Clinical and epidemiological characteristics of Madariaga and Venezuelan equine encephalitis virus infections.

Madariaga virus (MADV) and Venezuelan equine encephalitis virus (VEEV) are emerging arboviruses affecting rural and remote areas of Latin America. However, there are limited clinical and epidemiological reports available, and outbreaks are occurring at an increasing frequency. We addressed this gap by analyzing all the available clinical and epidemiological data of MADV and VEEV infections recorded since 1961 in Panama. A total of 168 of human alphavirus encephalitis cases were detected in Panama from 1961 to 2023. Here we describe the clinical signs and symptoms and epidemiological characteristics of these cases, and also explored signs and symptoms as potential predictors of encephalitic alphavirus infection when compared to those of other arbovirus infections occurring in the region. Our results highlight the challenges clinical diagnosis of alphavirus disease in endemic regions with overlapping circulation of multiple arboviruses.

Zoonotic mosquito-borne arboviruses: Spillover, spillback, and realistic mitigation strategies.

Emerging zoonotic mosquito-borne viruses pose increasing health threats because of growing mosquito population, geographic expansions, and control challenges. We emphasize the need for global preparedness to effectively mitigate the health, societal, and economic impacts of spillover by these viruses through proactive measures of prediction, surveillance, prevention, and treatment.

Madariaga and Venezuelan equine encephalitis virus seroprevalence in rodent enzootic hosts in Eastern and Western Panama.

While rodents are primary reservoirs of Venezuelan equine encephalitis virus (VEEV), their role in Madariaga virus (MADV) transmission remains uncertain, particularly given their overlapping geographic distribution. This study explores the interplay of alphavirus prevalence, rodent diversity, and land use within Darien and Western Panama provinces. A total of three locations were selected for rodent sampling in Darien province: Los Pavitos, El Real de Santa Maria and Santa Librada. Two sites were selected in Western Panama province: El Cacao and Cirí Grande. We used plaque reduction neutralization tests to assess MADV and VEEV seroprevalences in 599 rodents of 16 species across five study sites. MADV seroprevalence was observed at higher rates in Los Pavitos (Darien province), 9.0%, 95% CI: 3.6-17.6, while VEEV seroprevalence was elevated in El Cacao (Western Panama province), 27.3%, 95% CI: 16.1-40.9, and El Real de Santa María (Darien province), 20.4%, 95% CI: 12.6-29.7. Species like Oryzomys coesi, 23.1%, 95% CI: 5.0-53.8, and Transandinomys bolivaris, 20.0%, 95% CI: 0.5-71.6 displayed higher MADV seroprevalences than other species, whereas Transandinomys bolivaris, 80.0%, 95% CI: 28.3-99.4, and Proechimys semispinosus, 27.3%, 95% CI: 17.0-39.6, exhibited higher VEEV seroprevalences. Our findings provide support to the notion that rodents are vertebrate reservoirs of MADV and reveal spatial variations in alphavirus seropositivity among rodent species, with different provinces exhibiting distinct rates for MADV and VEEV. Moreover, specific rodent species are linked to unique seroprevalence patterns for these viruses, suggesting that rodent diversity and environmental conditions might play a significant role in shaping alphavirus distribution.

Experimental Yellow Fever in the Squirrel Monkey (Saimiri spp.): Hematological, Biochemical, and Immunological Findings.

Between 2016 and 2018, Brazil experienced the largest sylvatic epidemic of yellow fever virus (YFV). Despite to the magnitude and rapid spread of the epidemic, little is known about YFV dispersion. The study evaluated whether the squirrel monkey is a good model for yellow fever (YF) studies. Methods: Ten animals were infected with 1 × 106 PFU/mL of YFV, with one negative control. Blood samples were collected daily during the first 7 days and at 10, 20 and 30 days post infection (dpi) for detection of viral load and cytokines by RT-qPCR; measurements of AST, ALT, urea and creatinine were taken; IgM/IgG antibodies were detected by ELISA, and hemagglutination inhibition and neutralization tests were performed. The animals exhibited fever, flushed appearance, vomiting and petechiae, and one animal died. Viremia was detected between 1 and 10 dpi, and IgM/IgG antibodies appeared between 4 and 30 dpi. The levels of AST, ALT and urea increased. The immune responses were characterized by expression of S100 and CD11b cells; endothelial markers (VCAM-1, ICAM-1 and VLA-4), cell death and stress (Lysozyme and iNOS); and pro-inflammatory cytokines (IL-8, TNF-α, and IFN-γ) and anti-inflammatory cytokines (IL-10 and TGF-ß). The squirrel monkeys showed changes similar to those described in humans with YF, and are a good experimental model for the study of YF.

