Progression and Resolution of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Infection in Golden Syrian Hamsters.

To catalyze severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) research, including development of novel interventive and preventive strategies, the progression of disease was characterized in a robust coronavirus disease 2019 (COVID-19) animal model. In this model, male and female golden Syrian hamsters were inoculated intranasally with SARS-CoV-2 USA-WA1/2020. Groups of inoculated and mock-inoculated uninfected control animals were euthanized at 2, 4, 7, 14, and 28 days after inoculation to track multiple clinical, pathology, virology, and immunology outcomes. SARS-CoV-2-inoculated animals consistently lost body weight during the first week of infection, had higher lung weights at terminal time points, and developed lung consolidation per histopathology and quantitative image analysis measurements. High levels of infectious virus and viral RNA were reliably present in the respiratory tract at days 2 and 4 after inoculation, corresponding with widespread necrosis and inflammation. At day 7, when the presence of infectious virus was rare, interstitial and alveolar macrophage infiltrates and marked reparative epithelial responses (type II hyperplasia) dominated in the lung. These lesions resolved over time, with only residual epithelial repair evident by day 28 after inoculation. The use of quantitative approaches to measure cellular and morphologic alterations in the lung provides valuable outcome measures for developing therapeutic and preventive interventions for COVID-19 using the hamster COVID-19 model.

MEK/ERK activation plays a decisive role in Zika virus morphogenesis and release.

Brazil has experienced an increase in outbreaks caused by flaviviruses. The high incidence of dengue fever, the morbidity of Zika in children, and the high mortality of yellow fever have affected millions in recent years. Deciphering host-virus interactions is important for treating viral infections, and the mitogen-activated protein kinases (MAPK) are an interesting target because of their role in flavivirus replication. In particular, mitogen-activated protein kinase kinase (MEK), which targets extracellular-signal-regulated kinase (ERK), is necessary for dengue and yellow fever infections. In this study, we evaluated the role of the MEK/ERK pathway and the effect of the MEK inhibitor trametinib on the Asian ZIKV strain PE243 and the prototype African ZIKV strain MR766, addressing genome replication, morphogenesis, and viral release. ZIKV infection stimulated ERK phosphorylation in Vero cells at 12 and 18 hours postinfection (hpi). Trametinib showed sustained antiviral activity, inhibiting both ZIKV strains for at least four days, and electron microscopy showed probable inhibition of ZIKV morphogenesis. ZIKV PE243 can complete one cycle in Vero cells in 14 hours; genome replication was detected around 8 hpi, intracellular viral particles at 12 hpi, and extracellular progeny at 14 hpi. Treatments at 6-hour intervals showed that trametinib inhibited late stages of viral replication, and the titration of intra- or extracellular virions showed that the treatment especially affected viral morphogenesis and release. Thus, ZIKV stimulated ERK phosphorylation during viral morphogenesis and release, which correlated with trametinib inhibiting both the signaling pathway and viral replication.

Neurologic Manifestations of Noncongenital Zika Virus in Children.

We evaluated neurologic complications following noncongenital Zika virus infection in 11 children who presented with central nervous system signs. Zika virus RNA was detected by real-time reverse transcription-polymerase chain reaction in cerebrospinal fluid. Approximately one-quarter of patients required antiepileptic medication in follow-up, and 2 children progressed to learning difficulties or developmental delay.

Risk factors for neurological complications in children with Flavivirus infection.

This study aims to characterize the acute neurological manifestations caused by DENV, ZIKV, and YFV during hospitalization; identify the risk factors associated with persistent neurological complications after discharge; and evaluate the time to resolution during clinical follow-up. A prospective study evaluated 505 children, between March 2014 and July 2019, hospitalized with neurological manifestations and that doctors suspected infection of the central nervous system (CNS). Viral infection of collected cerebrospinal fluid (CSF) was confirmed by real-time reverse transcription-polymerase chain reaction (RT-PCR). Patients were clinically followed up after hospital discharge. Analysis of predictive factors and survival curves was performed. This study identified clinical symptoms and changes in the CSF laboratory, electroencephalogram (EEG), and CNS image as predictors of complications in children with confirmed infection in the CNS by DENV, ZIKV, or YFV. No statistical difference was found (p value 0.574) in the time to the resolution of complications in children after hospital discharge between the three types of flaviviruses. Children with YFV, detected in CSF samples, had a 53% higher risk of developing neurological complications. Performing etiological diagnosis by RT-PCR of CSF samples of children with neurological manifestations, especially during Flavivirus outbreaks, is an essential tool for improving the prognosis and clinical follow-up of these patients.

