In Vitro Antiviral Activity of Nordihydroguaiaretic Acid against SARS-CoV-2.

The coronavirus infectious disease 2019 (COVID-19) is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and has been spreading rapidly worldwide, creating a pandemic. This article describes the evaluation of the antiviral activity of nordihydroguaiaretic acid (NDGA), a molecule found in Creosote bush (Larrea tridentata) leaves, against SARS-CoV-2 in vitro. A 35 µM concentration of NDGA was not toxic to Vero cells and exhibited a remarkable inhibitory effect on the SARS-CoV-2 cytopathic effect, viral plaque formation, RNA replication, and expression of the SARS-CoV-2 spike glycoprotein. The 50% effective concentration for NDGA was as low as 16.97 µM. Our results show that NDGA could be a promising therapeutic candidate against SARS-CoV-2.

In vitro inhibition of SARS-CoV-2 Infection by dry algae powders.

Chlorella spp., Spirulina spp., and fucoidan dry powders, are commercialized as food supplements and are considered safe for human consumption. Their broad-spectrum antiviral properties have been studied, however, their effect against SARS-CoV-2 remains unknown. We investigated the potential antiviral activity of three algae powders: Chlorella vulgaris, Arthrospira maxima (Spirulina) and fucoidan purified from marine brown algae Sargassum spp. against SARS-CoV-2 infection in vitro. Vero cells were incubated with 70 µg/ml of each algae powder and either 50 or 100 TCID50/ml of SARS-CoV-2, in two types of experiments (pretreatment and simultaneous) and comparing two kinds of solvents (DMEM and DMSO). Chlorella vulgaris powder, inhibited SARS-CoV-2 infection in all assays; viral RNA was significantly reduced in supernatants at 24, 48, 72, and 96 h post-infection, the highest difference in viral load (8000-fold) was observed after 96 h. Arthrospira maxima powder inhibited SARS-CoV-2 infection using 50 TCID50/ml for both experimental schemes, but protection percent was lower when viral inoculum was increase to 100 TCID50/ml; viral RNA decreased 48 h after infection, reaching a 250-fold difference at 72 h. Fucoidan powder partially inhibited SARS-CoV-2 infection since no CPE was observed in 62.5% of trated cultures in DMEM, but the antiviral activity was increased to 100% of protection when DMSO was used as solvent. All the algae samples showed high antiviral activity against SARS-CoV-2 with a SI above of 18. These results suggest that all three algae samples are potential therapeutic candidates for the treatment of COVID-19.

Exclusive circulation of canine parvovirus type 2c in the Guadalajara metropolitan area in western Mexico: a five-year study.

Canine parvovirus type 2 (CPV-2) infection in dogs is associated with severe gastroenteritis, bloody diarrhea, and vomiting, resulting in high rates of death, especially in unvaccinated puppies within the first months of age. There are three variants, called CPV-2a, CPV-2b, and CPV-2c, co-circulating worldwide. Our group previously reported that the only circulating CPV-2 variant in the Guadalajara metropolitan area in western Mexico was type 2c. Now, a five-year study was performed in order to investigate the possible dominance of CPV-2c in our region. Rectal swabs were collected from 146 dogs with clinical gastroenteritis from May 2014 to August 2019 at the Veterinary Hospital of the University of Guadalajara. Of these, 90 dogs tested positive for canine parvovirus by PCR. Most of the infected dogs with CPV-2 had a partial or incomplete vaccination status (n = 88, 97.8%). Approximately 65% (n = 59) of them were mixed-breed dogs, 77.8% (n = 70) were under 6 months of age, and 37.8% (n = 34) of them died from clinical complications. RFLP analysis of amplicons derived from the vp2 gene showed that all 90 DNA samples corresponded to CPV-2c, with no evidence of the presence of CPV-2a or CPV-2b variants. Twenty-nine of the 90 DNA samples were selected for amplification of a portion of the vp2 gene, and sequencing of these amplicons showed that all of them had the sequence GAA at codon 426, encoding the amino acid glutamic acid, which is characteristic of CPV-2c. Phylogenetic analysis showed that the CPV-2c sequences were related to those of viruses from Europe and South America. The present study indicates that CPV-2c is still the only variant circulating in the dog population of the Guadalajara metropolitan area.

