Revisión rápida: monitoreo de la presencia e infectividad del virus SARS-CoV-2 y otros coronavirus en aguas residuales / Quick review: monitoring the presence and infectivity of SARS-CoV-2 and other coronaviruses in wastewater

Resumen: Objetivo: Describir la evidencia sobre la presencia e infectividad de SARS-CoV-2 y otros coronavirus en aguas residuales y su potencial uso como herramienta de vigilancia epidemiológica. Material y métodos: Búsqueda de publicaciones en PubMed y medRxiv desde enero 2003 hasta el 8 de junio de 2020 de acuerdo con la guía de revisiones rápidas de Cochrane. Resultados: Se incluyeron 29 publicaciones. El ARN de SARS-CoV-2 no infectivo se encontró en agua residual hospitalaria, agua residual cruda, tratada y lodos de plantas de tratamiento. Los niveles cuantitativos de ARN viral en agua residual presentan relación con el número de casos de Covid-19. SARS-CoV-1 y otros coronavirus permanecieron infectivos en agua residual cruda hasta por dos días. Conclusiones: Hasta esta revisión no existe evidencia sobre la presencia de virus infectivos de SARS-CoV-2 en agua residual cruda o tratada. La cuantificación de ARN de SARS-CoV-2 en agua residual es útil para la vigilancia epidemiológica.

Abstract: Objective: To describe the current evidence on the presence and infectivity of SARS-CoV-2 and other coronaviruses in wastewater; and its potential use as an epidemiological surveillance tool. Materials and methods: A search was performed in PubMed and medRxiv databases from January 2003 to June 8, 2020 according to the Cochrane Rapid Review Guide. Results: Twenty-nine publications were included. Non-infective RNA of SARS-CoV-2 has been detected in hospital sewage; raw and treated wastewater, and primary sludges from sewage treatment plants. Quantitative levels of viral RNA in wastewater are related with the number of Covid-19 cases. SARS-CoV-1 and other coronaviruses remained infective in wastewater up to two days. Conclusions: Currently, there is no evidence of the presence of infective SARS-CoV-2 in wastewater and its inactivation through treatment/disinfection has been proven. Quantification of SARS-CoV-2 RNA in wastewater can be a useful epidemiological surveillance tool.

Experimental infection and vector competence of Zika virus in Aedes aegypti mosquitoes from Acapulco, Guerrero, Mexico.

OBJECTIVE: To gain a better understanding of the Zika virus (ZIKV) vector transmission in Mexico, we determined the vector competence of a local population of Ae. aegypti (Acapulco, Guerrero) for a strain of ZIKV isolated from a Mexican febrile patient. MATERIALS AND METHODS: Eggs were hatched and larvae were reared under controlled conditions. After five days post-emergence, female mosquitoes were fed an infectious blood-meal containing ZIKV. Mosquitoes were analyzed at 4, 7 and 14-day post-infection (dpi). Infection (gut), dissemination (wings, legs and heads) and potential transmission (salivary glands) were assessed by RT-qPCR. The Rockefeller Ae. aegypti strain was used as ZIKV infection control. RESULTS: ZIKV infection, dissemination, and potential transmission rates were 96.2, 96.1 and 93.2%, respectively. CONCLUSIONS: Ae. aegypti (F1) from Acapulco were very susceptible to ZIKV infection, and showed similar vector competence to that of the susceptible Rockefeller strain. To our knowledge, this is the first report of vector competence for ZIKV performed in a Mexican laboratory.

OBJETIVO: Determinar la competencia vectorial de una población local de Ae. aegypti para transmitir el virus Zika (ZIKV) aislado de un paciente febril mexicano. MATERIAL Y MÉTODOS: Se desarrolló la primera generación (F1) de mosquitos Ae. aegypti en el insectario a partir de huevos colectados mediante ovitrampas en la Colonia Renacimiento, Acapulco, Guerrero. Después de cinco días de la emergencia, los mosquitos hembras fueron alimentados con sangre infecciosa con ZIKV. La infección (intestino), la diseminación (alas, piernas y cabezas) y la transmisión potencial (glándulas salivales) se evaluaron mediante RT-qPCR, a los 4, 7 y 14 días después de la alimentación. RESULTADOS: La infección por ZIKV, la diseminación y las tasas potenciales de transmisión fueron de 96.2, 96.1 y 93.2%, respectivamente. CONCLUSIONES: Los mosquitos Ae. aegypti (F1) de Acapulco presentan una alta competencia vectorial (93.2%). Según los autores de este estudio, este es el primer informe de competencia vectorial para ZIKV realizado en un laboratorio mexicano.

