Recent insights into SARS-CoV-2 omicron variant.

The SARS-CoV-2 omicron variant (B.1.1.529) was first identified in Botswana and South Africa, and its emergence has been associated with a steep increase in the number of SARS-CoV-2 infections. The omicron variant has subsequently spread very rapidly across the world, resulting in the World Health Organization classification as a variant of concern on 26 November 2021. Since its emergence, great efforts have been made by research groups around the world that have rapidly responded to fill our gaps in knowledge for this novel variant. A growing body of data has demonstrated that the omicron variant shows high transmissibility, robust binding to human angiotensin-converting enzyme 2 receptor, attenuated viral replication, and causes less severe disease in COVID-19 patients. Further, the variant has high environmental stability, high resistance against most therapeutic antibodies, and partial escape neutralisation by antibodies from convalescent patients or vaccinated individuals. With the pandemic ongoing, there is a need for the distillation of literature from primary research into an accessible format for the community. In this review, we summarise the key discoveries related to the SARS-CoV-2 omicron variant, highlighting the gaps in knowledge that guide the field's ongoing and future work.

Development of a New Multispecies Protein A-Based ELISA for Serodiagnosis of Hepatitis E Infection with Validation in Domestic Swine.

Hepatitis E virus (HEV) infects roughly 20 million people worldwide, causing self-limiting acute hepatic disease that can evolve into a chronic course. HEV-3, HEV-4, and HEV-7 genotypes are zoonotic and transmitted to humans by consuming raw or undercooked meat. Here, we developed an indirect ELISA based on the recombinant HEV-3 capsid and performed a seroprevalence study on domestic swine in northeastern Brazil. Our in-house ELISA was initially validated using a subset of 79 sera characterized by concordant results for two distinct commercial ELISA kits. Our ELISA exhibited excellent sensitivity (94%) and specificity (100%), with an area under the curve of 0.99 Further testing, including 212 swine sera, revealed a seroprevalence of 57.5% (95% confidence interval, 50.6-64.3%). Our findings indicate that the novel ELISA test could accurately detect specific anti-HEV antibodies in domestic pigs and should be further validated in humans and other mammals.

Uncovering neglected subtypes and zoonotic transmission of Hepatitis E virus (HEV) in Brazil.

Hepatitis E virus (HEV) circulation in humans and swine has been extensively studied in South America over the last two decades. Nevertheless, only 2.1% of reported HEV strains are available as complete genome sequences. Therefore, many clinical, epidemiological, and evolutionary aspects of circulating HEV in the continent still need to be clarified. Here, we conducted a retrospective evolutionary analysis of one human case and six swine HEV strains previously reported in northeastern, southern, and southeastern Brazil. We obtained two complete and four nearly complete genomic sequences. Evolutionary analysis comparing the whole genomic and capsid gene sequences revealed high genetic variability. This included the circulation of at least one unrecognized unique South American subtype. Our results corroborate that sequencing the whole capsid gene could be used as an alternative for HEV subtype assignment in the absence of complete genomic sequences. Moreover, our results substantiate the evidence for zoonotic transmission by comparing a larger genomic fragment recovered from the sample of the autochthonous human hepatitis E case. Further studies should continuously investigate HEV genetic diversity and zoonotic transmission of HEV in South America.

Widespread contamination of SARS-CoV-2 on highly touched surfaces in Brazil during the second wave of the COVID-19 pandemic.

Although SARS-CoV-2 surface contamination has been investigated in health care settings, little is known about the SARS-CoV-2 surface contamination in public urban areas, particularly in tropical countries. Here, we investigated the presence of SARS-CoV-2 on high-touch surfaces in a large city in Brazil, one of the most affected countries by the COVID-19 pandemic in the world. A total of 400 surface samples were collected in February 2021 in the City of Recife, Northeastern Brazil. A total of 97 samples (24.2%) tested positive for SARS-CoV-2 by RT-qPCR using the CDC-USA protocol. All the collection sites, except one (18/19, 94.7%) had at least one environmental surface sample contaminated. SARS-CoV-2 positivity was higher in public transport terminals (47/84, 55.9%), followed by health care units (26/84, 30.9%), beach areas (4/21, 19.0%), public parks (14/105, 13.3%), supply centre (2/21, 9.5%), and public markets (4/85, 4.7%). Toilets, ATMs, handrails, playgrounds and outdoor gyms were identified as fomites with the highest rates of SARS-CoV-2 detection. Taken together, our data provide a real-world picture of SARS-CoV-2 dispersion in highly populated tropical areas and identify critical control points that need to be targeted to break SARS-CoV-2 transmission chains.