Possible Routes for Zika Virus Vertical Transmission in Human Placenta: A Comprehensive Review.

Zika virus (ZIKV) infections have gained notoriety due to congenital abnormalities. Pregnant women have a greater risk of ZIKV infection and consequent transmission to their progeny due to the immunological changes associated with pregnancy. ZIKV has been detected in amniotic fluid, as well as in fetal and neonatal tissues of infected pregnant women. However, the mechanism by which ZIKV reaches the fetus is not well understood. The four dengue virus serotypes have been the most widely used flaviviruses to elucidate the host-cell entry pathways. Nevertheless, it is of increasing interest to understand the specific interaction between ZIKV and the host cell, especially in the gestation period. Herein, the authors describe the mechanisms of prenatal vertical infection of ZIKV based on results from in vitro, in vivo, and ex vivo studies, including murine models and nonhuman primates. It also includes up-to-date knowledge from ex vivo and natural infections in pregnant women explaining the vertical transmission along four tracks: transplacental, paracellular, transcytosis mediated by extracellular vesicles, and paraplacental route and the antibody-dependent enhancement process. A global understanding of the diverse pathways used by ZIKV to cross the placental barrier and access the fetus, along with a better comprehension of the pathogenesis of ZIKV in pregnant females, may constitute a fundamental role in the design of antiviral drugs to reduce congenital disabilities associated with ZIKV.

Development of a Platform for Noncovalent Coupling of Full Antigens to Tobacco Etch Virus-Like Particles by Means of Coiled-Coil Oligomerization Motifs.

Virus-like particles are excellent inducers of the adaptive immune response of humans and are presently being used as scaffolds for the presentation of foreign peptides and antigens derived from infectious microorganisms for subunit vaccine development. The most common approaches for peptide and antigen presentation are translational fusions and chemical coupling, but some alternatives that seek to simplify the coupling process have been reported recently. In this work, an alternative platform for coupling full antigens to virus-like particles is presented. Heterodimerization motifs inserted in both Tobacco etch virus coat protein and green fluorescent protein directed the coupling process by simple mixing, and the obtained complexes were easily taken up by a macrophage cell line.

Updating zika diagnostic methods: the point-of-care approach

Abstract Zika virus (ZIKV) has gained great importance worldwide since the past epidemic that occurred in 2015 in Brazil. Early identification of ZIKV is critical to minimize transmission and prevents potentially devastating consequences, including microcephaly in neonates of infected women, congenital blindness, or Guillain-Barré Syndrome. However, this is not an easy task, considering that approximately 80% of ZIKV infection cases are asymptomatic or oligosymptomatic, there are diverse modes of transmission (vertical transmission is through vectors and horizontal transmission through blood, saliva, semen, and urine from infected people), and the fact that ZIKV has a high identity percentage with other cocirculating Flaviviruses such as dengue. Here, we review ZIKV diagnostic methods, with special emphasis on the development of point-of-care diagnostic assays, since these devices commonly have two important advantages: they provide prompt screening and are affordable.

Updating Zika Diagnostic Methods: The Point-of-Care Approach.

Zika virus (ZIKV) has gained great importance worldwide since the past epidemic that occurred in 2015 in Brazil. Early identification of ZIKV is critical to minimize transmission and prevents potentially devastating consequences, including microcephaly in neonates of infected women, congenital blindness, or Guillain-Barré Syndrome. However, this is not an easy task, considering that approximately 80% of ZIKV infection cases are asymptomatic or oligosymptomatic, there are diverse modes of transmission (vertical transmission is through vectors and horizontal transmission through blood, saliva, semen, and urine from infected people), and the fact that ZIKV has a high identity percentage with other cocirculating Flaviviruses such as dengue. Here, we review ZIKV diagnostic methods, with special emphasis on the development of point-of-care diagnostic assays, since these devices commonly have two important advantages: they provide prompt screening and are affordable.

Vector competence of Aedes aegypti and Culex quinquefasciatus from the metropolitan area of Guadalajara, Jalisco, Mexico for Zika virus.

