Epidemiology Update of Hepatitis E Virus (HEV) in Uruguay: Subtyping, Environmental Surveillance and Zoonotic Transmission.

Hepatitis E Virus (HEV) infection is an emergent zoonotic disease of increasing concern in developed regions. HEV genotype 3 (HEV-3) is mainly transmitted through consumption of contaminated food in high-income countries and is classified into at least 13 subtypes (3a-3n), based on p-distance values from complete genomes. In Latin America, HEV epidemiology studies are very scant. Our group has previously detected HEV3 in clinical cases, swine, wild boars, captive white-collared peccaries, and spotted deer from Uruguay. Herein, we aimed to provide novel insights and an updated overview of the molecular epidemiology of zoonotic HEV in Uruguay, including data from wastewater-based surveillance studies. A thorough analysis of HEV whole genomes and partial ORF2 sequences from Uruguayan human and domestic pig strains showed that they formed a separate monophyletic cluster with high nucleotide identity and exhibited p-distance values over the established cut-off (0.093) compared with reference subtypes' sequences. Furthermore, we found an overall prevalence of 10.87% (10/92) in wastewater, where two samples revealed a close relationship with humans, and animal reservoirs/hosts isolates from Uruguay. In conclusion, a single, new HEV-3 subtype currently circulates in different epidemiological settings in Uruguay, and we propose its designation as 3o along with its reference sequence.

Wastewater surveillance of SARS-CoV-2 genomic populations on a country-wide scale through targeted sequencing.

SARS-CoV-2 surveillance of viral populations in wastewater samples is recognized as a useful tool for monitoring epidemic waves and boosting health preparedness. Next generation sequencing of viral RNA isolated from wastewater is a convenient and cost-effective strategy to understand the molecular epidemiology of SARS-CoV-2 and provide insights on the population dynamics of viral variants at the community level. However, in low- and middle-income countries, isolated groups have performed wastewater monitoring and data has not been extensively shared in the scientific community. Here we report the results of monitoring the co-circulation and abundance of variants of concern (VOCs) of SARS-CoV-2 in Uruguay, a small country in Latin America, between November 2020-July 2021 using wastewater surveillance. RNA isolated from wastewater was characterized by targeted sequencing of the Receptor Binding Domain region within the spike gene. Two computational approaches were used to track the viral variants. The results of the wastewater analysis showed the transition in the overall predominance of viral variants in wastewater from No-VOCs to successive VOCs, in agreement with clinical surveillance from sequencing of nasal swabs. The mutations K417T, E484K and N501Y, that characterize the Gamma VOC, were detected as early as December 2020, several weeks before the first clinical case was reported. Interestingly, a non-synonymous mutation described in the Delta VOC, L452R, was detected at a very low frequency since April 2021 when using a recently described sequence analysis tool (SAM Refiner). Wastewater NGS-based surveillance of SARS-CoV-2 is a reliable and complementary tool for monitoring the introduction and prevalence of VOCs at a community level allowing early public health decisions. This approach allows the tracking of symptomatic and asymptomatic individuals, who are generally under-reported in countries with limited clinical testing capacity. Our results suggests that wastewater-based epidemiology can contribute to improving public health responses in low- and middle-income countries.

Microbial Source Tracking Analysis Using Viral Indicators in Santa Lucía and Uruguay Rivers, Uruguay.

The aim of this study was to determine the origin (human, bovine or porcine) and the concentration of the fecal sources of contamination in waters from Santa Lucía basin and Uruguay River in Uruguay by using host-specific viral markers (adenoviruses and polyomaviruses) as microbial source tracking (MST). Between June 2015 and May 2016, monthly collections of surface water samples were performed in six sites in Santa Lucía basin and four sites in Uruguay River (n = 120 samples). Viral concentration was carried out using an absorption-elution method. Detection and quantification of human and porcine adenovirus (HAdV and PAdV, respectively) and human and bovine polyomavirus (HPyV and BoPyV, respectively) were performed by quantitative PCR (qPCR). To evaluate the infectivity of circulating HAdV, an integrated cell culture-qPCR (ICC-qPCR) was used. A logistic regression analysis was carried out to estimate the influence of environmental variables on the virus presence in surface waters. Overall, HAdV was the prevalent (18%; 21/120) followed by BoPyV (11%; 13/120) and HPyV (3%; 3/120), whereas PAdV was not detected in this study. The mean concentration ranged from 1.5 × 104 genomic copies/L (gc/L) for HAdV to 1.8 × 102 gc/L for HPyV. Infective HAdVs were observed in two out of ten analyzed samples. A significant effect of environmental temperature (p = 0.001) and river (p = 0.012) on the presence of human viruses was found. These results suggest that fecal contamination could affect the water quality of these rivers, showing deficiencies in the procedure of sewage discharge from regional cities, livestock and dairy farms.