Phylogenetic characterization of Orthobunyaviruses isolated from Trinidad shows evidence of natural reassortment.

The genus Orthobunyavirus is a diverse group of viruses in the family Peribunyaviridae, recently classified into 20 serogroups, and 103 virus species. Although most viruses within these serogroups are phylogenetically distinct, the absence of complete genome sequences has left several viruses incompletely characterized. Here we report the complete genome sequences for 11 orthobunyaviruses isolated from Trinidad, French Guiana, Guatemala, and Panama that were serologically classified into six serogroups and 10 species. Phylogenetic analyses of these 11 newly derived sequences indicate that viruses belonging to the Patois, Capim, Guama, and Group C serocomplexes all have a close genetic origin. We show that three of the 11 orthobunyaviruses characterized (belonging to the Group C and Bunyamwera serogroups) have evidence of histories of natural reassortment through the M genome segment. Our data also suggests that two distinct lineages of Group C viruses concurrently circulate in Trinidad and are transmitted by the same mosquito vectors. This study also highlights the importance of complementing serological identification with nucleotide sequencing when characterizing orthobunyaviruses.

Experimental Yellow Fever in Squirrel Monkey: Characterization of Liver In Situ Immune Response.

Non-human primates contribute to the spread of yellow fever virus (YFV) and the establishment of transmission cycles in endemic areas, such as Brazil. This study aims to investigate virological, histopathological and immunohistochemical findings in livers of squirrel monkeys (Saimiri spp.) infected with the YFV. Viremia occurred 1-30 days post infection (dpi) and the virus showed a predilection for the middle zone (Z2). The livers were jaundiced with subcapsular and hemorrhagic multifocal petechiae. Apoptosis, lytic and coagulative necrosis, steatosis and cellular edema were also observed. The immune response was characterized by the expression of S100, CD11b, CD57, CD4 and CD20; endothelial markers; stress and cell death; pro and anti-inflammatory cytokines, as well as Treg (IL-35) and IL-17 throughout the experimental period. Lesions during the severe phase of the disease were associated with excessive production of apoptotic pro-inflammatory cytokines, such as IFN-γ and TNF-α, released by inflammatory response cells (CD4+ and CD8+ T lymphocytes) and associated with high expression of molecules of adhesion in the inflammatory foci observed in Z2. Immunostaining of the local endothelium in vascular cells and the bile duct was intense, suggesting a fundamental role in liver damage and in the pathogenesis of the disease.

Etiologies of Acute Undifferentiated Febrile Illnesses in and near Iquitos from 1993 to 1999 in the Amazon River Basin of Peru.

The objective of this study was to determine the etiology of febrile illnesses among patients from October 1, 1993 through September 30, 1999, in the urban community of Iquitos in the Amazon River Basin of Peru. Epidemiological and clinical data as well as blood samples were obtained from consenting patients at hospitals, health clinics and private residences. Samples were tested for arboviruses in cell cultures and for IgM and IgG antibodies by ELISA. Blood smears were examined for malaria, and sera were tested for antibodies to Leptospira spp. by ELISA and microscopic agglutination. Among 6,607 febrile patients studied, dengue viruses caused 14.6% of the cases, and Venezuelan equine encephalitis virus caused 2.5%, Oropouche virus 1.0%, Mayaro virus 0.4%, and other arboviruses caused 0.2% of the cases. Also, 22.9% of 4,844 patients tested were positive for malaria, and of 400 samples tested, 9% had evidence of acute leptospirosis. Although the study was not designed to assess the importance of these pathogens as a cause of human morbidity in the total population, these results indicate that arboviruses, leptospirosis, and malaria were the cause of approximately 50% of the febrile cases. Although the arboviruses that were diagnosed can produce asymptomatic infections, our findings increased the overall understanding of the relative health burden of these infections, as well as baseline knowledge needed for designing and implementing further studies to better assess the health impact and threat of these pathogens in the Amazon Basin of Peru.

