Choclo virus (CHOV) recovered from deep metatranscriptomics of archived frozen tissues in natural history biorepositories.

BACKGROUND: Hantaviruses are negative-stranded RNA viruses that can sometimes cause severe disease in humans; however, they are maintained in mammalian host populations without causing harm. In Panama, sigmodontine rodents serve as hosts to transmissible hantaviruses. Due to natural and anthropogenic forces, these rodent populations are having increased contact with humans. METHODS: We extracted RNA and performed Illumina deep metatranscriptomic sequencing on Orthohantavirus seropositive museum tissues from rodents. We acquired sequence reads mapping to Choclo virus (CHOV, Orthohantavirus chocloense) from heart and kidney tissue of a two-decade old frozen museum sample from a Costa Rican pygmy rice rat (Oligoryzomys costaricensis) collected in Panama. Reads mapped to the CHOV reference were assembled and then validated by visualization of the mapped reads against the assembly. RESULTS: We recovered a 91% complete consensus sequence from a reference-guided assembly to CHOV with an average of 16X coverage. The S and M segments used in our phylogenetic analyses were nearly complete (98% and 99%, respectively). There were 1,199 ambiguous base calls of which 93% were present in the L segment. Our assembled genome varied 1.1% from the CHOV reference sequence resulting in eight nonsynonymous mutations. Further analysis of all publicly available partial S segment sequences support a clear relationship between CHOV clinical cases and O. costaricensis acquired strains. CONCLUSIONS: Viruses occurring at extremely low abundances can be recovered from deep metatranscriptomics of archival tissues housed in research natural history museum biorepositories. Our efforts resulted in the second CHOV genome publicly available. This genomic data is important for future surveillance and diagnostic tools as well as understanding the evolution and pathogenicity of CHOV.

Two Decades of Wildlife Pathogen Surveillance: Case Study of Choclo orthohantavirus and Its Wild Reservoir Oligoryzomys costaricensis.

The Costa Rican pygmy rice rat (Oligoryzomys costaricensis) is the primary reservoir of Choclo orthohantavirus (CHOV), the causal agent of hantavirus disease, pulmonary syndrome, and fever in humans in Panama. Since the emergence of CHOV in early 2000, we have systematically sampled and archived rodents from >150 sites across Panama to establish a baseline understanding of the host and virus, producing a permanent archive of holistic specimens that we are now probing in greater detail. We summarize these collections and explore preliminary habitat/virus associations to guide future wildlife surveillance and public health efforts related to CHOV and other zoonotic pathogens. Host sequences of the mitochondrial cytochrome b gene form a single monophyletic clade in Panama, despite wide distribution across Panama. Seropositive samples were concentrated in the central region of western Panama, consistent with the ecology of this agricultural commensal and the higher incidence of CHOV in humans in that region. Hantavirus seroprevalence in the pygmy rice rat was >15% overall, with the highest prevalence in agricultural areas (21%) and the lowest prevalence in shrublands (11%). Host-pathogen distribution, transmission dynamics, genomic evolution, and habitat affinities can be derived from the preserved samples, which include frozen tissues, and now provide a foundation for expanded investigations of orthohantaviruses in Panama.

Hantavirus in Panama: Twenty Years of Epidemiological Surveillance Experience.

Twenty years have passed since the emergence of hantavirus zoonosis in Panama at the beginning of this millennium. We provide an overview of epidemiological surveillance of hantavirus disease (hantavirus pulmonary syndrome and hantavirus fever) during the period 1999-2019 by including all reported and confirmed cases according to the case definition established by the health authority. Our findings reveal that hantavirus disease is a low-frequency disease, affecting primarily young people, with a relatively low case-fatality rate compared to other hantaviruses in the Americas (e.g., ANDV and SNV). It presents an annual variation with peaks every 4-5 years and an interannual variation influenced by agricultural activities. Hantavirus disease is endemic in about 27% of Panama, which corresponds to agroecological conditions that favor the population dynamics of the rodent host, Oligoryzomys costaricensis and the virus (Choclo orthohantavirus) responsible for hantavirus disease. However, this does not rule out the existence of other endemic areas to be characterized. Undoubtedly, decentralization of the laboratory test and dissemination of evidence-based surveillance guidelines and regulations have standardized and improved diagnosis, notification at the level of the primary care system, and management in intensive care units nationwide.

