Determinación de la seroprevalencia y factores de riesgo de diarrea viral bovina en una población de la provincia de Pamplona / Determination of seroprevalence and risk factors of bovine viral diarrhea in cows of the province of Pamplona

RESUMEN La diarrea viral bovina (DVB) es una enfermedad endémica de distribución mundial, responsable de ocasionar trastornos reproductivos con gran impacto económico y sanitario. Las pruebas serológicas permiten estimar la difusión del virus en una población no vacunada. Con el objeto de determinar la prevalencia de DVB en la zona lechera de alta montaña de la provincia de Pamplona, se desarrolló un estudio sero epidemiológico transversal, utilizando la técnica de ELISA. Adicionalmente, aplicando un cuestionario, se identificaron factores de riesgo relacionados. Siguiendo los requerimientos de la ley 1774, como se expuso ante el comité de ética de la Universidad de Pamplona, se recolectaron un total de 324 muestras de sangre, a partir de bovinos no vacunados, procedentes de 82 predios. 49 muestras de 18 predios fueron positivas, indicando una prevalencia individual de 15,12 % y para predios del 21,95 %. Con el cuestionario se determinó que, en general, los predios tenían menos de 20 animales, no se llevan registros y se usa la monta natural, como principal sistema reproductivo. El contacto de hembras con toros de otras explotaciones constituyó un factor de riesgo. La prevalencia encontrada para la provincia está por debajo del promedio nacional. Se deben mejorar algunas condiciones de manejo que eviten factores de riesgo, para prevenir la diseminación de la enfermedad. Se recomienda determinar la presencia de animales persistentemente infectados, para su posterior remplazo, así como implementar registros sistemáticos en los predios y hacer pruebas en animales de nueva adquisición.

ABSTRACT Bovine viral diarrhea (BVD) is an endemic disease distributed worldwide distribution, it is responsible for reproductive disorders with high economic and sanitary impact. Serological diagnostics allow for estimating the spread of the virus in an unvaccinated population. A cross-sectional study was developed to determine the prevalence of BVD in the dairy area of the high mountains of Pamplona, which included identifying related risk factors. Three hundred twenty-four blood samples were collected from non-vaccinated cattle from 82 farms, according to Law 1774 and the ethics committee of the University of Pamplona. Forty-nine samples from 18 farms were positive with an individual prevalence of 15.12% and for farms of 21.95%. The questionnaire indicated that most of the farms had less than 20 animals, had no records and natural mating was the main reproductive system. The contact of cows with bulls from other farms constituted a risk factor. The prevalence for the region was lower than found in other studies in the country. Some management conditions should be improved to avoid risk factors and prevent the spread of the disease. It is important to determine the presence of persistently infected animals, for their subsequent replacement, as well as to implement systematic records on farms and to test new animals arriving on farms.

Spatiotemporal Evolution of the Yellow Fever Epidemic in Southeast Brazil from 2016 to 2019.

Background: Yellow fever (YF) is a zoonotic disease transmitted by mosquitoes among humans and nonhuman primates. Although urban YF is eradicated, the sylvatic YF has reemerged in some areas of Brazil in the twenty-first century. From 2016 to 2019, a sylvatic YF epidemic occurred in Southeast Brazil, where it had been eradicated in the 1940s. Methods: This study's objective was to describe the epidemic in the states of the Southeast region, based on descriptive, cluster, and mobility analyses. Results: Both the descriptive and cluster analyses showed that the YF cases spread from the state of Minas Gerais southward, causing peaks in cases during the summer months. None of the state capitals was included in the clusters, but the connectivity between the municipalities in Greater Metropolitan São Paulo highlighted potential paths of spread. Despite differences in sociodemographic profiles between the Southeast and North of Brazil (the latter region considered endemic), the epidemiological profile was similar, except for patients' occupation, which was not related to rural work in the Southeast. Conclusion: The results contributed to our understanding of the paths by which YF spread across Southeast Brazil and the epidemiological profile in an area that had gone decades without autochthonous cases.

Dengue virus 3 genotype I (GI) lineage 1 (L1) isolates elicit differential cytopathic effect with syncytium formation in human glioblastoma cells (U251).

