Negevirus Piura Suppresses Zika Virus Replication in Mosquito Cells.

We investigated the interaction between the insect-specific virus, Piura virus (PIUV), and the arbovirus Zika virus (ZIKV) in Aedes albopictus cells. We performed coinfection experiments in C6/36 cells. Piura virus (Cor 33 strain, Colombia) and ZIKV (PRVABC58 strain, Puerto Rico) were co-inoculated into C6/36 cells using two multiplicity of infection (MOI) combinations: 0.1 for both viruses and 1.0 for ZIKV, 0.1 for PIUV. Wells were infected in triplicate with either PIUV and ZIKV coinfection, ZIKV-only, or PIUV-only. Mock infected cells served as control wells. The cell suspension was collected daily 7 days post-infection. Zika virus load was titrated by TCID50 on Vero 76 cells. The ZIKV-only infection and PIUV and ZIKV coinfection experiments were also quantified by RT-qPCR. We also investigated whether ZIKV interfered in the PIUV replication. PIUV suppressed the replication of ZIKV, resulting in a 10,000-fold reduction in ZIKV titers within 3 days post-infection. PIUV viral loads were not reduced in the presence of ZIKV. We conclude that, when concurrently infected, PIUV suppresses ZIKV in C6/36 cells while ZIKV does not interfere in PIUV replication.

Population-Based Serologic Survey of Vibrio cholerae Antibody Titers before Cholera Outbreak, Haiti, 2022.

A Vibrio cholerae O1 outbreak emerged in Haiti in October 2022 after years of cholera absence. In samples from a 2021 serosurvey, we found lower circulating antibodies against V. cholerae lipopolysaccharide in children <5 years of age and no vibriocidal antibodies, suggesting high susceptibility to cholera, especially among young children.

Comparison of West Nile Virus Disease in Humans and Horses: Exploiting Similarities for Enhancing Syndromic Surveillance.

West Nile virus (WNV) neuroinvasive disease threatens the health and well-being of horses and humans worldwide. Disease in horses and humans is remarkably similar. The occurrence of WNV disease in these mammalian hosts has geographic overlap with shared macroscale and microscale drivers of risk. Importantly, intrahost virus dynamics, the evolution of the antibody response, and clinicopathology are similar. The goal of this review is to provide a comparison of WNV infection in humans and horses and to identify similarities that can be exploited to enhance surveillance methods for the early detection of WNV neuroinvasive disease.

Increasing temperature denatures canine IgG reducing its ability to inhibit heartworm antigen detection.

BACKGROUND: Immune complexing of target antigen to high affinity host antibody is recognized to impact the sensitivity of commercial heartworm antigen tests. Published information describing the effect of heat on interfering canine host antibodies is lacking. Immune complex dissociation (ICD) by heat treatment of serum for samples initially testing negative for heartworm antigen increases sensitivity of commercial antigen tests, particularly for single sex or low adult infection intensities. In this study the stability and nature of the targeted epitope and mechanism of heat ICD were examined. METHODS: Canine IgG was isolated using protein-A columns from serum originating from four dogs evaluated after necropsy: one dog with evidence of previously cleared infection and three dogs with confirmed heartworm infections. These dogs were expected to have an excess of antibodies based on negative antigen test and to have no or low antigen optical density, respectively, following heat treatment. Interference of antigen detection on (non-heated) positive serum was evaluated, following 1:1 mixing of antibody/PBS solutions previously heated at 25 °C, 65 °C, 75 °C, 85 °C, 95 °C and 104 °C, compared to positive serum/PBS control measured by optical density using a commercial heartworm antigen ELISA and protein quantification. Live heartworms incubated in media for 72 h provided excretory/secretory antigen for antigen stability studies following heat, endopeptidase digestion and disulfide bond reduction. RESULTS: Mixing antigen-positive heartworm serum with antibody solutions demonstrated a significant inhibition of antigen detection for antibody solutions previously heated at 25 °C and 65 °C relative to positive serum/PBS control. Antigen detection optical density was restored at or above the control when positive serum was mixed with solutions previously heated at 75 °C, 85 °C, 95 °C and 104 °C. Significant changes occurred in protein levels for antibody solutions heated at 75 °C, 85 °C, 95 °C and 104 °C. Relative stability of antigen from live heartworms in culture was demonstrated following heat, chemical and enzymatic treatment. CONCLUSIONS: Significant changes in protein levels and antigen binding ability occurred in IgG solutions heated above 65 °C. The findings confirm heat denaturation of antibodies as the suspected mechanism of heat ICD at 104 °C for antigen diagnosis of heartworm. No significant change occurred in antigen detection following heat, chemical or enzymatic digestions supporting a heat-stable linear nature of the epitope.