Wild-Type Yellow Fever Virus RNA in Cerebrospinal Fluid of Child.

We report a 3-year-old child who was hospitalized because of severe manifestations of the central nervous system. The child died after 6 days of hospitalization. Analysis of postmortem cerebrospinal fluid showed the presence of yellow fever virus RNA. Nucleotide sequencing confirmed that the virus was wild-type yellow fever virus.

Central and peripheral nervous system involvement in Zika virus infection in a child.

A 7-year-old boy that presented an encephalomyeloradiculitis and no classic symptoms of arboviruses. Zika virus (ZIKV) was confirmed by molecular analyses of cerebrospinal fluid and 1 year later by plaque reduction neutralization test. This case demonstrates that ZIKV can be associated with diffuse nervous system infection in children.

NMR metabolomics to study the metabolic response of human osteoblasts to non-poled and poled poly (L-lactic) acid.

Untargeted nuclear magnetic resonance (NMR) metabolomics was employed, for the first time to our knowledge, to characterize the metabolome of human osteoblast (HOb) cells and extracts in the presence of non-poled or negatively poled poly-L-lactic acid (PLLA). The metabolic response of these cells to this polymer, extensively used in bone regeneration strategies, may potentially translate into useful markers indicative of in vivo biomaterial performance. We present preliminary results of multivariate and univariate analysis of NMR spectra, which have shown the complementarity of lysed cells and extracts in terms of information on cell metabolome, and unveil that, irrespective of poling state, PLLA-grown cells seem to experience enhanced oxidative stress and activated energy metabolism, at the cost of storage lipids and glucose. Possible changes in protein and nucleic acid metabolisms were also suggested, as well as enhanced membrane biosynthesis. Therefore, the presence of PLLA seems to trigger cell catabolism and anti-oxidative protective mechanisms in HOb cells, while directing them towards cellular growth. This was not sufficient, however, to lead to a visible cell proliferation enhancement in the presence of PLLA, although a qualitative tendency for negatively poled PLLA to be more effective in sustaining cell growth than non-poled PLLA was suggested. These preliminary results indicate the potential of NMR metabolomics in enlightening cell metabolism in response to biomaterials and their properties, justifying further studies of the fine effects of poled PLLA on these and other cells of significance in tissue regeneration strategies.

Meningitis Associated with Simultaneous Infection by Multiple Dengue Virus Serotypes in Children, Brazil.

To determine the causes of viral meningitis, we analyzed 22 cerebrospinal fluid samples collected during the 2014-2015 dengue epidemics in Brazil. We identified 3 serotypes of dengue virus (DENV-1, -2, and -3), as well as co-infection with 2 or 3 serotypes. We also detected the Asian II genotype of DENV-2.

Long-Term Low-Dose Pyrimethamine Use for the Prevention of Ocular Toxoplasmosis Recurrences: A Cohort Study.

PURPOSE: To describe the effect of long-term, low-dose pyrimethamine for the prevention of ocular toxoplasmosis (OT) recurrences. METHODS: Sixty-three consecutive patients with inactive ocular toxoplasmosis and positive toxoplasma IgG serology were included. Pyrimethamine (25 mg) + folinic acid (15 mg) were administered every other day (three times weekly) for 12 months. Eighteen patients received the treatment for an additional six months as part of an extension study. RESULTS: Thirty-eight patients (60.3%, n = 63) were female; 38 (60.3%) had a previous history of recurrence and 37 (58.7%) had active OT within the preceding 12 months. Three (4.8%) patients had unilateral recurrences at 8, 12 and 18 months after starting intermittent pyrimethamine treatment. Five patients (7.9%) were discontinued due to hematological, renal and hepatic changes. Treatment was considered successful in 42 patients (84%). CONCLUSION: Long-term, low-dose pyrimethamine can be considered as a treatment option for the prevention of ocular toxoplasmosis recurrence in selected patients, with only a few, mild and reversible systemic adverse events.

