Morphological and Molecular Changes in the Cortex and Cerebellum of Immunocompetent Mice Infected with Zika Virus.

Zika virus (ZIKV) disease continues to be a threat to public health, and it is estimated that millions of people have been infected and that there have been more cases of serious complications than those already reported. Despite many studies on the pathogenesis of ZIKV, several of the genes involved in the malformations associated with viral infection are still unknown. In this work, the morphological and molecular changes in the cortex and cerebellum of mice infected with ZIKV were evaluated. Neonatal BALB/c mice were inoculated with ZIKV intraperitoneally, and the respective controls were inoculated with a solution devoid of the virus. At day 10 postinoculation, the mice were euthanized to measure the expression of the markers involved in cortical and cerebellar neurodevelopment. The infected mice presented morphological changes accompanied by calcifications, as well as a decrease in most of the markers evaluated in the cortex and cerebellum. The modifications found could be predictive of astrocytosis, dendritic pathology, alterations in the regulation systems of neuronal excitation and inhibition, and premature maturation, conditions previously described in other models of ZIKV infection and microcephaly.

5'/3' RACE method for sequencing the 5' and 3' untranslated regions of Zika virus.

The spread of Zika virus (ZIKV) from the African continent to the Americas promoted its molecular evolution, as reflected by mutations in its RNA genome. Most of the ZIKV genome sequences in the GenBank database have incomplete 5' and 3' UTR sequences, reflecting the deficiency of whole-genome sequencing technologies to resolve the sequences of the genome ends. We modified a protocol for rapid amplification of cDNA ends (RACE) to determine the complete sequences of the 5' and 3' UTRs of a previously reported ZIKV isolate (GenBank no. MH544701.1). This strategy is useful for determining 5' and 3' UTR sequences of ZIKV isolates and will be useful for comparative genomics applications.

Fetal demise and SARS-CoV-2 infection during pregnancy: Histopathological and immunohistochemical findings of three cases referred to the Colombian National Institute of Health.

BACKGROUND: Severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) infection during pregnancy is related with adverse maternal, fetal, and neonatal outcomes. Placental SARS-CoV-2 involvement may include various degrees of inflammation and malperfusion leading to diverse pregnancy complications. METHODS: Placental, fetal and umbilical cord samples of three fetal demise cases that occurred in the context of maternal SARS-CoV-2 infections were analyzed. Cases were notified to the Colombian SARS-CoV-2 National Surveillance System. RT-PCR and immunohistochemistry (IHC) analysis were employed to identify potential tissue viral involvement. RESULTS: RT-PCR and IHC confirmed the presence of viral genomes and antigens in placental and umbilical cord tissues. Histopathological analysis revealed findings consistent with placental malperfusion and inflammation. CONCLUSIONS: SARS-CoV-2 infection during pregnancy can lead to placental dysfunction and damage compromising fetal survival. Many questions regarding SARS-CoV-2 dynamics during pregnancy including placental physiopathology and in utero transmission are still pending definitive answers.

Inmunohistochemical detection of pandemic SARSCoV- 2 antigens in lung tissue. / Detección inmunohistoquímica de antígenos de SARS-CoV-2 en tejido pulmonar

The COVID-19 pandemic caused by the SARS-CoV-2 virus has generated globally more than 110.7 million infections and 2.4 million deaths. The severity of this infection can range from asymptomatic, mild to severe. To know the possible associations between the presence of the virus and histopathological alterations found in tissues of fatal cases of COVID-19, the presence of the virus in the lung tissue of a patient with a clinical history of SARS-CoV-2 infection was evaluated. Lung tissue was histologically processed for immunohistochemical detection of SARSCoV-2. In the histopathological study, morphological changes associated with pneumonitis of viral origin were observed. Likewise, the location of the SARS-CoV-2 virus was observed mainly in the cytoplasm of the cells of the inflammatory infiltrate.

La pandemia de COVID-19 causada por el virus SARS-CoV-2 ha generado más de 110,7 millones de infecciones y 2,4 millones de muertes a nivel mundial. Esta infección puede ser asintomática y sus manifestaciones clínicas pueden variar entre leves y graves. Para conocer las posibles asociaciones entre la presencia del virus y las alteraciones histopatológicas encontradas en los tejidos de casos fatales de COVID-19, se evaluó la presencia del virus en el tejido pulmonar de un paciente con antecedentes clínicos de infección por SARS-CoV-2. La muestra se procesó para la detección inmunohistoquímica del virus. En el estudio histopatológico, se observaron cambios morfológicos asociados con neumonitis de origen viral. Asimismo, el virus se localizó principalmente en el citoplasma de las células del infiltrado inflamatorio.