Adaptive, diverse and de-centralized diagnostics are key to the future of outbreak response.

The global spread of SARS-CoV-2 has shaken our health care and economic systems, prompting re-evaluation of long-held views on how best to deliver care. This is especially the case for our global diagnostic strategy. While current laboratory-based centralized RT-qPCR will continue to serve as a gold standard diagnostic into the foreseeable future, the shortcomings of our dependence on this method have been laid bare. It is now clear that a robust diagnostics pandemic response strategy, like any disaster planning, must include adaptive, diverse and de-centralized solutions. Here we look at how the COVID-19 pandemic, and previous outbreaks, have set the stage for a new innovative phase in diagnostics and a re-thinking of pandemic preparedness.

Functional evaluation of human papillomavirus type 31 long control region variants.

Persistent infections by high-risk human papillomavirus (HR-HPV) are a necessary condition, but not sufficient for cervical cancer development. Genetic variants of HR-HPV appear to be related to the risk of persistent infections. The study performed a functional evaluation of variants of the HPV-31 promoter region (LCR). For this, cloning and subcloning of variants HPV-31/UFPE-21 HPV-31/UFPE-89, HPV-31/UFPE-66, E2 gene and prototype HPV-31 were performed. Transfection with different concentrations of E2 was done and the concentration of 25 ng was determined to be ideal for LCR activation. HPV-31/UFPE-21 and HPV-31/UFPE-89 have a greater ability to alter Nluc reporter gene expression levels and HPV-31/UFPE-66 showed decreased levels of gene expression of Nluc reporter gene compared to control. Statistical analysis showed a significant difference between the polymorphic LCR regions and the control (p < 0.0001). A more refined profile of variants of HPV-31 and its importance for the prognosis of cervical lesions begins to be drawn.

Searching Anti-Zika Virus Activity in 1H-1,2,3-Triazole Based Compounds.

Zika virus (ZIKV) is a mosquito-borne virus belonging to the Flaviviridae family and is responsible for an exanthematous disease and severe neurological manifestations, such as microcephaly and Guillain-Barré syndrome. ZIKV has a single strand positive-sense RNA genome that is translated into structural and non-structural (NS) proteins. Although it has become endemic in most parts of the tropical world, Zika still does not have a specific treatment. Thus, in this work we evaluate the cytotoxicity and antiviral activities of 14 hybrid compounds formed by 1H-1,2,3-triazole, naphthoquinone and phthalimide groups. Most compounds showed low cytotoxicity to epithelial cells, specially the 3b compound. After screening with all compounds, 4b was the most active against ZIKV in the post-infection test, obtaining a 50% inhibition concentration (IC50) of 146.0 µM and SI of 2.3. There were no significant results for the pre-treatment test. According to the molecular docking compound, 4b was suggested with significant binding affinity for the NS5 RdRp protein target, which was further corroborated by molecular dynamic simulation studies.

Insights into SARS-CoV-2, the Coronavirus Underlying COVID-19: Recent Genomic Data and the Development of Reverse Genetics Systems.