Toxoplasmosis outbreak in São Paulo, Brazil: Epidemiology and visual outcome.

PURPOSE: To describe a 2019 acute toxoplasmosis outbreak in the city of São Paulo, Brazil, and to evaluate the laboratory serological profile for toxoplasmosis for three consecutive years. The ophthalmological manifestations of the patients involved in the outbreak were also studied. METHODS: A cross-sectional descriptive study of a toxoplasmosis outbreak in São Paulo, Brazil, between February and May 2019. Epidemiological data were described, as were the observed ocular manifestations. As part of this study the number of patients with positive IgM toxoplasmosis serology was obtained from a large laboratory network (DASA) for three consecutive years, including the year of the outbreak (2018, 2019, 2020). RESULTS: Eighty-three individuals were identified in the outbreak and two clusters were studied. The clinical picture of at least 77% of the patients, the epidemiological analysis, and the short incubation period (5-8 days) suggested contamination by oocysts. Serological laboratory data analysis revealed an increase of positive toxoplasmosis IgM in 2019 of 73% compared to the previous year. Ophthalmological examination revealed that at least 4.8% of the patients developed toxoplasmic retinochoroiditis, none of whom had been treated during the acute systemic disease. CONCLUSION: Our findings indicate vegetable contamination as the possible source of this outbreak, a high prevalence of toxoplasmosis in São Paulo during the outbreak period, and a drop in the number of tests during the COVID-19 pandemic. Retinochoroiditis was observed in at least 4.8% of the cases. We confirm the need to implement effective means for the prevention, diagnosis, and treatment of the disease. This may involve raising awareness among the population of the importance of vegetable hygiene, and improved quality control of food and water.

2019-20 H1N1 clade A5a.1 viruses have better in vitro replication compared with the co-circulating A5a.2 clade.

Surveillance for emerging human influenza virus clades is important for identifying changes in viral fitness and assessing antigenic similarity to vaccine strains. While fitness and antigenic structure are both important aspects of virus success, they are distinct characteristics and do not always change in a complementary manner. The 2019-20 Northern Hemisphere influenza season saw the emergence of two H1N1 clades: A5a.1 and A5a.2. While several studies indicated that A5a.2 showed similar or even increased antigenic drift compared with A5a.1, the A5a.1 clade was still the predominant circulating clade that season. Clinical isolates of representative viruses from these clades were collected in Baltimore, Maryland during the 2019-20 season and multiple assays were performed to compare both antigenic drift and viral fitness between clades. Neutralization assays performed on serum from healthcare workers pre- and post-vaccination during the 2019-20 season show a comparable drop in neutralizing titers against both A5a.1 and A5a.2 viruses compared with the vaccine strain, indicating that A5a.1 did not have antigenic advantages over A5a.2 that would explain its predominance in this population. Plaque assays were performed to investigate fitness differences, and the A5a.2 virus produced significantly smaller plaques compared with viruses from A5a.1 or the parental A5a clade. To assess viral replication, low MOI growth curves were performed on both MDCK-SIAT and primary differentiated human nasal epithelial cell cultures. In both cell cultures, A5a.2 yielded significantly reduced viral titers at multiple timepoints post-infection compared with A5a.1 or A5a. Receptor binding was then investigated through glycan array experiments which showed a reduction in receptor binding diversity for A5a.2, with fewer glycans bound and a higher percentage of total binding attributable to the top three highest bound glycans. Together these data indicate that the A5a.2 clade had a reduction in viral fitness, including reductions in receptor binding, that may have contributed to the limited prevalence observed after emergence.

2019-2020 H1N1 clade A5a.1 viruses have better in vitro fitness compared with the co-circulating A5a.2 clade.