ICTV Virus Taxonomy Profile: Rhabdoviridae 2022.

The family Rhabdoviridae comprises viruses with negative-sense (-) RNA genomes of 10-16 kb. Virions are typically enveloped with bullet-shaped or bacilliform morphology but can also be non-enveloped filaments. Rhabdoviruses infect plants or animals, including mammals, birds, reptiles, amphibians or fish, as well as arthropods, which serve as single hosts or act as biological vectors for transmission to animals or plants. Rhabdoviruses include important pathogens of humans, livestock, fish or agricultural crops. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Rhabdoviridae, which is available at ictv.global/report/rhabdoviridae.

Emergence potential of mosquito-borne arboviruses from the Florida Everglades.

The Greater Everglades Region of South Florida is one of the largest natural wetlands and the only subtropical ecosystem found in the continental United States. Mosquitoes are seasonally abundant in the Everglades where several potentially pathogenic mosquito-borne arboviruses are maintained in natural transmission cycles involving vector-competent mosquitoes and reservoir-competent vertebrate hosts. The fragile nature of this ecosystem is vulnerable to many sources of environmental change, including a wetlands restoration project, climate change, invasive species and residential development. In this study, we obtained baseline data on the distribution and abundance of both mosquitos and arboviruses occurring in the southern Everglades region during the summer months of 2013, when water levels were high, and in 2014, when water levels were low. A total of 367,060 mosquitoes were collected with CO2-baited CDC light traps at 105 collection sites stratified among the major landscape features found in Everglades National Park, Big Cypress National Preserve, Fakahatchee State Park Preserve and Picayune State Forest, an area already undergoing restoration. A total of 2,010 pools of taxonomically identified mosquitoes were cultured for arbovirus isolation and identification. Seven vertebrate arboviruses were isolated: Everglades virus, Tensaw virus, Shark River virus, Gumbo Limbo virus, Mahogany Hammock virus, Keystone virus, and St. Louis encephalitis virus. Except for Tensaw virus, which was absent in 2013, the remaining viruses were found to be most prevalent in hardwood hammocks and in Fakahatchee, less prevalent in mangroves and pinelands, and absent in cypress and sawgrass. In contrast, in the summer of 2014 when water levels were lower, these arboviruses were far less prevalent and only found in hardwood hammocks, but Tensaw virus was present in cypress, sawgrass, pinelands, and a recently burned site. Major environmental changes are anticipated in the Everglades, many of which will result in increased water levels. How these might lead to the emergence of arboviruses potentially pathogenic to both humans and wildlife is discussed.

The Usefulness of a Duplex RT-qPCR during the Recent Yellow Fever Brazilian Epidemic: Surveillance of Vaccine Adverse Events, Epizootics and Vectors.

From 2016 to 2018, Brazil faced the biggest yellow fever (YF) outbreak in the last 80 years, representing a risk of YF reurbanization, especially in megacities. Along with this challenge, the mass administration of the fractionated YF vaccine dose in a naïve population brought another concern: the possibility to increase YF adverse events associated with viscerotropic (YEL-AVD) or neurological disease (YEL-AND). For this reason, we developed a quantitative real time RT-PCR (RT-qPCR) assay based on a duplex TaqMan protocol to distinguish broad-spectrum infections caused by wild-type yellow fever virus (YFV) strain from adverse events following immunization (AEFI) by 17DD strain during the vaccination campaign used to contain this outbreak. A rapid and more accurate RT-qPCR assay to diagnose YFV was established, being able to detect even different YFV genotypes and geographic strains that circulate in Central and South America. Moreover, after testing around 1400 samples from human cases, non-human primates and mosquitoes, we detected just two YEL-AVD cases, confirmed by sequencing, during the massive vaccination in Brazilian Southeast region, showing lower incidence than AEFI as expected.