Zika virus (ZIKV) is a mosquito-borne pathogen discovered in the late 40's in Uganda during a surveillance program for yellow fever. By 2014 the virus reached Eastern Island in the Americas, and two years later, the virus spread to almost all countries and territories of the Americas. The mosquito Aedes aegypti has been identified as the main vector of the disease, and several researchers have also studied the vector competence of Culex quinquefasciatus in virus transmission. The aim of the present study was to evaluate the vector competence of Ae. aegypti and Cx. quinquefasciatus in order to understand their roles in the transmission of ZIKV in Guadalajara, Jalisco, Mexico. In blood feeding laboratry experiments, we found that Ae. aegypti mosquitoes showed to be a competent vector able to transmit ZIKV in this area. On the other hand, we found that F0 Cx. quinquefasciatus mosquitoes are refractory to ZIKV infection, dissemination and transmission.

An Escherichia coli-Expressed Porcine Reproductive and Respiratory Syndrome Virus Chimeric Protein Induces a Specific Immunoglobulin G Response in Immunized Piglets.

Porcine reproductive and respiratory syndrome virus (PRRSV) still poses a threat to the swine industry worldwide. Currently, commercial vaccines against PRRSV, which consist of modified live or inactivated virus, reduce symptoms and viremia in immunized pigs, but efficacy against heterologous strains is variable. This has led to the development of subunit vaccines that contain viral antigens that show the highest variability. In this work, a chimeric protein comprising short amino acid sequences from glycoprotein 3 (GP3), glycoprotein 4 (GP4), glycoprotein 5 (GP5), and M (matrix protein) proteins of PRRSV was designed and expressed in Escherichia coli. This protein, designated as PRRSVchim, was purified by immobilized metal affinity chromatography and evaluated. PRRSVchim was identified by immunoglobulin G (IgG) presence in serum samples from PRRSV-positive pigs. Also, the protein probed to be antigenic in immunized mice and piglets and provided some degree of protection against challenge with a PRRSV field isolate. These results show the potential of PRRSVchim protein for both PRRSV diagnostic and immunoprophylaxis.

Author Correction: Zika Virus in Salivary Glands of Five Different Species of Wild-Caught Mosquitoes from Mexico.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Zika Virus in Salivary Glands of Five Different Species of Wild-Caught Mosquitoes from Mexico.

Zika virus (ZIKV) is a mosquito-borne pathogen, and Aedes aegypti has been identified as the main vector of the disease. Other mosquito species in the Aedes and Culex genera have been suggested to have the potential for being competent vectors based on experimental exposition of mosquitoes to an infectious blood meal containing ZIKV. Here, we report the isolation in cell culture of ZIKV obtained from different body parts of wild-caught female mosquitoes (Ae. aegypti, Ae. vexans, Cx. quinquefasciatus, Cx. coronator, and Cx. tarsalis) and whole male mosquitoes (Ae. aegypti and Cx. quinquefasciatus) in Mexico. Importantly, this is the first report that shows the presence of the virus in the salivary glands of the wild-caught female mosquitoes species, Cx. coronator, Cx. tarsalis, and Ae. vexans. Our findings strongly suggest that all the species reported herein are potential vectors for ZIKV.

Porcine circovirus type 2 protective epitope densely carried by chimeric papaya ringspot virus-like particles expressed in Escherichia coli as a cost-effective vaccine manufacture alternative.

Porcine circovirus type 2 (PCV2) still represents a major problem to the swine industry worldwide, causing high mortality rates in infected animals. Virus-like particles (VLPs) have gained attention for vaccine development, serving both as scaffolds for epitope expression and immune response enhancers. The commercial subunit vaccines against PCV2 consist of VLPs formed by the self-assembly of PCV2 capsid protein (CP) expressed in the baculovirus vector system. In this work, a PCV2 protective epitope was inserted into three different regions of papaya ringspot virus (PRSV) CP, namely, the N- and C-termini and a predicted antigenic region located near the N-terminus. Wild-type and chimeric CPs were modeled in silico, expressed in Escherichia coli, purified, and visualized by transmission electron microscopy. This is the first report that shows the formation of chimeric VLPs using PRSV as epitope-presentation scaffold. Moreover, it was found that PCV2 epitope localization strongly influences VLP length. Also, the estimated yields of the chimeric VLPs at a small-scale level ranged between 65 and 80 mg/L of culture medium. Finally, the three chimeric VLPs induced high levels of immunoglobulin G against the PCV2 epitope in immunized BALB/c mice, suggesting that these chimeric VLPs can be used for swine immunoprophylaxis against PCV2.