Cytokine Profiles and Antibody Response Associated to Choclo Orthohantavirus Infection.

Background: New World Hantaviruses (NWHs) are the etiological agent underlying hantavirus cardiopulmonary syndrome (HCPS), a severe respiratory disease with high mortality rates in humans. In Panama, infections with Choclo Orthohantavirus (CHOV) cause a much milder illness characterized by higher seroprevalence and lower mortality rates. To date, the cytokine profiles and antibody responses associated with this milder form of HCPS have not been defined. Therefore, in this study, we examined immune serological profiles associated with CHOV infections. Methods: For this retrospective study, sera from fifteen individuals with acute CHOV-induced HCPS, were analyzed alongside sera from fifteen convalescent phase individuals and thirty-three asymptomatic, CHOV-seropositive individuals. Cytokine profiles were analyzed by multiplex immunoassay. Antibody subclasses, binding, and neutralization against CHOV-glycoprotein (CHOV-GP) were evaluated by ELISA, and flow cytometry. Results: High titers of IFNγ, IL-4, IL-8, and IL-10 serum cytokines were found in the acute individuals. Elevated IL-4 serum levels were found in convalescent and asymptomatic seropositive individuals. High titers of IgG1 subclass were observed across the three cohorts analyzed. Neutralizing antibody response against CHOV-GP was detectable in few acute individuals but was strong in both convalescent and asymptomatic seropositive individuals. Conclusion: A Th1/Th2 cytokine signature is characteristic during acute mild HCPS caused by CHOV infection. High expression of Th2 and IL-8 cytokines are correlated with clinical parameters in acute mild HCPS. In addition, a strong IL-4 signature is associated with different cohorts, including asymptomatic individuals. Furthermore, asymptomatic individuals presented high titers of neutralizing antibodies.

Implementation of bamboo and monkey-pot traps for the sampling cavity-breeding mosquitoes in Darién, Panama.

Despite the importance of Aedes, Haemagogus and Sabethes in the transmission of yellow fever virus (YFV) and the public health impacts of recent YFV epidemics in the Americas, relatively little has been reported on the biology and ecology of these vectors. Many Aedes, Haemagogus and Sabethes spp. in the American tropics inhabit and develop in the forest canopy and are difficult to sample with conventional entomological surveillance methods. We tested the utility of two previously developed phytotelmata-style oviposition traps (bamboo Guadua angustifolia) and (monkey-pot Lecythis minor), for collecting immature forms of these mosquitoes in a forest near the community of Aruza Abajo, Darién Province, Panama. Our results showed distribution of mosquito species emerging from the two types of traps was found to be significantly different (X2 = 210.23; df = 14; P < 0.001), with significantly greater numbers of Sabethes (Peytonulus) aurescens (Lutz) and Sabethes (Peytonulus) undosus (Coquillett) emerging from the bamboo traps. More females of Sabethes (Sabethes) cyaneus (Fabricius) were captured in the monkey-pot traps, although the difference was not significant. No differences were observed in the average time to emergence for the two traps. These results suggest that various phytotelmata-style traps, including monkey-pot and bamboo, could be used to improve entomological surveillance of YFV vectors in the American tropic.

Endemic and Epidemic Human Alphavirus Infections in Eastern Panama: An Analysis of Population-Based Cross-Sectional Surveys.

Madariaga virus (MADV) has recently been associated with severe human disease in Panama, where the closely related Venezuelan equine encephalitis virus (VEEV) also circulates. In June 2017, a fatal MADV infection was confirmed in a community of Darien Province. We conducted a cross-sectional outbreak investigation with human and mosquito collections in July 2017, where sera were tested for alphavirus antibodies and viral RNA. In addition, by applying a catalytic, force-of-infection (FOI) statistical model to two serosurveys from Darien Province in 2012 and 2017, we investigated whether endemic or epidemic alphavirus transmission occurred historically. In 2017, MADV and VEEV IgM seroprevalences were 1.6% and 4.4%, respectively; IgG antibody prevalences were MADV: 13.2%, VEEV: 16.8%, Una virus (UNAV): 16.0%, and Mayaro virus: 1.1%. Active viral circulation was not detected. Evidence of MADV and UNAV infection was found near households, raising questions about its vectors and enzootic transmission cycles. Insomnia was associated with MADV and VEEV infections, depression symptoms were associated with MADV, and dizziness with VEEV and UNAV. Force-of-infection analyses suggest endemic alphavirus transmission historically, with recent increased human exposure to MADV and VEEV in Aruza and Mercadeo, respectively. The lack of additional neurological cases suggests that severe MADV and VEEV infections occur only rarely. Our results indicate that over the past five decades, alphavirus infections have occurred at low levels in eastern Panama, but that MADV and VEEV infections have recently increased-potentially during the past decade. Endemic infections and outbreaks of MADV and VEEV appear to differ spatially in some locations of eastern Panama.