The emergence and rapid worldwide spread of a novel pandemic of acute respiratory disease - eventually named coronavirus disease 2019 (COVID-19) by the World Health Organization (WHO) - across the human population has raised great concerns. It prompted a mobilization around the globe to study the underlying pathogen, a close relative of severe acute respiratory syndrome coronavirus (SARS-CoV) called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Numerous genome sequences of SARS-CoV-2 are now available and in-depth analyses are advancing. These will allow detailed characterization of sequence and protein functions, including comparative studies. Care should be taken when inferring function from sequence information alone, and reverse genetics systems can be used to unequivocally identify key features. For example, the molecular markers of virulence, host range and transmissibility of SARS-CoV-2 can be compared to those of related viruses in order to shed light on the biology of this emerging pathogen. Here, we summarize some recent insights from genomic studies and strategies for reverse genetics systems to generate recombinant viruses, which will be useful to investigate viral genome properties and evolution.

Norovirus-associated gastroenteritis, Pernambuco, Northeast Brazil, 2014-2017.

Norovirus (NoV) is currently the leading cause of nonbacterial gastroenteritis. In Brazil, few studies have characterized the molecular, epidemiological and clinical features of NoV-associated gastroenteritis. This study aimed to describe the molecular and clinicoepidemiological findings of NoV infections in patients of all ages throughout Pernambuco state, Northeast Brazil. Thus, 1135 stool samples were analyzed from patients with gastroenteritis from Pernambuco state. NoV was detected by enzyme immunoassay in 125 (11.01%) samples. Regarding gender distribution, 55 (44.00%) patients were female and 70 (56.00%) male. Their ages ranged from 5 days to 87 years, and the group most affected by NoV infection (88.00%) was children under 3 years. Complete clinical information was available for 88 out of 125 NoV-positive patients. Diarrhea was present in all patients and vomiting was reported in 60 patients (68.68%). Nine patients (10.22%) had bloody stools and 46 (52.27%) had a fever, with temperatures ranging from 37.90°C to 39.90°C (mean 38.20°C). NoV was detected mainly in the summer-autumn seasons. Genome sequencing and phylogenetic analyses identified four different NoV GII genotypes circulating in this area of the country. Therefore, our study provided valuable information about the clinics and epidemiology of NoV infection in tropical settings and will assist health authorities to develop better control strategies against this important pathogen.

Discovery of New Hydroxyethylamine Analogs against 3CLpro Protein Target of SARS-CoV-2: Molecular Docking, Molecular Dynamics Simulation, and Structure-Activity Relationship Studies.

The novel coronavirus, SARS-CoV-2, has caused a recent pandemic called COVID-19 and a severe health threat around the world. In the current situation, the virus is rapidly spreading worldwide, and the discovery of a vaccine and potential therapeutics are critically essential. The crystal structure for the main protease (Mpro) of SARS-CoV-2, 3-chymotrypsin-like cysteine protease (3CLpro), was recently made available and is considerably similar to the previously reported SARS-CoV. Due to its essentiality in viral replication, it represents a potential drug target. Herein, a computer-aided drug design (CADD) approach was implemented for the initial screening of 13 approved antiviral drugs. Molecular docking of 13 antivirals against the 3-chymotrypsin-like cysteine protease (3CLpro) enzyme was accomplished, and indinavir was described as a lead drug with a docking score of -8.824 and a XP Gscore of -9.466 kcal/mol. Indinavir possesses an important pharmacophore, hydroxyethylamine (HEA), and thus, a new library of HEA compounds (>2500) was subjected to virtual screening that led to 25 hits with a docking score more than indinavir. Exclusively, compound 16 with a docking score of -8.955 adhered to drug-like parameters, and the structure-activity relationship (SAR) analysis was demonstrated to highlight the importance of chemical scaffolds therein. Molecular dynamics (MD) simulation analysis performed at 100 ns supported the stability of 16 within the binding pocket. Largely, our results supported that this novel compound 16 binds with domains I and II, and the domain II-III linker of the 3CLpro protein, suggesting its suitability as a strong candidate for therapeutic discovery against COVID-19.

Multi-targeted gene silencing strategies inhibit replication of Canine morbillivirus.