Angiotensin-converting enzyme 2 in dogs with Dirofilaria immitis.

BACKGROUND: Infection by the canine heartworm, Dirofilaria immitis, causes significant cardiopulmonary disease, with progression impacted by increasing parasite numbers and duration of infection. The renin-angiotensin-aldosterone system (RAAS) is an important mediator of cardiac and pulmonary disease. Angiotensin-converting enzyme 2 (ACE2) mitigates the maladaptive effects of angiotensin II by converting it to angiotensin (1-7). We hypothesized that circulating ACE2 activity would be altered in dogs with high heartworm infection intensities relative to dogs without heartworms. METHODS: Frozen serum samples (-80 °C) from 30 dogs euthanized at Florida shelters were analyzed for ACE2 activity using liquid chromatography-mass spectrometry/mass spectroscopy and a kinetics approach with and without an ACE2 inhibitor. A convenience sample of 15 dogs without heartworms (HW0) and 15 dogs with > 50 heartworms (HW>50) was included. Heartworm number and microfilariae presence were determined at necropsy. The effects of heartworm status, body weight, and sex on ACE2 were evaluated using regression analysis. Values of P < 0.05 were considered significant. RESULTS: All HW0 dogs were D. immitis microfilariae-negative and all HW>50 dogs were D. immitis microfilariae-positive with a median adult worm count of 74 (minimum = 63, maximum = 137). The ACE2 activity of HW>50 dogs (median = 28.2 ng/ml; minimum = 13.6, maximum = 76.2) was not different from HW0 dogs (median 31.9 ng/ml; minimum = 14.1, maximum = 139.1; P = 0.53). The ACE2 activity was higher in dogs with high body weight (median 34.2 ng/ml minimum = 14.1, maximum = 76.2) than in dogs with low weight (median 27.5 ng/ml; minimum = 16.4, maximum = 139.1; P = .044). CONCLUSIONS: Heartworm infection did not impact ACE2 activity in shelter dogs with or without heartworms, but heavier dogs had higher ACE2 activity compared to lighter dogs. Comprehensive RAAS evaluation and additional clinical information would aid in understanding how ACE2 activity relates to the entire cascade and clinical status in dogs with heartworm disease.

The infected and the affected: A longitudinal study of the impact of the COVID-19 pandemic on schoolchildren in Florida.

Objectives: To identify risk factors associated with symptoms of anxiety, depression, and obsessive-compulsive disorder (OCD) among children during the 1st year of the COVID-19 pandemic. Methods: A longitudinal study with three cross-sectional timepoints [April 2020 (n = 273), October 2020 (n = 180), and April 2021 (n = 116)] was conducted at a K-12 public school in Florida. Infection and sero-positivity for SARS-CoV-2 was determined by molecular and serologic approaches. Adjusted odds ratios using mixed effect logistic regression models for symptom-derived indicators of anxiety, depression, and OCD in children in April 2021 are presented; past infection and seropositivity were included in the models. Results: The prevalence of anxiety, depression, or OCD moved from 47.1, to 57.2, to 42.2% across the three timepoints during the study. By endline of the study, in April 2021, non-white children were at higher risk for depression and OCD. Risk for anxiety, depression, and OCD was associated with students who lost a family member due to COVID-19 and who were identified as at-risk in previous timepoints. Rates of SARS-CoV-2 infection and seropositivity were low and not statistically associated with assessed outcomes. Conclusions: In situations like the COVID-19 pandemic, targeted mental health interventions and screenings are needed in children and adolescents, especially among minority children.