Surveillance for emerging human influenza virus clades is important for identifying changes in viral fitness and assessing antigenic similarity to vaccine strains. While fitness and antigenic structure are both important aspects of virus success, they are distinct characteristics and do not always change in a complementary manner. The 2019-2020 Northern Hemisphere influenza season saw the emergence of two H1N1 clades: A5a.1 and A5a.2. While several studies indicated that A5a.2 showed similar or even increased antigenic drift compared with A5a.1, the A5a.1 clade was still the predominant circulating clade that season. Clinical isolates of representative viruses from these clades were collected in Baltimore, Maryland during the 2019-2020 season and multiple assays were performed to compare both antigenic drift and viral fitness between clades. Neutralization assays performed on serum from healthcare workers pre- and post-vaccination during the 2019-2020 season show a comparable drop in neutralizing titers against both A5a.1 and A5a.2 viruses compared with the vaccine strain, indicating that A5a.1 did not have antigenic advantages over A5a.2 that would explain its predominance in this population. Plaque assays were performed to investigate fitness differences, and the A5a.2 virus produced significantly smaller plaques compared with viruses from A5a.1 or the parental A5a clade. To assess viral replication, low MOI growth curves were performed on both MDCK-SIAT and primary differentiated human nasal epithelial cell cultures. In both cell cultures, A5a.2 yielded significantly reduced viral titers at multiple timepoints post-infection compared with A5a.1 or A5a. Receptor binding was then investigated through glycan array experiments which showed a reduction in receptor binding diversity for A5a.2, with fewer glycans bound and a higher percentage of total binding attributable to the top three highest bound glycans. Together these data indicate that the A5a.2 clade had a reduction in viral fitness, including reductions in receptor binding, that may have contributed to the limited prevalence observed after emergence.

Postmortem Ultrastructural Analysis of the Retina from COVID-19 Deceased Patients.

PURPOSE: COVID-19 (coronavirus disease 2019) is an infectious disease caused by SARS-CoV-2, first reported in 2019 in Wuhan, China. Among the common complications is a pro-inflammatory and hypercoagulative response that compromises the vasculature among various organs. METHODS: In this report, we present the postmortem retinal findings of five patients observed by means of optical microscopy and transmission and scanning electron microscopy techniques. RESULTS: Clinical manifestations such as retinal hemorrhages and exacerbated inflammatory infiltrate, altered ultra structure with swollen mitochondria and pyknotic cells in both layers of the retina were observed in all analyzed eyes. CONCLUSION: Our data point to the fragility of this tissue in cases of severe COVID-19.

Vascular retinal findings after COVID-19 vaccination in 11 cases: a coincidence or consequence?

PURPOSE: The primary purpose of this study was to assess vascular retinal findings temporally related to COVID-19 vaccination. With greater information regarding all possible future adverse events, we hope to understand the real dimension and relevance of what was presented. METHODS: Eleven patients with visual complaints after COVID-19 vaccination were enrolled. Data on the following were included: age, sex, vaccine, time of symptom onset, systemic findings, medical history, best-corrected visual acuity, and ocular findings by slit-lamp biomicroscopy as well as multimodal retinal imaging (color fundus, red-free photography, spectral-domain optical coherence tomography, optical coherence tomography angiography, and fluorescein-angiography). Inclusion criteria were the presence of ophthalmologic signs within 30 days after the first or second dose of any COVID-19 vaccine. RESULTS: Of 11 patients, five had arterial occlusion (45.4%), four had venous occlusion (36.4%), and two (18.2%) had nonspecific vascular alterations suggestive of retinal ischemia such as cotton-wool spots. The mean age was 57 (SD = 16; range: 27-84) years. The mean time of symptoms onset was 10 (SD = 5.4; range: 3-16) days. Nine patients were female (81.8%). Systemic risk factors were observed in 36.4% of patients. Two patients had both neurological and visual symptoms, with arterial occlusion. Overall, 36.4% patients had COVID-19 in the previous year. Seven patients (63.6%) received ChAdOx1 nCoV-19 (AZD1222) vaccine. CONCLUSIONS: Our data suggest that retinal events temporally related to COVID-19 vaccination are possible but are very rare. The relationship of these events with post-COVID-19 vaccination warrants further attention to derive a meaningful conclusion.

COVID 19 repercussions in ophthalmology: a narrative review.