Genetic diversity of Orf virus isolated from sheep in Uruguay.

Orf virus (ORFV) is the etiological agent of contagious ecthyma, a disease widely spread in the world that occasionally causes zoonotic infections. This work is the first molecular characterization of ORFV in Uruguay, where we analyzed twenty-one sheep samples, eighteen of which were recovered from thirteen ORFV outbreaks that occurred during 2004 to 2011 as well as three strains from a national vaccine. Phylogenetic analysis and the derived amino acid sequences from the B2L gene suggest that the Uruguayan virus do not form a unique cluster, with most of them displaying similarities with worldwide ORFV isolates as well as our vaccine strains.

OH1 from Orf Virus: A New Tyrosine Phosphatase that Displays Distinct Structural Features and Triple Substrate Specificity.

Viral tyrosine phosphatases such as VH1 from Vaccinia and Variola virus are recognized as important effectors of host-pathogen interactions. While proteins sharing sequence to VH1 have been identified in other viruses, their structural and functional characterization is not known. In this work, we determined the crystal structure of the VH1 homolog in the Orf virus, herein named OH1. Similarly to Variola and Vaccinia VH1, the structure of OH1 shows a dimer with the typical dual-specificity phosphatase fold. In contrast to VH1, the OH1 dimer is covalently stabilized by a disulfide bond involving residue Cys15 in the N-terminal helix alpha-1 of both monomers, and Cys15 is a conserved residue within the Parapoxvirus genus. The in vitro functional characterization confirms that OH1 is a dual-specificity phosphatase and reveals its ability to dephosphorylate phosphatidylinositol 3,5-bisphosphate, a new activity potentially relevant in phosphoinositide recycling during virion maturation.

Detection of Common, Emerging and Uncommon VP4, and VP7 Human Group A Rotavirus Genotypes from Urban Sewage Samples in Uruguay.

Environmental approach has proven to be a useful tool for epidemiological studies demonstrating through environmental studies the diversity of viruses circulating in a given population. The aim of this study was to perform a phylogenetic characterization of the group A rotavirus (RVA) glycoprotein (G)- and protease-sensitive (P)-genotypes obtained from sewage samples (n = 116) collected in six cities of Uruguay during March 2011 to April 2013. A worldwide standardized semi-nested multiplex RT-PCR (SNM RT-PCR) protocol directed against VP4 and VP7 genes were conducted for RVA detection and consensual DNA fragments were submitted to nucleotide sequencing. P and/or G genotype was successfully determined by phylogenetic analysis in 61% (n = 37) of the positive samples obtained by SNM RT-PCR (n = 61). The RVA genotypes were as follow: G1 (n = 2), G2 (n = 14), G3 (n = 5), G12 (n = 2), P[4] (n = 4), P[8] (n = 16), and P[3] (n = 2). Interestingly, through phylogenetic analysis, emerging, and uncommon human genotypes could be detected. Results obtained from the comparison of RVA genotypes detected in the current study and Uruguayan RVA strains previously described for contemporary clinical pediatric cases showed that monitoring sewage may be a good screening option for a rapid and economical overview of the circulating genotypes in the surrounding human population and a useful approximation to study RVA epidemiology in a future vaccine monitoring program. The present study represents the first report in Uruguay that describes the phylogenetic diversity of RVA from urban sewage samples.

HSV-1 amplicon vectors launch the production of heterologous rotavirus-like particles and induce rotavirus-specific immune responses in mice.

Virus-like particles (VLPs) are promising vaccine candidates because they represent viral antigens in the authentic conformation of the virion and are therefore readily recognized by the immune system. As VLPs do not contain genetic material they are safer than attenuated virus vaccines. In this study, herpes simplex virus type 1 (HSV-1) amplicon vectors were constructed to coexpress the rotavirus (RV) structural genes VP2, VP6, and VP7 and were used as platforms to launch the production of RV-like particles (RVLPs) in vector-infected mammalian cells. Despite the observed splicing of VP6 RNA, full-length VP6 protein and RVLPs were efficiently produced. Intramuscular injection of mice with the amplicon vectors as a two-dose regimen without adjuvants resulted in RV-specific humoral immune responses and, most importantly, immunized mice were partially protected at the mucosal level from challenge with live wild-type (wt) RV. This work provides proof of principle for the application of HSV-1 amplicon vectors that mediate the efficient production of heterologous VLPs as genetic vaccines.

Induction of humoral responses to BHV-1 glycoprotein D expressed by HSV-1 amplicon vectors.