Isolation of a novel insect-specific flavivirus with immunomodulatory effects in vertebrate systems.

We describe the isolation and characterization of a novel insect-specific flavivirus (ISFV), tentatively named Aripo virus (ARPV), that was isolated from Psorophora albipes mosquitoes collected in Trinidad. The ARPV genome was determined and phylogenetic analyses showed that it is a dual host associated ISFV, and clusters with the main mosquito-borne flaviviruses. ARPV antigen was significantly cross-reactive with Japanese encephalitis virus serogroup antisera, with significant cross-reactivity to Ilheus and West Nile virus (WNV). Results suggest that ARPV replication is limited to mosquitoes, as it did not replicate in the sandfly, culicoides or vertebrate cell lines tested. We also demonstrated that ARPV is endocytosed into vertebrate cells and is highly immunomodulatory, producing a robust innate immune response despite its inability to replicate in vertebrate systems. We show that prior infection or coinfection with ARPV limits WNV-induced disease in mouse models, likely the result of a robust ARPV-induced type I interferon response.

Negeviruses Reduce Replication of Alphaviruses during Coinfection.

Negeviruses are a group of insect-specific viruses (ISVs) that have been found in many arthropods. Their presence in important vector species led us to examine their interactions with arboviruses during coinfections. Wild-type negeviruses reduced the replication of several alphaviruses during coinfections in mosquito cells. Negev virus (NEGV) isolates were also used to express green fluorescent protein (GFP) and anti-chikungunya virus (CHIKV) antibody fragments during coinfections with CHIKV. NEGV expressing anti-CHIKV antibody fragments was able to further reduce replication of CHIKV during coinfections, while reductions of CHIKV with NEGV expressing GFP were similar to titers with wild-type NEGV alone. These results are the first to show that negeviruses induce superinfection exclusion of arboviruses and to demonstrate a novel approach to deliver antiviral antibody fragments with paratransgenic ISVs. The ability to inhibit arbovirus replication and express exogenous proteins in mosquito cells makes negeviruses a promising platform for control of arthropod-borne pathogens. IMPORTANCE Negeviruses are a group of insect-specific viruses (ISVs), viruses known to infect only insects. They have been discovered over a wide geographical and species range. Their ability to infect mosquito species that transmit dangerous arboviruses makes negeviruses a candidate for a pathogen control platform. Coinfections of mosquito cells with a negevirus and an alphavirus demonstrated that negeviruses can inhibit the replication of alphaviruses. Additionally, modifying Negev virus (NEGV) to express a fragment of an anti-CHIKV antibody further reduced the replication of CHIKV in coinfected cells. This is the first evidence to demonstrate that negeviruses can inhibit the replication of important arboviruses in mosquito cells. The ability of a modified NEGV to drive the expression of antiviral proteins also highlights a method for negeviruses to target specific pathogens and limit the incidence of vector-borne diseases.

Experimental yellow fever virus infection in the squirrel monkey (Saimiri spp.) I: gross anatomical and histopathological findings in organs at necropsy.

BACKGROUND: Non-human primates contribute to the spread of the yellow fever virus (YFV) and the establishment of transmission cycles in endemic areas. OBJECTIVE: To describe the severe histopathological aspects of YFV infection, 10 squirrel monkeys were infected with YFV and blood, brain, liver, kidney, spleen, heart, lung, lymph node and stomach were collected at 1-7, 10, 20 and 30 days post-infection (dpi). METHODS: Histopathological analysis and detection of the genome and viral antigens and neutralising antibodies were performed by RT-PCR, immunohistochemistry and neutralisation test, respectively. FINDINGS: Only one animal died from the experimental infection. The genome and viral antigens were detected in all investigated organs (1-30 dpi) and the neutralising antibodies from seven to 30 dpi. The brain contained perivascular haemorrhage (6 dpi); in the liver, midzonal haemorrhage and lytic necrosis (6 dpi) were observed. The kidney had bleeding in the Bowman's capsule and tubular necrosis (6 dpi). Pyknotic lymphocytes were observed in the spleen (1-20 dpi), the lung had haemorrhage (2-6 dpi), in the endocardium it contained nuclear pyknosis and necrosis (2-3 dpi) and the stomach contained blood in the lumen (6 dpi). MAIN FINDINGS: Squirrel monkeys reliably reproduced the responses observed in human cases of yellow fever and, therefore, constitute an excellent experimental model for studies on the pathophysiology of the disease.