Experimental infection and vector competence of Zika virus in Aedes aegypti mosquitoes from Acapulco, Guerrero, Mexico / Infección experimental y competencia vectorial del virus Zika en mosquitos Aedes aegypti de Acapulco, Guerrero, México

Abstract: Objective: To gain a better understanding of the Zika virus (ZIKV) vector transmission in Mexico, we determined the vector competence of a local population ofAe. aegypti(Acapulco, Guerrero) for a strain of ZIKV isolated from a Mexican febrile patient. Materials and methods: Eggs were hatched and larvae were reared under controlled conditions. After five days post-emergence, female mosquitoes were fed an infectious blood-meal containing ZIKV. Mosquitoes were analyzed at 4, 7 and 14-day post-infection (dpi). Infection (gut), dissemination (wings, legs and heads) and potential transmission (salivary glands) were assessed by RT-qPCR. The RockefellerAe. aegyptistrain was used as ZIKV infection control. Results: ZIKV infection, dissemination, and potential transmission rates were 96.2, 96.1 and 93.2%, respectively. Conclusions: Ae. aegypti(F1) from Acapulco were very susceptible to ZIKV infection, and showed similar vector competence to that of the susceptible Rockefeller strain. To our knowledge, this is the first report of vector competence for ZIKV performed in a Mexican laboratory.

Resumen: Objetivo: Determinar la competencia vectorial de una población local deAe. aegyptipara transmitir el virus Zika (ZIKV) aislado de un paciente febril mexicano. Material y métodos: Se desarrolló la primera generación (F1) de mosquitosAe. aegyptien el insectario a partir de huevos colectados mediante ovitrampas en la Colonia Renacimiento, Acapulco, Guerrero. Después de cinco días de la emergencia, los mosquitos hembras fueron alimentados con sangre infecciosa con ZIKV. La infección (intestino), la diseminación (alas, piernas y cabezas) y la transmisión potencial (glándulas salivales) se evaluaron mediante RT-qPCR, a los 4, 7 y 14 días después de la alimentación. Resultados: La infección por ZIKV, la diseminación y las tasas potenciales de transmisión fueron de 96.2, 96.1 y 93.2%, respectivamente. Conclusiones: Los mosquitos Ae. aegypti (F1) de Acapulco presentan una alta competencia vectorial (93.2%). Según los autores de este estudio, este es el primer informe de competencia vectorial para ZIKV realizado en un laboratorio mexicano.

Revisión rápida: monitoreo de la presencia e infectividad del virus SARS-CoV-2 y otros coronavirus en aguas residuales.

Objetivo. Describir la evidencia sobre la presencia e infectividad de SARS-CoV-2 y otros coronavirus en aguas residuales y su potencial uso como herramienta de vigilancia epidemiológica. Material y métodos. Búsqueda de publicaciones en PubMed y medRxiv desde enero 2003 hasta el 8 de junio de 2020 de acuerdo con la guía de revisiones rápidas de Cochrane. Resultados. Se incluyeron 29 publicaciones. El ARN de SARS-CoV-2 no infectivo se encontró en agua residual hospitalaria, agua residual cruda, tratada y lodos de plantas de tratamiento. Los niveles cuantitativos de ARN viral en agua residual presentan relación con el número de casos de Covid-19. SARS-CoV-1 y otros coronavirus permanecieron infectivos en agua residual cruda hasta por dos días. Conclusiones. Hasta esta revisión no existe evidencia sobre la presencia de virus infectivos de SARS-CoV-2 en agua residual cruda o tratada. La cuantificación de ARN de SARS-CoV-2 en agua residual es útil para la vigilancia epidemiológica.

Ookinete-Specific Genes and 18S SSU rRNA Evidenced in Plasmodium vivax Selection and Adaptation by Sympatric Vectors.