Herpes simplex virus type-1 (HSV-1) amplicon vectors are versatile and useful tools for transferring genes into cells that are capable of stimulating a specific immune response to their expressed antigens. In this work, two HSV-1-derived amplicon vectors were generated. One of these expressed the full-length glycoprotein D (gD) of bovine herpesvirus 1 while the second expressed the truncated form of gD (gDtr) which lacked the trans-membrane region. After evaluating gD expression in the infected cells, the ability of both vectors to induce a specific gD immune response was tested in BALB/c mice that were intramuscularly immunized. Specific serum antibody responses were detected in mice inoculated with both vectors, and the response against truncated gD was higher than the response against full-length gD. These results reinforce previous findings that HSV-1 amplicon vectors can potentially deliver antigens to animals and highlight the prospective use of these vectors for treating infectious bovine rhinotracheitis disease.

Genetic characterization of respiratory syncytial viruses isolated from consecutive acute respiratory infections in a HIV infected child.

BACKGROUND: Human respiratory syncytial virus (HRSV) was isolated from a 2-year-old child suffering from perinatally transmitted AIDS in the course of three distinct episodes of respiratory infections. The first episode occurred in the winter of 1994, the following two episodes of cough and fever occurred two and 4 months after the initial episode. OBJECTIVE: To analyze the genetic variability of the child's HRSV strains, and with contemporary circulating HRSV isolates. RESULTS: The three child's HRSV isolates belonged to group B. Sequence analysis of the attachment (G) protein gene (which has the highest degree of antigenic and genetic diversity in HRSV), demonstrated no difference in the sequence obtained from the three isolates recovered from the child. Comparison of the child's HRSV strain with contemporary circulating group B HRSV isolates showed a close sequence similarity with one of them. CONCLUSIONS: The immunodeficiency in an HIV-positive child may have resulted in the recurrent isolation of one HRSV strain. Although it cannot be discarded the possibility that the recurrent episodes might be re-infections, it is unlikely in view of the lack of change in the HRSV glycoprotein G. This is the first study that analyzes the genetic variation in HRSV isolates from consecutive respiratory disease episodes in an immunosuppressed patient.

Genetic variation in the VP7 gene of human rotavirus isolated in Montevideo-Uruguay from 1996-1999.

The gene encoding the protein VP7 that induces the major neutralizing response has been sequenced from 34 human rotaviruses isolated from children with acute diarrhea in Montevideo (Uruguay) over a 4-year period (1996-1999). These sequences were analyzed and compared to representative corresponding sequences available on databases. In most years, serotype G1 was present as the single serotype, except in 1999 when serotypes G1 and G4 were present simultaneously. Two G1 VP7 lineages were identified. Serotype G2 was present in 1997. The G4 isolates are grouped with Argentine strains and emerged during 1998 in a recently defined sublineage. Neither serotype G3 nor the emerging serotype G9 were isolated during the study. Antigenic domains of isolates and of representative reference strains of each serotype were compared. Sequences of strains isolated during the same year, showed a high degree of homology among strains belonging to the same serotype.

Antigenic and genetic variability of human respiratory syncytial viruses (group A) isolated in Uruguay and Argentina: 1993-2001.

The antigenic and genetic diversity of G glycoprotein from 25 human respiratory viruses (group A) isolated during nine consecutive epidemics (1993-2001) in Montevideo, Uruguay, and 7 strains isolated in Buenos Aires, Argentina, in the same period were analyzed. Genetic variability was evaluated by partial sequence of the G protein gene. Phylogenetic analysis indicated that most Uruguayan and Argentinean group A isolates clustered into three genotypes: GA5, GA2, and GA1. Some strains clustered into the GA3 genotype characterized previously. The antigenic analysis was carried out with a panel of anti-G monoclonal antibodies that recognized conserved and strain-specific epitopes. A close correlation between the antigenic and genetic relatedness of the strains analyzed was observed.

Rotavirus nonstructural protein NSP5 interacts with major core protein VP2.

Rotavirus is a nonenveloped virus with a three-layered capsid. The inner layer, made of VP2, encloses the genomic RNA and two minor proteins, VP1 and VP3, with which it forms the viral core. Core assembly is coupled with RNA viral replication and takes place in definite cellular structures termed viroplasms. Replication and encapsidation mechanisms are still not fully understood, and little information is available about the intermolecular interactions that may exist among the viroplasmic proteins. NSP2 and NSP5 are two nonstructural viroplasmic proteins that have been shown to interact with each other. They have also been found to be associated with precore replication intermediates that are precursors of the viral core. In this study, we show that NSP5 interacts with VP2 in infected cells. This interaction was demonstrated with recombinant proteins expressed from baculovirus recombinants or in bacterial systems. NSP5-VP2 interaction also affects the stability of VP6 bound to VP2 assemblies. The data presented showed evidence, for the first time, of an interaction between VP2 and a nonstructural rotavirus protein. Published data and the interaction demonstrated here suggest a possible role for NSP5 as an adapter between NSP2 and the replication complex VP2-VP1-VP3 in core assembly and RNA encapsidation, modulating the role of NSP2 as a molecular motor involved in the packaging of viral mRNA.