Inmunohistochemical detection of pandemic SARS-CoV-2 antigens in lung tissue / Detección inmunohistoquímica de antígenos de SARS-CoV-2 en tejido pulmonar

The COVID-19 pandemic caused by the SARS-CoV-2 virus has generated globally more than 110.7 million infections and 2.4 million deaths. The severity of this infection can range from asymptomatic, mild to severe. To know the possible associations between the presence of the virus and histopathological alterations found in tissues of fatal cases of COVID-19, the presence of the virus in the lung tissue of a patient with a clinical history of SARS-CoV-2 infection was evaluated. Lung tissue was histologically processed for immunohistochemical detection of SARS- CoV-2. In the histopathological study, morphological changes associated with pneumonitis of viral origin were observed. Likewise, the location of the SARS-CoV-2 virus was observed mainly in the cytoplasm of the cells of the inflammatory infiltrate.

La pandemia de COVID-19 causada por el virus SARS-CoV-2 ha generado más de 110,7 millones de infecciones y 2,4 millones de muertes a nivel mundial. Esta infección puede ser asintomática y sus manifestaciones clínicas pueden variar entre leves y graves. Para conocer las posibles asociaciones entre la presencia del virus y las alteraciones histopatológicas encontradas en los tejidos de casos fatales de COVID-19, se evaluó la presencia del virus en el tejido pulmonar de un paciente con antecedentes clínicos de infección por SARS-CoV-2. La muestra se procesó para la detección inmunohistoquímica del virus. En el estudio histopatológico, se observaron cambios morfológicos asociados con neumonitis de origen viral. Asimismo, el virus se localizó principalmente en el citoplasma de las células del infiltrado inflamatorio.

Prolonged SARS-CoV-2 nucleic acid conversion time in military personnel outbreaks with presence of specific IgG antibodies.

Coronavirus disease 2019 (COVID-19) is transmitted person-to-person mainly by close contact or droplets from respiratory tract. However, the actual time of viral shedding is still uncertain as well as the different routes of transmission. We aimed to characterize RNA shedding from nasopharyngeal and rectal samples in prolonged cases of mild COVID-19 in young male soldiers. Seventy patients from three different military locations were monitored after recommending to follow more strict isolation measures to prevent the spread of the virus. Then, nasopharyngeal, rectal, and blood samples were taken. SARS-CoV-2 RNA was detected by RT-PCR and specific antibodies by chemiluminescent immunoassays. The median nucleic acid conversion time (NACT) was 60 days (IQR: 7-85 days). Rectal swabs were taken in 60 % of patients. Seven patients (10 %) were positive in nasopharyngeal and rectal swabs, and five (7.14 %) remained positive in rectal swabs, but negative in nasopharyngeal samples. Four patients (5.71 %) that had been discharged, were positive again after 15 days. No significant difference was found in nucleic acid conversion time between age groups nor clinical classification. Maintaining distancing among different positive patients is essential as a possible re-exposure to the virus could cause a longer nucleic acid conversion time in SARS-COV-2 infections.

CISH and IHC for the Simultaneous Detection of ZIKV RNA and Antigens in Formalin-Fixed Paraffin-Embedded Cell Blocks and Tissues.

Zika virus is an arthropod-borne virus that has recently emerged as a significant public health emergency due to its association with congenital malformations. Serological and molecular tests are typically used to confirm Zika virus infection. These methods, however, have limitations when the interest is in localizing the virus within the tissue and identifying the specific cell types involved in viral dissemination. Chromogenic in situ hybridization (CISH) and immunohistochemistry (IHC) are common histological techniques used for intracellular localization of RNA and protein expression, respectively. The combined use of CISH and IHC is important to obtain information about RNA replication and the location of infected target cells involved in Zika virus neuropathogenesis. There are no reports, however, of detailed procedures for the simultaneous detection of Zika virus RNA and proteins in formalin-fixed paraffin-embedded (FFPE) samples. Furthermore, the chromogenic detection methods for Zika virus RNA published thus far use expensive commercial kits, limiting their widespread use. As an alternative, we describe here a detailed and cost-effective step-by-step procedure for the simultaneous detection of Zika virus RNA and proteins in FFPE samples. First, we describe how to synthesize and purify homemade RNA probes conjugated with digoxygenin. Then, we outline the steps to perform the chromogenic detection of Zika virus RNA using these probes, and how to combine this technique with the immunodetection of viral antigens. To illustrate the entire workflow, we use FFPE samples derived from infected Vero cells as well as from human and mouse brain tissues. These methods are highly adaptable and can be used to study Zika virus or even other viruses of public health relevance, providing an optimal and economical alternative for laboratories with limited resources. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Synthesis of RNA probes conjugated with digoxigenin (DIG) Basic Protocol 2: Simultaneous detection of ZIKV RNA and proteins in FFPE cell blocks and tissues.