BACKGROUND: Dengue virus (DENV) is a Flaviviridae member classified into four antigenically distinct serotypes (DENV 1, 2, 3, and 4) and further subdivided genotypes. DENV3 is subdivided into four or five genotypes, depending on the classification adopted. Despite their high genetic proximity, as revealed by phylogenetic complete polyprotein analysis, DENV3 MG-20 and DENV3 PV_BR showed different neurovirulence in mice models. Our group identified six amino acid mutations in protein E, including the E62K and E123Q, which may affect interactions of hydrophobic clusters on domain II, thus leading to the observed differences in the studied viruses. METHODS: Human glioblastoma cells (U251) derived from a malignant glioblastoma tumor by explant technique were infected by the DENV3 GIL1 isolates DENV3 MG-20 and DENV3 PV_BR and analyzed by plaque assays and titration, optical, immunofluorescence, and transmission electronic microscopy. RESULTS: The two isolates showed different cytopathic effects (CPE) and fusogenic patterns, further confirmed by indirect immunofluorescence. Transmission electron microscopy revealed intense cytopathic effects in DENV3 MG-20 infected U251 cells, displaying endoplasmic reticulum hypertrophy and turgid vesicles with proteins and multiple viruses, distinct from DENV3 PV_BR infected cells. It is hypothesized that the different amino acids in the DENV3 MG-20 isolate are related to an increased membrane fusion ability in viral infection, thus facilitating immune system evasion and increased chances of central nervous system cell infection. CONCLUSION: These results emphasize the biological differences between the isolates, which could be a critical factor in host-virus interaction and severe dengue development. Our study presents comparative results of highly similar isolates with the potential to generate more subsidies for a deeper understanding of the DENV pathogenesis. The neurotropism of the isolate DENV3 MG-20 (belonging to the DENV3 GI L1 genotype) showing infection of nervous system cells (U251) could contribute to understanding neurological dengue disease.

Northern and central Chile still free of emerging flaviviruses in mosquitoes (Diptera: Culicidae).

Geographic isolation and strict control limits in border areas have kept Chile free from various pathogens, including Flavivirus. However, the scenario is changing mainly due to climate change, the reintroduction of more aggressive mosquitoes, and the great wave of migration of people from endemic countries in recent years. Hence, it is necessary to surveillance mosquitoes to anticipate a possible outbreak in the population and take action to control it. This study aimed to investigate the presence of Flavivirus RNA by molecular tools with consensus primers in mosquitoes collected in the extreme north and central Chile. From 2019 to 2021, a prospective study was carried out in localities of Northern and part of Central Chile. Larvae, pupae, and adults of mosquitoes were collected in rural and urban sites in each locality. The collected samples were pooled by species and geographical location and tested using RT-PCR and RT-qPCR to determine presence of Flavivirus. 3085 specimens were collected, the most abundant specie Culex quinquefasciatus in the North and Aedes (Ochlerotatus) albifasciatus in the Center of Chile. Both genera are associated with Flavivirus transmission. However, PCR and RT-PCR did not detect Flavivirus RNA in the mosquitoes studied. These negative results indicate we are still a free Flavivirus country, which is reaffirmed by the non-existence of endemic human cases. Despite this, routine surveillance of mosquitoes and the pathogens they carry is highly recommended to evaluate each area-specific risk of vector-borne transmission.

Zika Virus Strains and Dengue Virus Induce Distinct Proteomic Changes in Neural Stem Cells and Neurospheres.

Brain abnormalities and congenital malformations have been linked to the circulating strain of Zika virus (ZIKV) in Brazil since 2016 during the microcephaly outbreak; however, the molecular mechanisms behind several of these alterations and differential viral molecular targets have not been fully elucidated. Here we explore the proteomic alterations induced by ZIKV by comparing the Brazilian (Br ZIKV) and the African (MR766) viral strains, in addition to comparing them to the molecular responses to the Dengue virus type 2 (DENV). Neural stem cells (NSCs) derived from induced pluripotent stem (iPSCs) were cultured both as monolayers and in suspension (resulting in neurospheres), which were then infected with ZIKV (Br ZIKV or ZIKV MR766) or DENV to assess alterations within neural cells. Large-scale proteomic analyses allowed the comparison not only between viral strains but also regarding the two- and three-dimensional cellular models of neural cells derived from iPSCs, and the effects on their interaction. Altered pathways and biological processes were observed related to cell death, cell cycle dysregulation, and neurogenesis. These results reinforce already published data and provide further information regarding the biological alterations induced by ZIKV and DENV in neural cells.

Towards the Laboratory Maintenance of Haemagogus janthinomys (Dyar, 1921), the Major Neotropical Vector of Sylvatic Yellow Fever.