In the southern Pacific coast of Chiapas, Mexico (SM), the two most abundant vector species, Nyssorhynchus albimanus and Anopheles pseudopunctipennis, were susceptible to different Plasmodium vivax Pvs25/28 haplotypes. To broaden our understanding of the existing P. vivax in the area, genes encoding proteins relevant for ookinete development and the 18S rRNA were studied. P. vivax infectivity (percentage of infected mosquitoes and oocyst numbers) was evaluated by simultaneously feeding infected blood samples from patients to Ny. albimanus and An. pseudopunctipennis female mosquitoes. Three infectivity patterns were identified: one group of parasites were more infective to An. pseudopunctipennis than to Ny. albimanus, another group was more infective to Ny. albimanus, while a third group infected both vectors similarly. In 29 parasite isolates, the molecular variations of ookinete-specific genes and the 18S rRNA-type S were analyzed. Using concatenated sequences, phylogenetic trees, and Structure analysis, parasite clustering within SM isolates and between these and those from other geographical origins were investigated. A ML phylogenetic tree resolved two parasite lineages: PvSM-A and PvSM-B. They were associated to a different 18S rRNA variant. PvSM-A parasites had 18S rRNA variant rV2 and correspond to parasites causing high oocyst infection in Ny. albimanus. A new ML tree and Structure analysis, both comprising global sequences, showed PvSM-A clustered with Latin American parasites. Meanwhile, all isolates of PvSM-B had 18S rRNA variant rV1 and remained as unique genetic cluster comprising two subgroups: PvSM-Ba, producing high infection in An. pseudopunctipennis, and PvSM-Bb, causing similar oocyst infection in both vector species. PvSM-A parasites were genetically similar to parasites from South America. Meanwhile, PvSM-B were exclusive to southern Mexico and share ancestry with Asian parasites. The results suggest that these lineages evolved separately, likely by geographic and vector restriction.

Non-coding Class Switch Recombination-Related Transcription in Human Normal and Pathological Immune Responses.

Antibody class switch recombination (CSR) to IgG, IgA, or IgE is a hallmark of adaptive immunity, allowing antibody function diversification beyond IgM. CSR involves a deletion of the IgM/IgD constant region genes placing a new acceptor Constant gene, downstream of the VDJH exon. CSR depends on non-coding (CSRnc) transcription of donor Iµ and acceptor IH exons, located 5' upstream of each CH coding gene. Although, our knowledge of the role of CSRnc transcription has advanced greatly, its extension and importance in healthy and diseased humans is scarce. We analyzed CSRnc transcription in 70,603 publicly available RNA-seq samples, including GTEx, TCGA, and the Sequence Read Archive using recount2, an online resource consisting of normalized RNA-seq gene and exon counts, as well as, coverage BigWig files that can be programmatically accessed through R. CSRnc transcription was validated with a qRT-PCR assay for Iµ, Iγ3, and Iγ1 in humans in response to vaccination. We mapped IH transcription for the human IGH locus, including the less understood IGHD gene. CSRnc transcription was restricted to B cells and is widely distributed in normal adult tissues, but predominant in blood, spleen, MALT-containing tissues, visceral adipose tissue and some so-called "immune privileged" tissues. However, significant Iγ4 expression was found even in non-lymphoid fetal tissues. CSRnc expression in cancer tissues mimicked the expression of their normal counterparts, with notable pattern changes in some common cancer subsets. CSRnc transcription in tumors appears to result from tumor infiltration by B cells, since CSRnc transcription was not detected in corresponding tumor-derived immortal cell lines. Additionally, significantly increased Iδ transcription in ileal mucosa in Crohn's disease with ulceration was found. In conclusion, CSRnc transcription occurs in multiple anatomical locations beyond classical secondary lymphoid organs, representing a potentially useful marker of effector B cell responses in normal and pathological immune responses. The pattern of IH exon expression may reveal clues of the local immune response (i.e., cytokine milieu) in health and disease. This is a great example of how the public recount2 data can be used to further our understanding of transcription, including regions outside the known transcriptome.

Longitudinal analysis of the peripheral B cell repertoire reveals unique effects of immunization with a new influenza virus strain.