Unusual pattern of chikungunya virus epidemic in the Americas, the Panamanian experience.

BACKGROUND: Chikungunya virus (CHIKV) typically causes explosive epidemics of fever, rash and polyarthralgia after its introduction into naïve populations. Since its introduction in Panama in May of 2014, few autochthonous cases have been reported; most of them were found within limited outbreaks in Panama City in 2014 and Puerto Obaldia town, near the Caribbean border with Colombia in 2015. In order to confirm that Panama had few CHIKV cases compared with neighboring countries, we perform an epidemiological analysis of chikungunya cases reported from May 2014 to July 2015. Moreover, to understand this paucity of confirmed CHIKV cases, a vectorial analysis in the counties where these cases were reported was performed. METHODS: Chikungunya cases were identified at medical centers and notified to health authorities. Sera samples were analyzed at Gorgas Memorial Institute for viral RNA and CHIKV-specific antibody detection. RESULTS: A total of 413 suspected cases of CHIKV infections were reported, with incidence rates of 0.5 and 0.7 per 100,000 inhabitants in 2014 and 2015, respectively. During this period, 38.6% of CHIKV cases were autochthonous with rash and polyarthralgia as predominant symptoms. CHIKV and DENV incidence ratios were 1:306 and 1:34, respectively. A phylogenetic analysis of E1/E2 genomic segment indicates that the outbreak strains belong to the Asian genotype and cluster together with CHIKV isolates from other American countries during the same period. Statistical analysis of the National Vector Control program at the district level shows low and medium vector infestation level for most of the counties with CHIKV cases. This index was lower than for neighboring countries. CONCLUSIONS: Previous training of clinical, laboratory and vector workers allowed a good caption and detection of the chikungunya cases and fast intervention. It is possible that low/medium vector infestation level could explain in part the paucity of chikungunya infections in Panama.

Spatial-Temporal Distribution of Hantavirus Rodent-Borne Infection by Oligoryzomys fulvescens in the Agua Buena Region--Panama.

BACKGROUND: Hotspot detection and characterization has played an increasing role in understanding the maintenance and transmission of zoonotic pathogens. Identifying the specific environmental factors (or their correlates) that influence reservoir host abundance help increase understanding of how pathogens are maintained in natural systems and are crucial to identifying disease risk. However, most recent studies are performed at macro-scale and describe broad temporal patterns of population abundances. Few have been conducted at a microscale over short time periods that better capture the dynamical patterns of key populations. These finer resolution studies may better define the likelihood of local pathogen persistence. This study characterizes the landscape distribution and spatio-temporal dynamics of Oligoryzomys fulvescens (O. fulvescens), an important mammalian reservoir in Central America. METHODS: Information collected in a longitudinal study of rodent populations in the community of Agua Buena in Tonosí, Panama, between April 2006 and December 2009 was analyzed using non-spatial analyses (box plots) and explicit spatial statistical tests (correlograms, SADIE and LISA). A 90 node grid was built (raster format) to design a base map. The area between the nodes was 0.09 km(2) and the total study area was 6.43 km(2) (2.39 x 2.69 km). The temporal assessment dataset was divided into four periods for each year studied: the dry season, rainy season, and two months-long transitions between seasons (the months of April and December). RESULTS: There were heterogeneous patterns in the population densities and degrees of dispersion of O. fulvescens that varied across seasons and among years. The species typically was locally absent during the late transitional months of the season, and re-established locally in subsequent years. These populations re-occurred in the same area during the first three years but subsequently re-established further south in the final year of the study. Spatial autocorrelation analyses indicated local populations encompassed approximately 300-600 m. The borders between suitable and unsuitable habitats were sharply demarcated over short distances. CONCLUSION: Oligoryzomys fulvescens showed a well-defined spatial pattern that evolved over time, and led to a pattern of changing aggregation. Thus, hot spots of abundance showed a general shifting pattern that helps explain the intermittent risk from pathogens transmitted by this species. This variation was associated with seasonality, as well as anthropogenic pressures that occurred with agricultural activities. These factors help define the characteristics of the occurrence, timing, intensity and duration of synanthropic populations affected by human populations and, consequently, possible exposure that local human populations experience.