Haemagogus (Haemagogus) janthinomys (Dyar, 1921), the major neotropical vector of sylvatic yellow fever virus, is notoriously difficult to maintain in captivity. It has never been reared beyond an F1 generation, and almost no experimental transmission studies have been performed with this species since the 1940s. Herein we describe installment hatching, artificial blood feeding, and forced-mating techniques that enabled us to produce small numbers of F3 generation Hg. janthinomys eggs for the first time. A total of 62.8% (1562/2486) F1 generation eggs hatched during ≤10 four-day cycles of immersion in a bamboo leaf infusion followed by partial drying. Hatching decreased to 20.1% (190/944) in the F2 generation for eggs laid by mosquitoes copulated by forced mating. More than 85% (79/92) female F2 mosquitoes fed on an artificial blood feeding system. While we were unable to maintain a laboratory colony of Hg. janthinomys past the F3 generation, our methods provide a foundation for experimental transmission studies with this species in a laboratory setting, a critical capacity in a region with hyper-endemic transmission of dengue, Zika, and chikungunya viruses, all posing a risk of spillback into a sylvatic cycle.

Insights into the Host Specificity of Mosquito-Borne Flaviviruses Infecting Wild Mammals.

Mosquito-borne flaviviruses (MBFVs) are of public and animal health concern because they cause millions of human deaths annually and impact domestic animals and wildlife globally. MBFVs are phylogenetically divided into two clades, one is transmitted by Aedes mosquitoes (Ae-MBFVs) associated with mammals and the other by Culex mosquitoes (Cx-MBFVs) associated with birds. However, this assumption has not been evaluated. Here, we synthesized 79 published reports of MBFVs from wild mammals, estimating their host. Then, we tested whether the host specificity was biased to sampling and investigation efforts or to phylogenetic relationships using a viral phylogenetic tree drawn from analyzing whole flavivirus genomes obtained in GenBank. We found in total 18 flaviviruses, nine related to Aedes spp. and nine to Culex spp. infecting 129 mammal species. Thus, this supports that vectors are transmitting MBFV across available host clades and that ornithophilic mosquitoes are readily infecting mammals. Although most of the mosquito species are generalists in their host-feeding preferences, we also found a certain degree of MBFV's specificity, as most of them infect closely related mammal species. The present study integrates knowledge regarding MBFVs, and it may help to understand their transmission dynamics between viruses, vectors, and mammal hosts.

Sofosbuvir use for yellow fever: a new perspective treatment.

Yellow fever is an acute febrile illness for which there is no specific antiviral treatment. Since 2016, Brazil has experienced two outbreaks, and collective health measures have been adopted to contain these grievances. However, published data about the drug sofosbuvir against flaviviruses are promising, suggesting the relevance of conducting future clinical trials.

Nucleoside Analogs with Antiviral Activity against Yellow Fever Virus.

Yellow fever virus (YFV) is a human Flavivirus reemerging in parts of the world. While a vaccine is available, large outbreaks have recently occurred in Brazil and certain African countries. Development of an effective antiviral against YFV is crucial, as there is no available effective drug against YFV. We have identified several novel nucleoside analogs with potent antiviral activity against YFV with 50% effective concentration (EC50) values between 0.25 and 1 µM with selectivity indices over 100 in culture.

Boletín epidemiológico semanal 30: Arbovirus / Weekly epidemiological bulletin 30

En Colombia los Arbovirus con la mayor incidencia estimada son dengue, chikungunya y zika, esta condición dada por las características geográficas del país, las condiciones de transmisión de los virus y la presencia del vector en la mayoría de municipios.

In Colombia, the Arboviruses with the highest estimated incidence are dengue, chikungunya and zika, this condition given by the geographical characteristics of the country, the conditions of virus transmission and the presence of the vector in most municipalities.

Cross-Protection of Dengue Virus Infection against Congenital Zika Syndrome, Northeastern Brazil.

The Zika virus outbreak in Latin America resulted in congenital malformations, called congenital Zika syndrome (CZS). For unknown reasons, CZS incidence was highest in northeastern Brazil; one potential explanation is that dengue virus (DENV)-mediated immune enhancement may promote CZS development. In contrast, our analyses of historical DENV genomic data refuted the hypothesis that unique genome signatures for northeastern Brazil explain the uneven dispersion of CZS cases. To confirm our findings, we performed serotype-specific DENV neutralization tests in a case-control framework in northeastern Brazil among 29 Zika virus-seropositive mothers of neonates with CZS and 108 Zika virus-seropositive control mothers. Neutralization titers did not differ significantly between groups. In contrast, DENV seroprevalence and median number of neutralized serotypes were significantly lower among the mothers of neonates with CZS. Supported by model analyses, our results suggest that multitypic DENV infection may protect from, rather than enhance, development of CZS.

A Novel Hepacivirus in Wild Rodents from South America.