Hayes Yard virus: a novel ephemerovirus isolated from a bull with severe clinical signs of bovine ephemeral fever is most closely related to Puchong virus.

Bovine ephemeral fever is a vector-borne disease of ruminants that occurs in tropical and sub-tropical regions of Africa, Asia and Australia. The disease is caused by a rhabdovirus, bovine ephemeral fever virus (BEFV), which occurs as a single serotype globally. Although several other closely related ephemeroviruses have been isolated from cattle and/or arthropods, only kotonkan virus from Nigeria and (tentatively) Mavingoni virus from Mayotte Island in the Indian Ocean have been previously associated with febrile disease. Here, we report the isolation of a novel virus (Hayes Yard virus; HYV) from blood collected in February 2000 from a bull (Bos indicus) in the Northern Territory of Australia. The animal was suffering from a severe ephemeral fever-like illness with neurological involvement, including recumbency and paralysis, and was euthanised. Histological examination of spinal cord and lung tissue identified extensive haemorrhage in the dura mata with moderate perineuronal oedema and extensive emphysema. HYV displayed cone-shaped morphology, typical of rhabdoviruses, and was found to be most closely related antigenically to Puchong virus (PUCV), isolated in 1965 from mosquitoes in Malaysia. Analysis of complete genome sequences of HYV (15 025 nt) and PUCV (14 932 nt) indicated that each has a complex organisation (3' N-P-M-G-GNS-α1-α2-ß-γ-L 5') and expression strategy, similar to that of BEFV. Based on an alignment of complete L protein sequences, HYV and PUCV cluster with other rhabdoviruses in the genus Ephemerovirus and appear to represent two new species. Neutralising antibody to HYV was also detected in a retrospective survey of cattle sera collected in the Northern Territory.

Genomic characterization of Changuinola viruses from Panama: evidence for multiple genome segment reassortment.

The complete coding sequences of five divergent strains of Changuinola virus (CGLV), collected over a 16-year period in Panama, were determined, using viral metagenomics. Each strain had 10 RNA segments that encoded structural and non-structural proteins with amino acid identities ranging from 33 to 99% with sequences of other 15 members of the Changuinola virus (Reoviridae: Orbivirus) species group. Genetic analyses of the five Panamanian virus strains revealed probable reassortment among multiple segments of the viruses.

Characterization of Port Bolivar Virus, a Novel Entomobirnavirus (Birnaviridae) Isolated from Mosquitoes Collected in East Texas, USA.

This report describes and characterizes a novel entomobirnavirus, designated Port Bolivar virus (PTBV), that was isolated from a pool of Aedes sollicitans mosquitoes collected in a saltwater marsh in East Texas, USA. Full genome sequencing and phylogenetic analyses indicate that PTBV is distinct but genetically related to Drosophila X virus and mosquito X virus, which are assigned to species in the genus Entomobirnavirus, family Birnaviridae. PTBV produced cytopathic effect (CPE) in cultures of mosquito (C6/36) cells, but not in Vero cell cultures. Ultrastructural studies of PTBV in infected C6/36 cells demonstrated unenveloped virus particles about 55 nm in diameter.

Molecular Characterization of Hamster-Adapted Yellow Fever Virus.

We previously reported two hamster models for viscerotropic yellow fever virus (YFV) infection: one using a YFV strain (Jiménez), isolated from a fatal human case in Panama in 1974, and the other using the prototype YFV strain (Asibi). Asibi hamster passage 7 (P7) was associated with accumulation of seven amino acid substitutions, including five in the envelope protein. In this study we report the genome sequences of the hamster Jiménez P0 and P10 viruses in which we identified only two amino acid substitutions during passage, one each in the nonstructural proteins NS3 and NS5, indicating a role for the nonstructural proteins in increased YFV viscerotropism in the Jiménez hamster model. Thus, there are multiple molecular mechanisms involved in viscerotropism of YFV in the hamster model. Neither Asibi P7 nor Jiménez P10 viruses were viscerotropic in mice or guinea pigs. Thus, the hamster viscerotropic phenotype did not translate to other laboratory rodent species.