BACKGROUND: Canine morbilivirus (canine distemper virus, CDV) is a highly contagious pathogen associated with high morbidity and mortality in susceptible carnivores. Although there are CDV vaccines available, the disease poses a huge threat to dogs and wildlife hosts due to vaccine failures and lack of effective treatment. Thus, the development of therapeutics is an urgent need to achieve rapid outbreak control and reduce mortality in target species. Gene silencing by RNA interference has emerged as a promising therapeutic approach against different human and animal viruses. In this study, plasmid-based short hairpin RNAs (shRNAs) against three different regions in either CDV nucleoprotein (N), or large polymerase (L) genes and recombinant adenovirus-expressing N-specific multi-shRNAs were generated. Viral cytopathic effect, virus titration, plaque-forming unit reduction, and real-time quantitative RT-PCR analysis were used to check the efficiency of constructs against CDV. RESULTS: In CDV-infected VerodogSLAM cells, shRNA-expressing plasmids targeting the N gene markedly inhibited the CDV replication in a dose-dependent manner, with viral genomes and titers being decreased by over 99%. Transfection of plasmid-based shRNAs against the L gene displayed weaker inhibition of viral RNA level and virus yield as compared to CDV N shRNAs. A combination of shRNAs targeting three sites in the N gene considerably reduced CDV RNA and viral titers, but their effect was not synergistic. Recombinant adenovirus-expressing multiple shRNAs against CDV N gene achieved a highly efficient knockdown of CDV N mRNAs and successful inhibition of CDV replication. CONCLUSIONS: We found that this strategy had strong silencing effects on CDV replication in vitro. Our findings indicate that the delivery of shRNAs using plasmid or adenovirus vectors potently inhibits CDV replication and provides a basis for the development of therapeutic strategies for clinical trials.

Tri- and Diterpenoids from Stillingia loranthacea as Inhibitors of Zika Virus Replication.

This study represents the first phytochemical analysis of Stillingia loranthacea (S. loranthacea) and describes new terpenoids obtained from the root bark of this species. The fractionation of the hexane extract from the root bark led to the isolation of two new 28-nor-taraxarenes derivatives, loranthones A and B (1 and 2), four new tigliane diterpenes (5-8), three known tigliane diterpenes (9-11), and three known flexibilene diterpenes, tonantzitlolones A-C (12-14). The investigation of these compounds and the use of a molecular networking-based prioritization approach afforded two other new 28-nor-taraxarenes, loranthones C and D (3 and 4). The cytotoxicity of compounds 1, 2, and 5-14 was evaluated against Vero cells, and their 20% cytotoxic concentration (CC20) values varied from 8.7 to 328 µM; antiviral activity was tested against an epidemic Zika virus (ZIKV) strain circulating in Brazil. Six out of 12 compounds (2, 5, 9-11, and 14) exhibited significant antiviral effects against ZIKV. Specifically, compounds 2 and 5 offered the most promise as lead compounds as they had a 1.7 and 1.8 log10 TCID50/mL reduction in ZIKV replication, respectively. Together, the present findings have identified S. loranthacea terpenoids as potent anti-ZIKV inhibitors and pave the way to the development of possible new treatments against this devastating pathogen.

Evolutionary study of potentially zoonotic hepatitis E virus genotype 3 from swine in Northeast Brazil.

Hepatitis E virus (HEV), an emerging virus associated with acute hepatic disease, leads to thousands of deaths worldwide. HEV has already been reported in Brazil; however, there is a lack of epidemiological and molecular information on the genetic variability, taxonomy, and evolution of HEV. It is thus unclear whether hepatitis E is a neglected disease in Brazil or it has low relevance for public health in this country. Here, for the first time, we report the presence of HEV in Northeast Brazil. A total of 119 swine faecal samples were screened for the presence of HEV RNA using real-time polymerase chain reaction (RT-PCR) and further confirmed by conventional RT-PCR; among these, two samples were identified as positive. Molecular evolution analyses based on capsid sequences revealed that the samples had close proximities to HEV sequences belonging to genotype 3 and were genetically related to subtype 3f isolated in humans. Parsimony ancestral states analysis indicated gene flow events from HEV cross-species infection, suggesting an important role of pig hosts in viral spillover. HEV's ability for zoonotic transmission by inter-species host switching as well as its possible adaptation to new animal species remain important issues for human health.