Review of the current and potential use of biological and molecular methods for the estimation of the postmortem interval in animals and humans.

We provide here an overview of the state of applied techniques in the estimation of the early period of the postmortem interval (PMI). The biological methods included consist of body cooling, CSF potassium, body cooling combined with CSF potassium, and tissue autolysis. For each method, we present its application in human and veterinary medicine and provide current methodology, strengths, and weaknesses, as well as target areas for improvement. We examine current and future molecular methods as they pertain to DNA and primarily to messenger RNA degradation for the estimation of the PMI, as well as the use of RNA in aging wounds, aging blood stains, and the identification of body fluids. Various types of RNA have different lengths, structures, and functions in cells. These differences in RNAs determine various intrinsic properties, such as their half-lives in cells, and, hence, their decay rate as well as their unique use for specific forensic tests. Future applications and refinements of RNA-based techniques provide opportunities for the use of molecular methods in the estimation of PMI and other general forensic applications.

A population-based serological survey of Vibrio cholerae antibody titers in Ouest Department, Haiti in the year prior to the 2022 cholera outbreak.

After three years with no confirmed cholera cases in Haiti, an outbreak of Vibrio cholerae O1 emerged in October 2022. Levels of pre-existing antibodies provide an estimate of prior immunologic exposure, reveal potentially relevant immune responses, and set a baseline for future serosurveillance. We analyzed dried blood spots collected in 2021 from a population-weighted representative cross-sectional serosurvey in two communes in the Ouest Department of Haiti. We found lower levels of circulating IgG and IgA antibodies against V. cholerae lipopolysaccharide (LPS, IgG and IgA p<0.0001) in those below 5 years of age compared to those five years and older. Among a subset of patients with higher titers of antibodies, we were unable to detect any functional (vibriocidal) antibodies. In conclusion, the lack of detectable functional antibodies, and age-discordant levels of V. cholerae LPS IgG, suggest that populations in Haiti may be highly susceptible to cholera disease, especially among young children.

Everglades virus evolution: Genome sequence analysis of the envelope 1 protein reveals recent mutation and divergence in South Florida wetlands.

Everglades virus (EVEV) is a subtype (II) of Venezuelan equine encephalitis virus (VEEV), endemic in southern Florida, USA. EVEV has caused clinical encephalitis in humans, and antibodies have been found in a variety of wild and domesticated mammals. Over 29,000 Culex cedecei females, the main vector of EVEV, were collected in 2017 from Big Cypress and Fakahatchee Strand Preserves in Florida and pool-screened for the presence of EVEV using reverse transcription real-time polymerase chain reaction. The entire 1 E1 protein gene was successfully sequenced from fifteen positive pools. Phylogenetic analysis showed that isolates clustered, based on the location of sampling, into two monophyletic clades that diverged in 2009. Structural analyses revealed two mutations of interest, A116V and H441R, which were shared among all isolates obtained after its first isolation of EVEV in 1963, possibly reflecting adaptation to a new host. Alterations of the Everglades ecosystem may have contributed to the evolution of EVEV and its geographic compartmentalization. This is the first report that shows in detail the evolution of EVEV in South Florida. This zoonotic pathogen warrants inclusion into routine surveillance given the high natural infection rate in the vectors. Invasive species, increasing urbanization, the Everglades restoration, and modifications to the ecosystem due to climate change and habitat fragmentation in South Florida may increase rates of EVEV spillover to the human population.