BACKGROUND: The new coronavirus of 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread globally and has repercussions within ophthalmological care. It has caused ocular manifestations in some patients, which can spread through eye secretions. OBJECTIVES: The purpose of this review was to summarize the currently available evidence on COVID-19 with regard to its implications for ophthalmology. DESIGN AND SETTING: Narrative review developed by a research group at Universidade Federal de São Paulo (UNIFESP), São Paulo (SP), Brazil, and at Ludwig-Maximilians-Universität, Munich, Germany. METHODS: We searched the literature on the repercussions of COVID-19 within ophthalmological care, using the MEDLINE and LILACS databases, with the keywords "COVID-19", "ophthalmology" and "coronavirus", from January 1, 2020, to March 27, 2021. Clinical trials, meta-analysis, randomized controlled trials, reviews and systematic reviews were identified. RESULTS: We retrieved 884 references, of which 42 were considered eligible for intensive review and critical analysis. Most of the studies selected reported the evidence regarding COVID-19 and its implications for ophthalmology. CONCLUSIONS: Knowledge of eye symptoms and ocular transmission of the virus remains incomplete. New clinical trials with larger numbers of patients may answer these questions in the future. Moreover, positively, implementation of innovative changes in medicine such as telemedicine and artificial intelligence may assist in diagnosing eye diseases and in training and education for students.

Retinal autofluorescence findings after COVID-19.

The main purpose of this study was to investigate the presence of retinal autofluorescence findings in COVID-19 patients. Observational study conducted in São Paulo in 2020. Demographic, medical history, and concomitant events, as well as medications used, hospitalization details, and laboratory test results, were obtained. Patients underwent eye examination and multimodal imaging, including color, red-free, autofluorescence fundus photography and optical coherence tomography. Eighteen patients had autofluorescence findings (6 females; average age 54 years, range 31 to 86 years; 26 eyes). Hyper-autofluorescence findings were present in 6 patients, Hypo-autofluorescence in 14 patients, and 6 patients had mixed pattern lesions. Retinal autofluorescence abnormalities were present in COVID-19 patients and may be secondary to primary or secondary changes caused by the SARS-CoV-2.

Neurological manifestations due to dengue virus infection in children: clinical follow-up.

The aim was to assess neurological complications in children with an invasive neurological disease by dengue virus (DENV) and the time to resolve symptoms after hospital discharge. A prospective study was conducted at a referral hospital for infectious diseases in Brazil between March 2014 and July 2019. All children hospitalized with neurologic manifestations and DENV RNA detected by real-time reverse transcription-polymerase chain reaction (RT-qPCR) in cerebrospinal fluid (CSF) were followed up until complete resolution of neurological complications. On average, they were followed up for 16 months. Among 56 DENV-positive children, 39% had some neurologic complications after hospital discharge and found that 19.6% were discharged with anticonvulsants due to seizures, 10.7% developed motor complications (e.g. muscle weakness, paresis, ataxia, and walking disability), 5.4% had headaches, and 14.3% had sleep disorders. Among the 56 children, only three had a clinical diagnosis of dengue because the symptoms are nonspecific and 35% showed no change in cerebrospinal fluid (CSF). The average time to resolve complications was 5.9 months (ranging from 1 m to 32 m). These results should alert physicians to the difficulties of a clinical diagnosis of an infection that causes neurological complications after discharge in a significant number of children. RT-qPCR's etiological diagnosis of DENV infection enabled better clinical follow-up for early intervention in children with neurological complications.

The impact of viral infections on childhood central nervous system infections.

BACKGROUND: Viruses are a common cause of central nervous system (CNS) infections. However, studies of CNS viral pathogens in pediatric patients are poorly explored because viral infections are often erroneously diagnosed as bacterial infections. METHODS: 299 CNS samples were collected from pediatric patients aged from one month to 14 years old. A total of 140 viral meningitis cases that met the inclusion criteria were included in this study. In 38 of the 140 cerebral spinal fluid (CSF) samples (27.1%), conventional and real-time PCR were used to identify viruses commonly associated with CNS infections. RESULTS: Among them, 23 patients (16.5%) tested positive for flaviviruses such as dengue, Zika, and yellow fever virus, eight patients (5.7%) were positive for enterovirus (ENTV), and six patients (4.3%) were positive for human herpesvirus 1/2. We also identified one case of dengue virus and ENTV co-infection. CONCLUSIONS: A correlation between clinical symptoms and laboratory findings for the viruses was identified. Our study also reinforces the importance of including viruses in the laboratory diagnosis of CNS infections especially flaviviruses, which assists public health authorities in implementing early interventions.