Pandemic 2009 H1N1 influenza was associated with pediatric death rates that were 10 times the rates for seasonal influenza in previous years.

As part of any plan to lift or ease the confinement restrictions that are in place in many different countries, there is an urgent need to increase the capacity of laboratory testing for SARS CoV-2. Detection of the viral genome through RT-qPCR is the golden standard for this test, however, the high demand of the materials and reagents needed to sample individuals, purify the viral RNA, and perform the RT-qPCR test has resulted in a worldwide shortage of several of these supplies. Here, we show that directly lysed saliva samples can serve as a suitable source for viral RNA detection that is cheaper and can be as efficient as the classical protocol that involves column purification of the viral RNA. In addition, it surpasses the need for swab sampling, decreases the risk of the healthcare personnel involved in this process, and accelerates the diagnostic procedure.

Human astroviruses (HAstVs) cause severe diarrhea and represent an important health problem in children under two years of age. Despite their medical importance, the study of these pathogens has been neglected. To better understand the astrovirus antigenic structure and the basis of protective immunity, in this work we produced a panel of neutralizing monoclonal antibodies (Nt-MAbs) to HAstV serotypes 1, 2, and 8 and identified the mutations that allow the viruses to escape neutralization. We first tested the capacity of the recombinant HAstV capsid core and spike domains to elicit Nt-Abs. Hyperimmunization of animals with the two domains showed that although both induced a potent immune response, only the spike was able to elicit antibodies with neutralizing activity. Based on this finding, we used a mixture of the recombinant spike domains belonging to the three HAstV serotypes to immunize mice. Five Nt-MAbs were isolated and characterized; all of them were serotype specific, two were directed to HAstV-1, one was directed to HAstV-2, and two were directed to HAstV-8. These antibodies were used to select single and double neutralization escape variant viruses, and determination of the amino acid changes that allow the viruses to escape neutralization permitted us to define the existence of four potentially independent neutralization epitopes on the HAstV capsid. These studies provide the basis for development of subunit vaccines that induce neutralizing antibodies and tools to explore the possibility of developing a specific antibody therapy for astrovirus disease. Our results also establish a platform to advance our knowledge on HAstV cell binding and entry. IMPORTANCE Human astroviruses (HAstVs) are common etiological agents of acute gastroenteritis in children, the elderly, and immunocompromised patients; some virus strains have also been associated with neurological disease. Despite their medical importance, the study of these pathogens has advanced at a slow pace. In this work, we produced neutralizing antibodies to the virus and mapped the epitopes they recognize on the virus capsid. These studies provide the basis for development of subunit vaccines that induce neutralizing antibodies, as well as tools to explore the development of a specific antibody therapy for astrovirus disease. Our results also establish a platform to advance our knowledge on HAstV cell binding and entry.

Several molecules have been identified as receptors or coreceptors for rotavirus infection, including glycans, integrins, and hsc70. In this work we report that the tight junction proteins JAM-A, occludin, and ZO-1 play an important role during rotavirus entry into MA104 cells. JAM-A was found to function as coreceptor for rotavirus strains RRV, Wa, and UK, but not for rotavirus YM. Reassortant viruses derived from rotaviruses RRV and YM showed that the virus spike protein VP4 determines the use of JAM-A as coreceptor.

Viruses are the most frequent cause of respiratory disease in children. However, despite the advanced diagnostic methods currently in use, in 20 to 50% of respiratory samples a specific pathogen cannot be detected. In this work, we used a metagenomic approach and deep sequencing to examine respiratory samples from children with lower and upper respiratory tract infections that had been previously found negative for 6 bacteria and 15 respiratory viruses by PCR. Nasal washings from 25 children (out of 250) hospitalized with a diagnosis of pneumonia and nasopharyngeal swabs from 46 outpatient children (out of 526) were studied. DNA reads for at least one virus commonly associated to respiratory infections was found in 20 of 25 hospitalized patients, while reads for pathogenic respiratory bacteria were detected in the remaining 5 children. For outpatients, all the samples were pooled into 25 DNA libraries for sequencing. In this case, in 22 of the 25 sequenced libraries at least one respiratory virus was identified, while in all other, but one, pathogenic bacteria were detected. In both patient groups reads for respiratory syncytial virus, coronavirus-OC43, and rhinovirus were identified. In addition, viruses less frequently associated to respiratory infections were also found. Saffold virus was detected in outpatient but not in hospitalized children. Anellovirus, rotavirus, and astrovirus, as well as several animal and plant viruses were detected in both groups. No novel viruses were identified. Adding up the deep sequencing results to the PCR data, 79.2% of 250 hospitalized and 76.6% of 526 ambulatory patients were positive for viruses, and all other children, but one, had pathogenic respiratory bacteria identified. These results suggest that at least in the type of populations studied and with the sampling methods used the odds of finding novel, clinically relevant viruses, in pediatric respiratory infections are low.