Evaluation of the Immunogenicity of a Potyvirus-Like Particle as an Adjuvant of a Synthetic Peptide.

Improvement of current vaccines is highly necessary to increase immunogenicity levels and protection against several pathogens. Virus-like particles (VLPs) are promising approaches for vaccines because they emulate infectious virus structure, but lack any genetic material needed for replication. Plant viruses have emerged as a potential framework for VLP design, mainly because there is no preexisting immunity in mammals. In this study, we evaluated the scaffold of the papaya ringspot virus (PRSV) as a VLP adjuvant for a short synthetic peptide derived from the Hemagglutinin protein of AH1 N1 influenza virus-hemagglutinin (VLP-HA). Our results demonstrated that the adjuvant property of this VLP is highly similar to the trivalent influenza vaccine, showing comparable levels of IgG- and IgA-specific antibodies to HA-derived peptide in serum and feces of vaccinated mice, respectively. Furthermore, VLP-HA-immunized mice showed Th1-biased immune response as suggested by measuring IgG subclasses in comparison with the predominance of Th2-biased immune response in trivalent influenza vaccine dose-vaccinated mice. VLP-HA administration in mice induced comparable levels of activated CD4+- and CD8+-specific T lymphocytes for the HA-derived peptide. These results suggest the potential adjuvant capacity of the PRSV-VLP as a carrier for short synthetic peptides.

Detection of dengue, west Nile virus, rickettsiosis and leptospirosis by a new real-time PCR strategy.

Dengue virus (DENV) infection causes sudden fever along with several nonspecific signs and symptoms and in severe cases, death. DENV is transmitted to people by Aedes aegypti and Ae. albopictus mosquitoes, whose populations increase during rainy season. West Nile Virus (WNV), Rickettsia spp. and Leptospira spp. are fever-causing pathogens that share many of the initial symptoms of DENV infection and also thrive in the rainy season. Outbreaks in some regions may be due to any of these pathogens that can co-circulate. Plus, they are clinically indistinguishable until severe symptoms appear, even though these diseases should be treated differently. An effective differential diagnosis would help clinicians and vector control departments to make right decisions for control and treatment of these diseases. Therefore, we developed four different SYBR green (®) -based reverse transcription quantitative PCR (RT-qPCR) assays for simultaneous detection of DENV, WNV, Rickettsia spp. and Leptospira spp. The assay has been optimized to yield results in less than 1 h; and in order to reduce contamination risk, all reagents were premixed and lyophilized on 96 well plates and thus only requires the addition of water and total nucleic acids from the sample. Sensitivities of the assays were less than 100 copies of nucleic acid targeted for these four pathogens. Assays did not show cross reactivity with any of the four pathogens nor to human nucleic acids. We are presenting a sensitive and selective kit that detects four relevant pathogens from tropical regions, that is quick, cost-effective and easy to use.

Genotyping of Canine parvovirus in western Mexico.

Canine parvovirus (CPV) is one of the most common infectious agents related to high morbidity rates in dogs. In addition, the virus is associated with severe gastroenteritis, diarrhea, and vomiting, resulting in high death rates, especially in puppies and nonvaccinated dogs. To date, there are 3 variants of the virus (CPV-2a, CPV-2b, and CPV-2c) circulating worldwide. In Mexico, reports describing the viral variants circulating in dog populations are lacking. In response to this deficiency, a total of 41 fecal samples of suspected dogs were collected from October 2013 through April 2014 in the Veterinary Hospital of the University of Guadalajara in western Mexico. From these, 24 samples resulted positive by polymerase chain reaction, and the viral variant was determined by restriction fragment length polymorphism. Five positive diagnosed samples were selected for partial sequencing of the vp2 gene and codon analysis. The results demonstrated that the current dominant viral variant in Mexico is CPV-2c. The current study describes the genotyping of CPV strains, providing valuable evidence of the dominant frequency of this virus in a dog population from western Mexico.

The adenylyl cyclase Rv2212 modifies the proteome and infectivity of Mycobacterium bovis BCG.

