.4 (16/20-80%) were identified as norovirus recombinants by phylogeny and bioinformatic analyzes. The GII.P7-GII.6 (62.5%) and GII.Pe-GII.4 (25%) genotypes had recombination point's upstream ORF1/2 overlapping region, whereas GII.P16-GII.3 (12.5%) genotype had the recombination point in the overlapping region. Furthermore, the GII.P7-GII.6, from samples collected in 2009-2011 had different recombinant points than the GII.P7-GII.6 from samples obtained in 2014-2015, forming two different clusters in the phylogenetic analysis. Our study brings information on the circulation of recombinant norovirus genotypes in Mid-West of Brazil, including recombinants with atypical recombination breakpoints, and provides evidence for the circulation of different lineages of the same recombinant genotype.

Sapovirus in Rectal and Nasopharyngeal Swab Samples of Children with Symptoms of Acute Gastroenteritis.

The study included 102 hospitalized children 0-72 months of age, with symptoms of acute gastroenteritis. One fecal and one nasopharyngeal swab sample were obtained from each child. Samples were screened for sapovirus and viral loads were determined. Sapovirus was detected in 18.6% of fecal samples and in 36.3% of nasopharyngeal swab samples. High viral loads were detected.

Norovirus in feces and nasopharyngeal swab of children with and without acute gastroenteritis symptoms: First report of GI.5 in Brazil and GI.3 in nasopharyngeal swab.

BACKGROUND: Noroviruses (NoVs) are an important cause of acute gastroenteritis (AGE), worldwide. OBJECTIVES: To evaluate the frequency, viral load and molecular profile of NoV in fecal and nasopharyngeal swab samples from hospitalized children, and to determine children's secretor status. STUDY DESIGN: From May 2014 to May 2015, 219 children were included in the study, 96 with gastroenteric symptoms and 123 without gastroenteric symptoms. All fecal and nasopharyngeal swab samples were screened by TaqMan RT-qPCR duplex (GI/GII NoV) and quality samples were characterized by genomic sequencing. RESULTS: Norovirus positivity rate in feces was 15.4% in asymptomatic and 18.8% in the symptomatic group. The median viral loads in feces were 2.69×108GC/g and 4.32×107GC/g from children with or without AGE symptoms, respectively. In nasopharyngeal swab samples, the NoV positivity was 11.4% in symptomatic children, with a median viral load of 2.20×107GC/mL and 6.5% in asymptomatic children, with an average viral load of 1.73×106GC/mL. In only two cases NoV was detected in both samples. A considerable genomic variability was observed in feces, with six genotypes being detected, as follows: GII.4, GII.6, GI.3 and GII.3, GI.2 and GI.5. Two GI.3 was detected in nasopharyngeal swab. CONCLUSIONS: Our data reveal considerable NoV frequencies in both nasopharyngeal and fecal samples from symptomatic and asymptomatic children. Higher viral loads were detected in samples from AGE symptomatic children, when compared to asymptomatic children. High genomic variability was observed, with this being the first report of GI.5 NoV in Brazil and of GI.3 in nasopharyngeal swab samples.