Characterization of the emerging B.1.621 variant of interest of SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genetic diversity has the potential to impact the virus transmissibility and the escape from natural infection- or vaccine-elicited neutralizing antibodies. Here, representative samples from circulating SARS-CoV-2 in Colombia between January and April 2021, were processed for genome sequencing and lineage determination following the nanopore amplicon ARTIC network protocol and PANGOLIN pipeline. This strategy allowed us to identify the emergence of the B.1.621 lineage, considered a variant of interest (VOI) with the accumulation of several substitutions affecting the Spike protein, including the amino acid changes I95I, Y144T, Y145S and the insertion 146 N in the N-terminal domain, R346K, E484K and N501Y in the Receptor binding Domain (RBD) and P681H in the S1/S2 cleavage site of the Spike protein. The rapid increase in frequency and fixation in a relatively short time in Magdalena, Atlantico, Bolivar, Bogotá D.C, and Santander that were near the theoretical herd immunity suggests an epidemiologic impact. Further studies will be required to assess the biological and epidemiologic roles of the substitution pattern found in the B.1.621 lineage.

Novel Highly Divergent SARS-CoV-2 Lineage With the Spike Substitutions L249S and E484K.

COVID-19 pandemics has led to genetic diversification of SARS-CoV-2 and the appearance of variants with potential impact in transmissibility and viral escape from acquired immunity. We report a new and highly divergent lineage containing 21 distinctive mutations (10 non-synonymous, eight synonymous, and three substitutions in non-coding regions). The amino acid changes L249S and E484K located at the CTD and RBD of the Spike protein could be of special interest due to their potential biological role in the virus-host relationship. Further studies are required for monitoring the epidemiologic impact of this new lineage.

Epidemiology of cytomegalovirus Infection among mothers and infants in Colombia.

We assessed maternal and infant cytomegalovirus (CMV) infection in Colombia. Maternal serum was tested for CMV immunoglobulin G antibodies at a median of 10 (interquartile range: 8-12) weeks gestation (n = 1501). CMV DNA polymerase chain reaction was performed on infant urine to diagnose congenital (≤21 days of life) and postnatal (>21 days) infection. Maternal CMV seroprevalence was 98.1% (95% confidence interval [CI]: 97.5%-98.8%). Congenital CMV prevalence was 8.4 (95% CI: 3.9%-18.3%; 6/711) per 1000 live births. Among 472 infants without confirmed congenital CMV infection subsequently tested at age 6 months, 258 (54.7%, 95% CI: 50.2%-59.1%) had postnatal infection.

Characterization of Gnrh/Gnih elements in the olfacto-retinal system and ovary during zebrafish ovarian maturation.

Gonadotropin releasing hormone (GnRH) is one of the key players of brain-pituitary-gonad axis, exerting overall control over vertebrate reproduction. In zebrafish, two variants were characterized and named as Gnrh2 and Gnrh3. In this species, Gnrh3, the hypohysiotropic form, is expressed by neurons of the olfactory-retinal system, where it is related with food detection, intra/interspecific recognition, visual acuity and retinal processing modulation. Previous studies have reported the presence of Gnrh receptors in the zebrafish retina, but not yet in the zebrafish olfactory epithelium. The current study analyzed the presence of gnrh2 and gnrh3, their receptors (gnrhr 1,2,3 and 4) and gnih (gonadotropin inhibitory hormone) transcripts, as well as the Gnrh3 protein in the olfactory epithelium (OE), olfactory bulb (OB), retina and ovary during zebrafish ovarian maturation. We found an increase of gnrh receptors transcripts in the OE at the final stages of ovarian maturation. In the OE, Gnrh3 protein was detected in the olfactory receptor neurons cilia and in the olfactory nerve fibers. Interestingly, in the OB, we found an inverse expression pattern between gnih and gnrh3. In the retina, gnrhr4 mRNA was found in the nuclei of amacrine, bipolar, and ganglion cells next to Gnrh3 positive fibers. In the ovary, gnrh3, gnrhr2 and gnrhr4 transcripts were found in perinucleolar oocytes, while gnih in oocytes at the cortical alveolus stage. Our results suggested that Gnrh/Gnih elements are involved in the neuromodulation of the sensorial system particularly at the final stages of maturation, playing also a paracrine role in the ovary.