BACKGROUND: Despite the potential to produce antibodies that can neutralize different virus (heterotypic neutralization), there is no knowledge of why vaccination against influenza induces protection predominantly against the utilized viral strains (homotypic response). Identification of structural patterns of the B cell repertoire associated to heterotypic neutralization may contribute to identify relevant epitopes for a universal vaccine against influenza. METHODS: Blood samples were collected from volunteers immunized with 2008/2009 trivalent inactivated vaccine (TIV), pandemic H1N1 (pdmH1N1) monovalent inactivated vaccine (MIV) and the 2014/2015 TIV. Neutralization was assessed by hemagglutination and microneutralization test. IgG V(H) amplicons derived from peripheral blood RNA from pre-immune and 7 days post vaccination were subjected to 454-Roche sequencing. Full reconstruction of the sampled repertoires was done with ImmunediveRsity. RESULTS: The TIV induced a predominantly homotypic neutralizing serologic response, while the 09 MIV induced a heterotypic neutralizing seroconversion in 17% of the individuals. Both the 08/09 and the 14/15 TIV were associated with a reduction in clonotypic diversity, whereas 09 MIV was the opposite. Moreover, TIV and MIV induced distinctive patterns of IGHV segment use that are consistent with B cell selection by conserved antigenic determinants shared by the pre-pandemic and the pandemic strains. However, low somatic hypermutation rates in IgG after 09 MIV immunization, but not after 08/09 and 14/15 TIV immunization were observed. Furthermore, no evidence of the original antigenic sin was found in the same individuals after vaccination with the three vaccines. CONCLUSIONS: Immunization with a new influenza virus strain (2009 pdmH1N1) induced unique effects in the peripheral B cell repertoire clonal structure, a stereotyped response involving distinctive IGHV segment use and low somatic hypermutation levels. These parameters were contrastingly different to those observed in response to pre-pandemic and post-pandemic vaccination, and may be the result of clonal selection of common antigenic determinants, as well as germinal center-independent responses that wane as the pandemic strain becomes seasonal. Our findings may contribute in the understanding of the structural and cellular basis required to develop a universal influenza vaccine.

Hybridization-based antibody cDNA recovery for the production of recombinant antibodies identified by repertoire sequencing.

High-throughput sequencing of the antibody repertoire is enabling a thorough analysis of B cell diversity and clonal selection, which may improve the novel antibody discovery process. Theoretically, an adequate bioinformatic analysis could allow identification of candidate antigen-specific antibodies, requiring their recombinant production for experimental validation of their specificity. Gene synthesis is commonly used for the generation of recombinant antibodies identified in silico. Novel strategies that bypass gene synthesis could offer more accessible antibody identification and validation alternatives. We developed a hybridization-based recovery strategy that targets the complementarity-determining region 3 (CDRH3) for the enrichment of cDNA of candidate antigen-specific antibody sequences. Ten clonal groups of interest were identified through bioinformatic analysis of the heavy chain antibody repertoire of mice immunized with hen egg white lysozyme (HEL). cDNA from eight of the targeted clonal groups was recovered efficiently, leading to the generation of recombinant antibodies. One representative heavy chain sequence from each clonal group recovered was paired with previously reported anti-HEL light chains to generate full antibodies, later tested for HEL-binding capacity. The recovery process proposed represents a simple and scalable molecular strategy that could enhance antibody identification and specificity assessment, enabling a more cost-efficient generation of recombinant antibodies.

Transcriptome of the adult female malaria mosquito vector Anopheles albimanus.