Experimental yellow fever virus infection in the squirrel monkey (Saimiri spp. ) I: gross anatomical and histopathological findings in organs at necropsy

BACKGROUND Non-human primates contribute to the spread of the yellow fever virus (YFV) and the establishment of transmission cycles in endemic areas. OBJECTIVE To describe the severe histopathological aspects of YFV infection, 10 squirrel monkeys were infected with YFV and blood, brain, liver, kidney, spleen, heart, lung, lymph node and stomach were collected at 1-7, 10, 20 and 30 days post-infection (dpi). METHODS Histopathological analysis and detection of the genome and viral antigens and neutralising antibodies were performed by RT-PCR, immunohistochemistry and neutralisation test, respectively. FINDINGS Only one animal died from the experimental infection. The genome and viral antigens were detected in all investigated organs (1-30 dpi) and the neutralising antibodies from seven to 30 dpi. The brain contained perivascular haemorrhage (6 dpi); in the liver, midzonal haemorrhage and lytic necrosis (6 dpi) were observed. The kidney had bleeding in the Bowman's capsule and tubular necrosis (6 dpi). Pyknotic lymphocytes were observed in the spleen (1-20 dpi), the lung had haemorrhage (2-6 dpi), in the endocardium it contained nuclear pyknosis and necrosis (2-3 dpi) and the stomach contained blood in the lumen (6 dpi). MAIN FINDINGS Squirrel monkeys reliably reproduced the responses observed in human cases of yellow fever and, therefore, constitute an excellent experimental model for studies on the pathophysiology of the disease.

Characterization of Three Novel Viruses from the Families Nyamiviridae, Orthomyxoviridae, and Peribunyaviridae, Isolated from Dead Birds Collected during West Nile Virus Surveillance in Harris County, Texas.

This report describes and characterizes three novel RNA viruses isolated from dead birds collected during West Nile virus surveillance in Harris County, TX, USA (the Houston metropolitan area). The novel viruses are identified as members of the families Nyamaviridae, Orthomyxoviridae, and Peribunyaviridae and have been designated as San Jacinto virus, Mason Creek virus, and Buffalo Bayou virus, respectively. Their potential public health and/or veterinary importance are still unknown.

Inter- and intra-lineage genetic diversity of wild-type Zika viruses reveals both common and distinctive nucleotide variants and clusters of genomic diversity.

Zika virus (ZIKV) strains belong to the East African, West African, and Asian/American phylogenetic lineages. RNA viruses, like ZIKV, exist as populations of genetically-related sequences whose heterogeneity may impact viral fitness, evolution, and virulence. Genetic diversity of representative ZIKVs from each lineage was examined using next generation sequencing (NGS) paired with downstream entropy and single nucleotide variant (SNV) analysis. Comparisons showed that inter-lineage diversity was statistically supported, while intra-lineage diversity. Intra-lineage diversity was significant for East but not West Africa strains. Furthermore, intra-lineage diversity for the Asian/American lineage was not supported for human serum isolates; however, a placenta isolate differed significantly. Relative in the pre-membrane/membrane (prM/M) gene of several ZIKV strains. Additionally, the East African lineage contained a greater number of synonymous SNVs, while a greater number of non-synonymous SNVs were identified for American strains. Further, inter-lineage SNVs were dispersed throughout the genome, whereas intra-lineage non-synonymous SNVs for Asian/American strains clustered within prM/M and NS1 gene. This comprehensive analysis of ZIKV genetic diversity provides a repository of SNV positions across lineages. We posit that increased non-synonymous SNV populations and increased relative genetic diversity of the prM/M and NS1 proteins provides more evidence for their role in ZIKV virulence and fitness.