BackgroundAcute respiratory tract infections are the leading cause of morbidity and mortality in children worldwide. Many studies have described the frequency of viruses in hospitalized patients, but studies describing the prevalence of viruses in the community setting are limited, particularly in developing countries, where most of the deaths from serious respiratory diseases occur. The aim of this study was to evaluate the diversity of respiratory viruses in the community setting using molecular diagnostic tools, as well as the clinical characteristics of respiratory viral infections in the general pediatric practice in Mexico.MethodsChildren with respiratory tract infections attending private pediatric practices during a 10-month period in five cities of the state of Veracruz were included. Nasal swabs were taken and processed by a multiplex detection kit for 15 respiratory viruses.Results525 children were included from July 2011 to May 2012; 44% were female, mean age was 45 months. The 3 most frequent clinical diagnosis were: rhinopharyngitis 68%, pharyngitis 18%, and 3.3% influenza-like illness. 71.5% of the samples were positive for virus. The five most frequent pathogens were respiratory syncycitial virus in 18.3% of the children, rhinovirus in 17.5%, influenza A 9.1%, adenovirus 7.2%, and enterovirus 3.4%, although all 15 viruses were detected; there were viral coinfections in 14.1%, and 28.5% of the samples were negative.ConclusionsA large proportion of respiratory infections in the community setting in Mexico was associated to viruses. Although testing for common respiratory pathogens in children with acute respiratory tract infections may lead to a better understanding of the role of viral pathogens in, and eventually to improvement in the management of, individual patients, additional prospective studies are required to study the need of routinely using such tests in general pediatric practices in resource-limited countries.

In many countries a second wave of infections caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has occurred, triggering a shortage of reagents needed for diagnosis and compromising the capacity of laboratory testing. There is an urgent need to develop methods to accelerate the diagnostic procedures. Pooling samples represents a strategy to overcome the shortage of reagents, since several samples can be tested using one reaction, significantly increasing the number and speed with which tests can be carried out. We have reported the feasibility to use a direct lysis procedure of saliva as source for RNA to SARS-CoV-2 genome detection by reverse transcription quantitative-PCR (RT-qPCR). Here, we show that the direct lysis of saliva pools, of either five or ten samples, does not compromise the detection of viral RNA. In addition, it is a sensitive, fast, and inexpensive method that can be used for massive screening, especially considering the proximity of the reincorporation of activities in universities, offices, and schools.

BackgroundMost of the studies characterizing the incidence of rhinovirus (RV) have been carried out in hospitalized children and in developed countries. In those studies, RV-C has been associated with more severe respiratory tract infections than RV species A and B. In this study we determined the frequency and diversity of RV strains associated with upper and lower respiratory tract infections (URTI, LRTI) in Mexico, and describe the clinical characteristics of the illness associated with different RV species.MethodsA prospective surveillance of 526 and 250 children with URTI and LRTI was carried out. Respiratory samples were analyzed by RT-PCR for viruses. The 5′ untranslated region of the RV genome was amplified and sequenced.ResultsIn the case of URTI, 17.5% were positive for RV, while this virus was found in 24.8% of LRTI. The RV species was determined in 73 children with URTI: 61.6% were RV-A, 37% RV-C and, 1.4% RV-B; and in 43 children with LRTI: 51.2% were RV-A, 41.8% RV-C, and 7% RV-B. No significant differences in clinical characteristics were found in patients with RV-A or RV-C infections. A high genetic diversity of RV strains was found in both URTI and LRTI.ConclusionsBoth RV-A and RV-C species were frequently found in hospitalized as well as in outpatient children. This study underlines the high prevalence and genetic diversity of RV strains in Mexico and the potential severity of disease associated with RV-A and RV-C infections.