BACKGROUND: Human Malaria is transmitted by mosquitoes of the genus Anopheles. Transmission is a complex phenomenon involving biological and environmental factors of humans, parasites and mosquitoes. Among more than 500 anopheline species, only a few species from different branches of the mosquito evolutionary tree transmit malaria, suggesting that their vectorial capacity has evolved independently. Anopheles albimanus (subgenus Nyssorhynchus) is an important malaria vector in the Americas. The divergence time between Anopheles gambiae, the main malaria vector in Africa, and the Neotropical vectors has been estimated to be 100 My. To better understand the biological basis of malaria transmission and to develop novel and effective means of vector control, there is a need to explore the mosquito biology beyond the An. gambiae complex. RESULTS: We sequenced the transcriptome of the An. albimanus adult female. By combining Sanger, 454 and Illumina sequences from cDNA libraries derived from the midgut, cuticular fat body, dorsal vessel, salivary gland and whole body, we generated a single, high-quality assembly containing 16,669 transcripts, 92% of which mapped to the An. darlingi genome and covered 90% of the core eukaryotic genome. Bidirectional comparisons between the An. gambiae, An. darlingi and An. albimanus predicted proteomes allowed the identification of 3,772 putative orthologs. More than half of the transcripts had a match to proteins in other insect vectors and had an InterPro annotation. We identified several protein families that may be relevant to the study of Plasmodium-mosquito interaction. An open source transcript annotation browser called GDAV (Genome-Delinked Annotation Viewer) was developed to facilitate public access to the data generated by this and future transcriptome projects. CONCLUSIONS: We have explored the adult female transcriptome of one important New World malaria vector, An. albimanus. We identified protein-coding transcripts involved in biological processes that may be relevant to the Plasmodium lifecycle and can serve as the starting point for searching targets for novel control strategies. Our data increase the available genomic information regarding An. albimanus several hundred-fold, and will facilitate molecular research in medical entomology, evolutionary biology, genomics and proteomics of anopheline mosquito vectors. The data reported in this manuscript is accessible to the community via the VectorBase website (http://www.vectorbase.org/Other/AdditionalOrganisms/).

Sequence variation of ookinete surface proteins Pvs25 and Pvs28 of Plasmodium vivax isolates from Southern Mexico and their association to local anophelines infectivity.

The polymorphism of Pvs25 and Pvs28 ookinete surface proteins, their association to circumsporozoite protein repeat (CSPr) genotypes (Vk210 and Vk247) and their infectivity to local Anopheles albimanus and Anopheles pseudopunctipennis were investigated in Plasmodium vivax-infected blood samples obtained from patients in Southern Mexico. The pvs25 and pvs28 complete genes were amplified, cloned and sequenced; and the CSPr genotype was determined by PCR amplification and hybridization. The amino acid Pvs25 and Pvs28 polymorphisms were mapped to their corresponding protein structure. Infected blood samples were simultaneously provided through artificial feeders to both mosquito species; the ratio of infected mosquitoes and oocyst numbers were recorded. The polymorphism of pvs25 and pvs28 was limited to few nucleotide positions, and produced three haplotypes: type A/A parasites presented Pvs25 and Pvs28 amino acid sequences identical to that of Sal I reference strain; parasites type B1 presented a mutation 130 Ile-->Thr in Pvs25, while type B2 presented 87 Gln-->Lys/130 Ile-->Thr in the same molecule. Both types B1 and B2 parasites presented changes in Pvs28 at 87 Asn-->Asp, 110 Tyr-->Asn and five GSGGE/D repeat sequences between the fourth EGF-like domain and the GPI. Most P. vivaxparasites from the coastal plains and the overlapping region were Pvs25/28 A/A, CSPrVk210 and were infective only to An. albimanus (p< or =0.0001). Parasites originating in foothills were Pvs25/28 type B1/B or B2/B and CSPrVk210 or Vk247, and were more infective to An. pseudopunctipennis than to An. albimanus (p< or =0.001). These results and the analysis of Pvs25/28 from other parts of the world indicated that non-synonymous variations in these proteins occur in amino acid residues exposed on the surface of the proteins, and are likely to interact with midgut mosquito ligands. We hypothesize that these molecules have been shaped by co-evolutionary adaptations of parasites to their susceptible vectors.

Antimalarial 4-phenylcoumarins from the stem bark of Hintonia latiflora.

The EtOAc extract of the stem bark of Hintonia latiflora showed the suppression of total parasitemia and the chemosuppression of schizont numbers, when tested in vivo against Plasmodium berghei infection in mice. Bioassay-directed fractionation of the EtOAc extract, using the in vitro 16 h and the in vivo 4-day suppression tests on P. berghei schizont numbers, led to the isolation of the new compound 5-O-beta-D-glucopyranosyl-7,4'-dimethoxy-3'-hydroxy-4-phenylcoumarin (1), along with the known 5-O-beta-D-glucopyranosyl-7-methoxy-3',4'-dihydroxy-4-phenylcoumarin (2). The structure of compound 1 was established on the basis of spectroscopic data interpretation. Compounds 1 and 2 suppressed the development of P. berghei schizonts in vitro with IC50 values of 24.7 and 25.9 microM, respectively. Compound 2 suppressed the development of schizonts at the dose of 40 mg/kg by 70.8% in the in vivo assay.