SARS-CoV-2 infections in infants in Haiti 2020-2021; evidence from a seroepidemiological cohort.

Few data are available on frequency of SARS-CoV-2 infection among very young children in low- to middle-income countries (LMIC), with the studies that are available biased towards higher income countries with low reported infection and seroconversion rates. Between February 2019 and March 2021, 388 dried blood spot (DBS) samples were obtained from 257 children less than 30 months of age as part of a prospective observational cohort study of pregnant women and their infants in Haiti; longitudinal samples were available for 107 children. In a subsequent retrospective analysis, DBS samples were tested by ELISA for antibody targeting the receptor binding domain of the SARS-CoV-2 S1 protein. Over the course of the study, 16·7% of the infants became seropositive. All seropositive samples were collected after March 19, 2020 (the date of the first reported COVID-19 case in Haiti) with the highest hazards measured in August 2020. Sampling date was the only covariate associated with the hazard of seroconversion. Our data provide an estimate of SARS-CoV-2 infection rates among very young children without prior SARS-CoV-2 exposure during the initial pandemic waves in Haiti, and demonstrate that these children mount a detectable serological response which is independent of patient age.

Understanding the epidemiology and impact of the COVID-19 pandemic on domestic violence and child abuse in Saudi Arabia: A cross-sectional study.

Objectives: The purpose of this paper is to highlight the impact of a pandemic on the lives of vulnerable members of the community who have experienced or are 'at risk' of experiencing intimate family violence and child abuse in Saudi Arabia. By reviewing the experience in Saudi Arabia in the context of the international literature, the study explores similarities and differences in the impact of the pandemic on family violence. Methods: The study investigated the impact of the COVID-19 pandemic on family violence and child abuse in Saudi Arabia by conducting a comparative analysis of the prevalence of cases, types of abuse, and geographical location of those experiencing violence between the years 2019 and 2020. Data were obtained from the Family Violence Reporting Center 1919 in Saudi Arabia. Results: The study found that the risk of family violence and child abuse increased during the COVID-19 pandemic in Saudi Arabia, similar to findings in other countries. Conversely, there was a decrease in the number of reports due to factors associated with COVID-19. Conclusions: The study highlighted the importance of ensuring that existing policies and services are sufficient to ensure the risks arising from the impact of COVID-19 responses and minimize the negative consequences.

Chikungunya outbreak in Karachi Pakistan 2016-2017: An analysis of viral isolates.

In December 2016 physicians in Karachi, Pakistan,witnessed an increase in patients presenting with febrile illness and severe polyarthralgia. Subsequently, chikungunya virus (CHIKV)) was isolated from three patients. This virus was sequenced and compared with other isolates of CHIKV obtained in India and Pakistan during recent outbreaks. Phylogenetic analysis indicated that the Karachi isolates were most similar to the East Central South African CHIKV lineage and showed sequence homology to isolates obtained in other parts of Pakistan and India. More importantly, two of the CHIKV isolates had a nucleotide substitution in the E1 gene corresponding to an amino acid change at chain F portion of the E1 protein.

Invasive Burmese pythons alter host use and virus infection in the vector of a zoonotic virus.

The composition of wildlife communities can have strong effects on transmission of zoonotic vector-borne pathogens, with more diverse communities often supporting lower infection prevalence in vectors (dilution effect). The introduced Burmese python, Python bivittatus, is eliminating large and medium-sized mammals throughout southern Florida, USA, impacting local communities and the ecology of zoonotic pathogens. We investigated invasive predator-mediated impacts on ecology of Everglades virus (EVEV), a zoonotic pathogen endemic to Florida that circulates in mosquito-rodent cycle. Using binomial generalized linear mixed effects models of field data at areas of high and low python densities, we show that increasing diversity of dilution host (non-rodent mammals) is associated with decreasing blood meals on amplifying hosts (cotton rats), and that increasing cotton rat host use is associated with increasing EVEV infection in vector mosquitoes. The Burmese python has caused a dramatic decrease in mammal diversity in southern Florida, which has shifted vector host use towards EVEV amplifying hosts (rodents), resulting in an indirect increase in EVEV infection prevalence in vector mosquitoes, putatively elevating human transmission risk. Our results indicate that an invasive predator can impact wildlife communities in ways that indirectly affect human health, highlighting the need for conserving biological diversity and natural communities.