All organisms have the capacity to sense and respond to environmental changes. These signals often involve the use of second messengers such as cyclic adenosine monophosphate (cAMP). This second messenger is widely distributed among organisms and coordinates gene expression related with pathogenesis, virulence, and environmental adaptation. Genomic analysis in Mycobacterium tuberculosis has identified 16 adenylyl cyclases (AC) and one phosphodiesterase, which produce and degrade cAMP, respectively. To date, ten AC have been biochemically characterized and only one (Rv0386) has been found to be important during murine infection with M. tuberculosis. Here, we investigated the impact of hsp60-driven Rv2212 gene expression in Mycobacterium bovis Bacillus Calmette-Guerin (BCG) during growth in vitro, and during macrophage and mice infection. We found that hsp60-driven expression of Rv2212 resulted in an increased capacity of replication in murine macrophages but an attenuated phenotype in lungs and spleen when administered intravenously in mice. Furthermore, this strain displayed an altered proteome mainly affecting proteins associated with stress conditions (bfrB, groEL-2, DnaK) that could contribute to the attenuated phenotype observed in mice.

West nile virus and its theories, a big puzzle in Mexico and latin america.

It has been 13 years since the first outbreak of West Nile Virus (WNV) occurred in the Americas. Since then, thousands of human cases have been reported in the United States. In contrast, there has not yet been an outbreak of WNV in any Latin American countries, including Mexico where <20 cases have been reported. We aimed to review publications to gather the main theories related to the fact that not all the countries of the continent reported human cases or that they have reported few cases since the introduction of WNV in the Western Hemisphere. We identified relevant publications using the PubMed database. Furthermore, we present on-line published information from Mexico. We found that researchers have tried to explain this phenomenon using several theories, like pre-existing antibodies against a heterotypical virus that have conferred cross protection in the population. Another explanation is that the strains circulating in Latin America are attenuated or that they came from a different origin of introduction in the continent. Another theory is that a conclusive diagnostic in regions where more than one Flavivirus is circulating results in cross-reaction in serological tests. Probably the sum of factors described by researchers in these theories in order to explain the behavior of the virus has resulted in the low number of reported cases in Latin America.

Dengue virus-infected Aedes aegypti in the home environment.

We determined abundance of Aedes aegypti mosquitoes and presence of dengue virus (DENV) in females collected from premises of laboratory-confirmed dengue patients over a 12-month period (March 2007 to February 2008) in Merida, Mexico. Backpack aspiration from 880 premises produced 1,836 females and 1,292 males indoors (predominantly from bedrooms) and 102 females and 108 males from patios/backyards. The mean weekly indoor catch rate per home peaked at 7.8 females in late August. Outdoor abundances of larvae or pupae were not predictive of female abundance inside the home. DENV-infected Ae. aegypti females were recovered from 34 premises. Collection of DENV-infected females from homes of dengue patients up to 27 days after the onset of symptoms (median, 14 days) shows the usefulness of indoor insecticide application in homes of suspected dengue patients to prevent their homes from becoming sources for dispersal of DENV by persons visiting and being bitten by infected mosquitoes.

Use of Google Earth to strengthen public health capacity and facilitate management of vector-borne diseases in resource-poor environments.

OBJECTIVE: Novel, inexpensive solutions are needed for improved management of vector-borne and other diseases in resource-poor environments. Emerging free software providing access to satellite imagery and simple editing tools (e.g. Google Earth) complement existing geographic information system (GIS) software and provide new opportunities for: (i) strengthening overall public health capacity through development of information for city infrastructures; and (ii) display of public health data directly on an image of the physical environment. METHODS: We used freely accessible satellite imagery and a set of feature-making tools included in the software (allowing for production of polygons, lines and points) to generate information for city infrastructure and to display disease data in a dengue decision support system (DDSS) framework. FINDINGS: Two cities in Mexico (Chetumal and Merida) were used to demonstrate that a basic representation of city infrastructure useful as a spatial backbone in a DDSS can be rapidly developed at minimal cost. Data layers generated included labelled polygons representing city blocks, lines representing streets, and points showing the locations of schools and health clinics. City blocks were colour-coded to show presence of dengue cases. The data layers were successfully imported in a format known as shapefile into a GIS software. CONCLUSION: The combination of Google Earth and free GIS software (e.g. HealthMapper, developed by WHO, and SIGEpi, developed by PAHO) has tremendous potential to strengthen overall public health capacity and facilitate decision support system approaches to prevention and control of vector-borne diseases in resource-poor environments.