The Hepacivirus genus comprises single-stranded positive-sense RNA viruses within the family Flaviviridae. Several hepaciviruses have been identified in different mammals, including multiple rodent species in Africa, Asia, Europe, and North America. To date, no rodent hepacivirus has been identified in the South American continent. Here, we describe an unknown hepacivirus discovered during a metagenomic screen in Akodon montensis, Calomys tener, Oligoryzomys nigripes, Necromys lasiurus, and Mus musculus from São Paulo State, Brazil. Molecular detection of this novel hepacivirus by RT-PCR showed a frequency of 11.11% (2/18) in Oligoryzomys nigripes. This is the first identification of hepavivirus in sigmondonine rodents and in rodents from South America. In sum, our results expand the host range, viral diversity, and geographical distribution of the Hepacivirus genus.

Hemofagocitosis secundaria a dengue / Hemophagocytosis secondary to dengue fever

La infección por el virus del dengue constituye un problema de salud pública mundial. Causada por un virus de la familia Flaviviridae, presenta un amplio espectro clínico, desde formas asintomáticas frecuentes hasta las formas graves de fiebre hemorrágica y shock por fuga capilar. Existen cuatro serotipos; los serotipos 2 y 3 están asociados a las formas graves de la enfermedad. El diagnóstico definitivo de infección por dengue depende del aislamiento del virus en sangre, de la detección del antígeno viral o el ARN viral en suero o tejido, o detección de anticuerpos específicos. El síndrome hemofagocítico se produce ante la desregulación del sistema inmune que lleva a la activación macrofágica descontrolada, y se manifiesta con alteraciones clínicas, hematológicas, bioquímicas e histológicas. La asociación entre ambas entidades se encuentra descripta como una forma inusual y grave de presentación que responde a la tormenta de citocinas liberadas durante la enfermedad. Presentamos el caso de un paciente adulto con dengue, hemofagocitosis y valores inusualmente elevados de ferritina en sangre que evolucionó favorablemente con tratamiento de soporte.

Dengue virus infection constitutes a major public health problem worldwide. It is caused by a virus belonging to the Flaviviridae family. It produces a wide range of clinical presentations, from asymptomatic infection to severe forms of the disease with hemorrhagic fever or shock secondary to capillary leak syndrome. Four serotypes have been described; serotype 2 and serotype 3 are associated with the most severe forms of the disease. The diagnosis is based on laboratory tests aimed to detect antibodies, viral RNA, or antigens in serum. The hemophagocytic syndrome is generated by a dysfunction of the immune system with clinical, hematological, biochemical and histological manifestations. The association between these two entities is described as an unusual and severe presentation of dengue fever. We present a case of an adult patient with this association and very high blood levels of ferritin, who responded favorably to supportive care.

Improved reverse transcription-polymerase chain reaction assay for the detection of flaviviruses with semi-nested primers for discrimination between dengue virus serotypes and Zika virus

BACKGROUND The genus Flavivirus includes a variety of medically important viruses, including dengue virus (DENV) and Zika virus (ZIKV), which are most prevalent in Brazil. Because the clinical profile of patients affected by different DENV serotypes or ZIKV may be similar, the development of new methods that facilitate a rapid and accurate diagnosis is crucial. OBJECTIVES The current study aimed to develop an improved reverse transcription-polymerase chain reaction (RT-PCR) protocol for universal detection of flaviviruses by using semi-nested primers that discriminate between DENV serotypes and ZIKV. METHODS The bioinformatics workflow adopted for primer design included: (1) alignment of 1,442 flavivirus genome sequences, (2) characterisation of 27 conserved regions, (3) generation of a primer set comprising 77 universal primers, and (4) selection of primer pairs with greatest coverage and specificity. Following primer design, the reaction was validated in vitro. The same approach was applied to the design of primers specific for DENV and ZIKV, using a species-specific sequence database. FINDINGS The new assay amplified an 800-806 nt variable region of the NS5 gene and allowed discrimination of virtually all flavivirus species using reference-sequence comparison. The 800-806 nt fragment was validated as a template for a semi-nested multiplex PCR using five additional primers for the detection of DENV and ZIKV. These primers were designed to generate amplicons of different sizes, allowing differentiation of the four serotypes of DENV, and ZIKV using agarose gel electrophoresis. MAIN CONCLUSIONS The bioinformatics pipeline allowed efficient primer design, making it possible to identify the best targets within the coding region of the NS5 protein. The multiplex system proved effective in differentiation of DENV1-4 and ZIKV on a 2% agarose gel. The possibility of discriminating DENV serotypes and ZIKV in the same reaction provided a faster result consuming less sample. In addition, this simplified approach ensured the reduction of the cost per analysis.

Longitudinal follow-up of Zika virus RNA in semen of a traveller returning from Barbados to the Netherlands with Zika virus disease, March 2016.