In vitro and in vivo models for studying Zika virus biology.

The emergence and rapid spread of Zika virus (ZIKV) in the Americas has prompted the development of in vitro and in vivo models to understand several aspects of ZIKV biology and boost the development of vaccines and antivirals. In vitro model studies include reverse genetics systems, two-dimensional (2D) cell models, such as primary cells and cell lines, and ex vivo three-dimensional (3D) models derived from skin, brain and placenta. While these models are cost-effective and allow rigorous control of experimental variables, they do not always recapitulate in vivo scenarios. Thus, a number of in vivo models have been developed, including mosquitoes (Aedes sp. and Culex sp.), embryonated chicken eggs, immunocompetent and immunodeficient mice strains, hamsters, guinea pigs, conventional swine and non-human primates. In this review, we summarize the main research systems that have been developed in recent years and discuss their advantages, limitations and main applications.

Lessons Learned at the Epicenter of Brazil's Congenital Zika Epidemic: Evidence From 87 Confirmed Cases.

Congenital Zika virus infection has stimulated great international concern. A prospective case series of 87 infants with laboratory-confirmed congenital Zika syndrome (CZS) at the epicenter of the Brazilian Zika epidemic in Pernambuco state is presented. Mothers were interviewed for symptoms of possible Zika virus (ZIKV) infection during pregnancy, and fetal ultrasounds were obtained. Infant cerebrospinal fluid (CSF) samples were tested for ZIKV-specific antibodies, and sera were screened for other congenital infections. Neuroimaging and ophthalmologic evaluations were also performed. Sixty-six mothers (76%) reported symptoms of ZIKV infection during gestation. Fetal ultrasounds were available from 90% of the mothers, and all demonstrated brain structural abnormalities. All of the CSF samples tested positive for ZIKV immunoglobulin M. The majority of infants (89%) were term; the mean birth weight was 2577 ± 260 g, and the mean head circumference was 28.1 ± 1.8 cm. Severe microcephaly, defined as head circumference 3 SD below the mean for sex and gestational age, was found in 72 (82%) infants. All infants had an abnormal neurological exam, and 18 (20.7%) had arthrogryposis. The main abnormalities detected in computed tomography scans were calcifications (99%), followed by ventricular enlargement (94%), cortical hypogyration (81%), and less commonly, cerebellar hypoplasia (52%). Unilateral diaphragm paralysis was identified in 3 infants. Maternal young age, term infant, small for gestational age, and the presence of ophthalmologic abnormalities were significantly associated with a smaller head circumference Z score. Our findings, based on laboratory-confirmed ZIKV infection, add valuable evidence for the understanding of CZS.

6-methylmercaptopurine riboside, a thiopurine nucleoside with antiviral activity against canine distemper virus in vitro.

BACKGROUND: Canine distemper (CD) is a widespread infectious disease that can severely impact a variety of species in the order Carnivora, as well as non-carnivore species such as non-human primates. Despite large-scale vaccination campaigns, several fatal outbreaks have been reported in wild and domestic carnivore populations. This, in association with expansion of the disease host range and the development of vaccine-escape strains, has contributed to an increased demand for therapeutic strategies synergizing with vaccine programs for effectively controlling canine distemper. 6-methylmercaptopurine riboside (6MMPr) is a modified thiopurine nucleoside with known antiviral properties against certain RNA viruses. METHODS: We tested the inhibitory effects of 6MMPr against a wild-type CDV strain infection in cell culture. We measured infectious particle production and viral RNA levels in treated and untreated CDV-infected cells. Ribavirin (RIB) was used as a positive control. RESULTS: Here, we report for the first time the antiviral effects of 6MMPr against canine distemper virus (CDV) in vitro. 6MMPr was able to reduce viral RNA levels and to inhibit the production of infectious CDV particles. The therapeutic selectivity of 6MMPr was approximately six times higher than that of ribavirin. CONCLUSION: Our results indicate that 6MMPr has high anti-CDV potential and warrants further testing against other paramyxoviruses, as well as clinical testing of the compound against CDV.