Epidemiology of Emergent Madariaga Encephalitis in a Region with Endemic Venezuelan Equine Encephalitis: Initial Host Studies and Human Cross-Sectional Study in Darien, Panama.

BACKGROUND: Neurotropic arboviral infections are an important cause of encephalitis. A zoonotic, vector-borne alphavirus, Madariaga virus (MADV; formerly known as South American eastern equine encephalitis virus), caused its first documented human outbreak in 2010 in Darien, Panama, where the genetically similar Venezuelan equine encephalitis virus (VEEV) is endemic. We report the results of a seroprevalence survey of animals and humans, illustrating contrasting features of MADV and VEEV ecology and epidemiology. METHODS: Small mammals were trapped in 42 sites in Darien, Panama, using Sherman traps, Tomahawk traps, and mist nets for bats. Blood was tested for the presence of neutralizing antibodies to MADV and VEEV. In addition, bird sera collected in 2007 in Chagres, Panama, were tested for MADV and VEEV neutralizing antibodies. Viremia was ascertained by RT-PCR. Human exposure to these two viruses was determined by IgG ELISA, followed by plaque reduction neutralization tests. To identify relevant risk factors for MADV or VEEV exposure, logistic regression analysis was performed, and the most parsimonious model was selected based on the Akaike information criterion. RESULTS: The animal survey yielded 32 bats (16 species), 556 rodents (12 species), and 20 opossums (4 species). The short-tailed cane mouse (Zygodontomys brevicauda) found abundantly in pasture and farms, had the highest MADV seroprevalence (8.3%). For VEEV, the shrub and forest-dwelling long-whiskered rice rat (Transandinomys bolivaris) had the highest seroprevalence (19.0%). Viremia was detected in one animal (Z. brevicauda). Of the 159 bird sera (50 species) tested, none were positive for either virus. In humans (n = 770), neutralizing antibodies to MADV and VEEV were present in 4.8% and 31.5%, respectively. MADV seropositivity was positively associated with cattle ranching, farming, and fishing. Having VEEV antibodies and shrubs near the house diminished risk. Age, forest work, farming and fishing were risk factors for VEEV, while having MADV antibodies, glazed windows, waste pick-up and piped water were protective. CONCLUSION: Our findings suggest that the short-tailed cane mouse and the long-whiskered rice rat serve as hosts for MADV and VEEV, respectively. The preferred habitat of these rodent species coincides with areas associated with human infection risk. Our findings also indicate that MADV emerged recently in humans, and that the transmission cycles of these two sympatric alphaviruses differ spatially and in host utilization.

[Detection of Rickettsia in ectoparasites of wild and domestic mammals from the Cerro Chucanti private reserve and from neighboring towns, Panamá, 2007-2010]. / Detección de Rickettsia spp. en ectoparásitos de animales domésticos y silvestres de la Reserva Natural Privada Cerro Chucantí y comunidades aledañas, Panamá, 2007-2010.

INTRODUCTION: Ectoparasites are the main vectors of rickettsiosis. In Panama, however, limited data are available concerning the arthropod species that serve as vectors or reservoirs. OBJECTIVES: Data are presented concerning the presence of Rickettsia in ectoparasites of wildlife and domestic animals in the Cerro Chucantí private nature reserve and in neighboring villages. MATERIALS AND METHODS: Nine humans, 95 domestic mammals and 48 wild mammals were examined. Twenty-one species of ectoparasites were obtained, including fleas, lice, ticks and mites. These were preserved in 95% ethanol. Later, the DNA was extracted from the ticks and fleas and analyzed by molecular techniques to detect presence of Rickettsia. RESULTS: Of a total of 425 PCR reactions, 270 were positive for Rickettsia and 155 negative. Among the positive samples, 86 PCR amplified for the gltA gene (55% of positives) and 41 of these also amplified the ompA gene. DNA of Rickettsia amblyommii was found in horses ticks (Amblyomma cajennense, Dermacentor nitens), dogs ticks (Rhipicephalus sanguineus) and free living nymphs in the forest. Additionally, DNA of R. felis was found in fleas from dogs Ctenocephalides felis. CONCLUSIONS: The presence of R. amblyommii and R. felis was detected in ticks and fleas of domestic animals in villages near Cerro Chucanti; however no Rickettsia DNA was found in ectoparasites of non-domestic wildlife.