We report the longitudinal follow-up of Zika virus (ZIKV) RNA in semen of a traveller who developed ZIKV disease after return to the Netherlands from Barbados, March 2016. Persistence of ZIKV RNA in blood, urine, saliva and semen was followed until the loads reached undetectable levels. RNA levels were higher in semen than in other sample types and declined to undetectable level at day 62 post onset of symptoms.

Transcriptomic analysis of responses to cytopathic bovine viral diarrhea virus-1 (BVDV-1) infection in MDBK cells.

The bovine viral diarrhea virus (BVDV) is responsible for significant economic losses in the dairy and cattle industry; however, little is known about the protective and pathological responses of hosts to infection. The present study determined the principal molecular markers implicated in viral infection through meta-transcriptomic analysis using MDBK cells infected for two hours with a field isolate of BVDV-1. While several immune regulator genes were induced, genes involved in cell signaling, metabolic processes, development, and integrity were down-regulated, suggesting an isolation of infected cells from cell-to-cell interactions and responses to external signals. Analysis through RT-qPCR confirmed the expression of more than one hundred markers. Interestingly, there was a significant up-regulation of two negative NF-κB regulators, IER3 and TNFAIP3, indicating a possible blocking of this signaling pathway mediated by BVDV-1 infection. Additionally, several genes involved in the metabolism of reactive oxygen species were down-regulated, suggesting increased oxidative stress. Notably, a number of genes involved in cellular growth and development were also regulated during infection, including MTHFD1L, TGIF1, and Brachyury. Moreover, there was an increased expression of the genes ß-catenin, caprin-2, GSK3ß, and MMP-7, all of which are crucial to the Wnt signaling pathway that is implicated in the embryonic development of a variety of organisms. This meta-transcriptomic analysis provides the first data towards understanding the infection mechanisms of cytopathic BVDV-1 and the putative molecular relationship between viral and host components.

The effects of reference genes in qRT-PCR assays for determining the immune response of bovine cells (MDBK) infected with the Bovine Viral Diarrhea Virus 1 (BVDV-1).

The bovine viral diarrhea virus (BVDV) causes significant economic losses to the dairy industry worldwide, and understanding its infection mechanisms would be extremely useful in designing new and efficient treatments. Due to the limited number of specific antibodies against bovine proteins, differential gene expression analyses are vital for researching host immune responses to viral infection. qRT-PCR provides a sensitive platform to conduct such gene expression analyses, but suitable housekeeping genes are needed for accurate transcript normalization. The present study assessed nine reference genes in bovine kidney cells under conditions of BVDV-1 infection, incubation with pathogen-associated molecular patterns, and co-incubation with BAY117085, a pharmacological inhibitor of the NF-κB signaling pathway. Analyses of Ct values using the BestKeeper and Normfinder programs ranked CD81, RPL4, and GAPDH as the most reliable reference genes. This determination of a stable set of reference genes in this culture system will facilitate analyses of expression levels for genes of interest.

Powassan virus in mammals, Alaska and New Mexico, U.S.A., and Russia, 2004-2007.

Powassan virus is endemic to the United States, Canada, and the Russian Far East. We report serologic evidence of circulation of this virus in Alaska, New Mexico, and Siberia. These data support further studies of viral ecology in rapidly changing Arctic environments.

Dos nuevos registros de vectores potenciales de Fiebre Amarilla selvática y Mayaro para el Ecuador / Two new records of potential Sylvan Yellow Fever and Mayaro species vectors from Ecuador

Se registran por primera vez en el Ecuador dos especies de mosquitos (Diptera: Culicidae), vectores potenciales de los virus de la Fiebre Amarilla y Mayaro. Sabethes amazonicus Gordon and Evans y Haemagogus anastasionis Dyar fueron colectados en fase adulta en una localidad de la provincia de Zamora-Chinchipe, en la cual se han presentado casos de Fiebre Amarilla selvática con anterioridad. Este hallazgo muestra la importancia de profundizar los estudios de linea base de vectores en el país, distribución geográfica y aspectos ecológicos y su posible relación epidemiológica con enfermedades emergentes y re-emergentes.

Two new records of mosquitoes (Diptera: Culicidae) are presented from Ecuador, and both potential vectors of yellow fever virus and Mayaro. Sabethes amazonicus Gordon and Evans and Haemagogus anastasionis Dyar adults were collected in a locality in the province of Zamora-Chinchipe, in which there have been cases of yellow fever previously. This finding shows the importance of further studies of baseline vector in the country, geographical distribution and ecological aspects and possible epidemiological link with emerging and re-emerging diseases.