Feasibility of using tissue autolysis to estimate the postmortem interval in horses.

Estimation of the postmortem interval (PMI) is a poorly studied field in veterinary pathology. The development of field-applicable methods is needed given that animal cruelty investigations are increasing continually. We evaluated various histologic criteria in equine brain, liver, and muscle tissue to aid the estimation of PMI in horses, which is central to forensic investigations of suspicious death. After death, autolysis proceeds predictably, depending on environmental conditions. Currently, no field-applied methods exist that accurately estimate the PMI using histology in animals or humans through quantification of autolysis. Brain, liver, and skeletal muscle from 12 freshly euthanized horses were held at 22°C and 8°C for 72 h. Tissues were sampled at T0h, T1h, T2h, T4h, T6h, T12h, T24h, T36h, T48h, T60h, and T72h. For each tissue, we quantified 5 to 7 criteria associated with autolysis, based on the percentage of microscopic field involved. Each criterion was modeled, with temperature and time as independent variables. Changes were most predictable in liver and muscle over the first 72 h postmortem. The criteria for autolysis that were present most extensively at both temperatures were hepatocyte individualization and the separation of bile duct epithelium from the basement membrane. The changes that were present next most extensively were disruption of myofiber continuity, hypereosinophilia, and loss of striation. Brain changes were highly variable. The high statistical correlation between the parameter "autolysis" and the variables "time/temperature", indicates that autolysis is progressive and predictable. Further investigation of these criteria is needed to establish histologic algorithms for PMI.

Perspectives on New Vaccines against Arboviruses Using Insect-Specific Viruses as Platforms.

Arthropod-borne viruses (arboviruses) are global pathogens circulating endemically with local explosive outbreaks and constant encroachment into new locations. Few vaccines against arboviruses exist; most for humans are in development or clinical trials. Insect-specific viruses (ISVs) offer a unique platform for expression of arbovirus proteins, through the creation of ISV/arbovirus chimeras. Studies have shown promising results of these vaccines with several advantages over their wild-type counterparts. In this review, we discuss the current status of these potential vaccines using ISVs.

Insect-Specific Viruses: An overview and their relationship to arboviruses of concern to humans and animals.

The group of Insect-specific viruses (ISVs) includes viruses apparently restricted to insects based on their inability to replicate in the vertebrates. Increasing numbers of ISVs have been discovered and characterized representing a diverse number of viral families. However, most studies have focused on those ISVs belonging to the family Flaviviridae, which highlights the importance of ISV study from other viral families, which allow a better understanding for the mechanisms of transmission and evolution used for this diverse group of viruses. Some ISVs have shown the potential to modulate arboviruses replication and vector competence of mosquitoes. Based on this, ISVs may be used as an alternative tool for biological control, development of vaccines, and diagnostic platforms for arboviruses. In this review, we provide an update of the general characteristics of ISVs and their interaction with arboviruses that infect vertebrates.

Detection of heartworm antigen without cross-reactivity to helminths and protozoa following heat treatment of canine serum.