Results of a Zika Virus (ZIKV) Immunoglobulin M-Specific Diagnostic Assay Are Highly Correlated With Detection of Neutralizing Anti-ZIKV Antibodies in Neonates With Congenital Disease.

BACKGROUND: Usually, immunoglobulin M (IgM) serologic analysis is not sufficiently specific to confirm Zika virus (ZIKV) infection. However, since IgM does not cross the placenta, it may be a good marker of infection in neonates. METHODS: We tested blood from 42 mothers and neonates with microcephaly and collected cerebrospinal fluid (CSF) specimens from 30 neonates. Molecular assays were performed for detection of ZIKV, dengue virus, and chikungunya virus; IgM enzyme-linked immunosorbent assays and plaque-reduction neutralization tests (PRNTs) were performed to detect ZIKV and dengue virus. No control neonates without microcephaly were evaluated. RESULTS: Among neonates, all 42 tested positive for ZIKV IgM: 38 of 42 serum specimens (90.5%) were positive, whereas 30 of 30 CSF specimens (100%) were positive. ZIKV IgM-specific ELISA ratios, calculated as the mean optical density (OD) of the test sample when reacted on viral antigen divided by the mean OD of the negative control when reacted with viral antigen, were higher in CSF specimens (median, 14.9 [range, 9.3-16.4]) than in serum (median, 8.9 [range, 2.1-20.6]; P = .0003). All ZIKV IgM-positive results among the neonates were confirmed by the detection of neutralizing antibodies. Mother/neonate pairs with primary ZIKV infection had neutralizing antibodies to ZIKV only, and mother/neonate pairs with ZIKV virus infection secondary to infection with another flavivirus had high titers of neutralizing antibodies to ZIKV. Among secondary infections, median titers in serum were 2072 (range, 232-12 980) for mothers and 2730 (range, 398-12 980) for neonates (P < .0001), and the median titer in CSF was 93 (range, 40-578) among neonates (P < .0001). CONCLUSIONS: Among neonates, detection of ZIKV IgM in serum is confirmatory of congenital ZIKV infection, and detection of ZIKV IgM in CSF is confirmatory of neurologic infection. Therefore, we recommend testing for ZIKV IgM in neonates suspected of having congenital ZIKV infection and performance of PRNTs in equivocal cases.

Polymorphisms in the haemagglutinin gene influenced the viral shedding of pandemic 2009 influenza virus in swine.

Interactions between the viral surface glycoprotein haemagglutinin (HA) and the corresponding receptors on host cells is one important aspect of influenza virus infection. Mutations in HA have been described to affect pathogenicity, antigenicity and the transmission of influenza viruses. Here, we detected polymorphisms present in HA genes of two pandemic 2009 H1N1 (H1N1pdm09) isolates, A/California/04/2009 (Ca/09) and A/Mexico/4108/2009 (Mx/09), that resulted in amino acid changes at positions 186 (S to P) and 194 (L to I) of the mature HA1 protein. Although not reported in the published H1N1pdm09 consensus sequence, the P186 genotype was more readily detected in primary infected and contact-naïve pigs when inoculated with a heterogeneous mixed stock of Ca/09. Using reverse genetics, we engineered Ca/09 and Mx/09 genomes by introducing Ca/09 HA with two naturally occurring variants expressing S186/I194 (HA-S/I) and P186/L194 (HA-P/L), respectively. The Ca/09 HA with the combination of P186/L194 with either the Ca/09 or Mx/09 backbone resulted in higher and prolonged viral shedding in naïve pigs. This efficiency appeared to be more likely through an advantage in cell surface attachment rather than replication efficiency. Although these mutations occurred within the receptor-binding pocket and the Sb antigenic site, they did not affect serological cross-reactivity. Relative increases of P186 in publicly available sequences from swine H1N1pdm09 viruses supported the experimental data, indicating this amino acid substitution conferred an advantage in swine.