BACKGROUND: Detection of Dirofilaria immitis, or heartworm, through antigen in sera is the primary means of diagnosing infections in dogs. In recent years, the practice of heat-treating serum prior to antigen testing has demonstrated improved detection of heartworm infection. While the practice of heat-treating serum has resulted in earlier detection and improved sensitivity for heartworm infections, it has been suggested that heat treatment may cause cross reactivity with A. reconditum and intestinal helminth infections of dogs. No studies have assessed the potential cross-reactivity of these parasites with heartworm tests before and after heat treatment using blood products and an appropriate gold standard reference. METHODS: Canine sera (n=163) was used to evaluate a heartworm antigen-ELISA (DiroCHEK®) and potential cross-reactivity with common parasitic infections. The heartworm status and additional parasite infections were confirmed by necropsy and adult helminth species verified morphologically or by PCR, and feces evaluated by centrifugal fecal flotation. RESULTS: Intestinal parasites were confirmed in 140 of the dogs by necropsy, and 130 by fecal flotation. Acanthocheilonema reconditum microfilariae were confirmed in 22 dogs. Prevalence of heartworm infection confirmed by necropsy was 35.6% (58/163). In the 105 dogs without heartworms, specificity remained unchanged at 100% both before and after heat treatment despite confirmed infections with A. reconditum, Ancylostoma caninum, Ancylostoma brasiliense, Trichuris vulpis, Toxocara canis, Dipylidium caninum, Spirometra mansonoides, Macracanthorynchus ingens, Cystoisospora sp., Giardia sp., and Sarcocystis sp. CONCLUSIONS: These findings suggest that the use of heat treatment improves sensitivity of heartworm tests and is unlikely to cause false positive antigen results due to Acanthocheilonema reconditum, intestinal helminths, and protozoal parasites in dogs.

West Nile Virus-Induced Neurologic Sequelae-Relationship to Neurodegenerative Cascades and Dementias.

PURPOSE OF REVIEW: West Nile virus (WNV) emerged from Central Africa in the 1990s and is now endemic throughout much of the world. Twenty years after its introduction in the USA, it is becoming apparent that neurological impairments can persist for years following infection. Here, we review the epidemiological data in support of such long-term deficits and discuss possible mechanisms that drive these persistent manifestations. RECENT FINDINGS: Focusing on the recently discovered antimicrobial roles of amyloid and alpha-synuclein, we connect WNV late pathology to overlapping features encountered in neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. We also summarize new research on microglial activation and engulfment of neural synapses seen in recovered WNV as well as in neurodegenerative diseases, and discuss how loss of integrity of the blood-brain barrier (BBB) may exacerbate this process. SUMMARY: Neuroinvasive viral infections such as WNV may be linked epidemiologically and mechanistically to neurodegeneration. This may open doors to therapeutic options for hitherto untreatable infectious sequelae; additionally, it may also shed light on the possible infectious etiologies of age-progressive neurodegenerative dementias.

The Clinical Features of Co-circulating Dengue Viruses and the Absence of Dengue Hemorrhagic Fever in Pakistan.

Dengue virus (DENV) is the most common and widespread arboviral infection worldwide. Though all four DENV serotypes cocirculate in nature, the clinicopathological framework of these serotypes is undefined in Pakistan. A cross-sectional, observational study was performed to document the circulation of various arboviruses in the Sindh region of Pakistan. Here we describe a population of patients diagnosed with DENV spanning a 2-year period. This study used an orthogonal system of NS1 antigen ELISA followed by RT-PCR for DENV detection and subtyping. A total of 168 NS1 positive patients were evaluated of which 91 patients were serotyped via RT-PCR. There was no significant difference between sex or age for infection risk and peak transmission occurred during the Autumn months. DENV2 was the most common serotype followed by DENV1 then DENV3, then DENV4. The data show that DENV1 patients were more likely to have abnormal liver function tests; DENV2 infected patients were more likely to exhibit arthralgia and neurological symptoms; DENV3 patients were more likely to complain of burning micturition and have elevated lymphocyte counts and low hematocrit; and DENV4 patients were more likely to report headaches and rash. Notably, no dengue hemorrhagic fever or other manifestations of severe dengue fever were present in patients with primary or secondary infections. We were able to identify significantly more NS1 antigen positive patients than RT-PCR. This study demonstrates that all four DENV serotypes are co-circulating and co-infecting in Pakistan.