Hantavirus infection and habitat associations among rodent populations in agroecosystems of Panama: implications for human disease risk.

Hantavirus cardiopulmonary syndrome (HCPS), which is caused by infection with Choclo virus, is uncommon in Panama, yet seropositivity among rural residents is as high as 60%. To clarify the environmental risk factors favoring rodent-to-human transmission, we tested serum from 3,067 rodents captured over a five-year period for antibodies against recombinant N protein of hantavirus by enzyme immunoassay and strip immunoblot. Among 220 seropositive rodents, Oligoryzomys fulvescens, the reservoir of Choclo virus, had the highest overall seroprevalence (23.5%); more abundant rodents (Zygodontomys brevicauda and Sigmodon hirsutus) had lower seroprevalences. In the mixed (combined modern and traditional) productive agroecosystem, the highest seroprevalence was among O. fulvescens captured in residences and in crops grown within 40 meters of a residence, with significantly lower seroprevalence in adjacent pasture and non-productive vegetation. Thus, crop habitats may serve as refugia for invasion into adjacent human residences and suggests several interventions to reduce human infection.

Detección de Rickettsia spp. en ectoparásitos de animales domésticos y silvestres de la Reserva Natural Privada Cerro Chucantí y comunidades aledañas, Panamá, 2007-2010 / Detection of Rickettsia in ectoparasites of wild and domestic mammals from the Cerro Chucanti private reserve and from neighboring towns, Panamá, 2007-2010

Introducción. Los ectoparásitos son los principales vectores de rickettsiosis. En Panamá se tienen escasos datos sobre los artrópodos que pudieran considerarse vectores o reservorios. Objetivos. Presentar datos sobre la presencia de Rickettsia spp. en ectoparásitos de fauna silvestre y animales domésticos en la Reserva Natural Privada Cerro Chucantí y poblados vecinos. Materiales y métodos. Se revisaron 9 personas, 95 mamíferos domésticos y 48 silvestres. Los animales domésticos se examinaron con anuencia del propietario, mientras que la fauna silvestre se capturó con trampas Sherman y Tomahawk. Se extrajeron 21 especies de ectoparásitos: pulgas, piojos, garrapatas y otros ácaros, los cuales se preservaron en etanol al 95 %. Se extrajo material genético de garrapatas y pulgas para ser analizado por técnicas moleculares en la detección de Rickettsia spp. Resultados. Se practicaron 425 reacciones de PCR, de las cuales, 270 resultaron negativas y 155 positivas. De las positivas, 86 amplificaron para el gen gltA (55 % de las positivas); de estos también amplificaron 41 (26 %) para ompA. Se encontró material genético de Rickettsia amblyommii, en garrapatas de caballos (Amblyomma cajennense, Dermacentor nitens), de perros (Rhipicephalus sanguineus) y ninfas de Amblyomma recolectadas en el bosque. Además, se detectó ADN de R. felis en pulgas Ctenocephalides felis de perros. Conclusiones. Se pudo detectar la presencia de R. amblyommii y R. felis en garrapatas y pulgas de animales domésticos de los poblados cercanos a Cerro Chucantí, aun cuando no se pudo encontrar material genético de Rickettsia en ectoparásitos de la fauna silvestre.

Introduction. Ectoparasites are the main vectors of rickettsiosis. In Panama, however, limited data are available concerning the arthropod species that serve as vectors or reservoirs. Objectives. Data are presented concerning the presence of Rickettsia in ectoparasites of wildlife and domestic animals in the Cerro Chucantí private nature reserve and in neighboring villages. Materials and methods. Nine humans, 95 domestic mammals and 48 wild mammals were examined. Twenty-one species of ectoparasites were obtained, including fleas, lice, ticks and mites. These were preserved in 95% ethanol. Later, the DNA was extracted from the ticks and fleas and analyzed by molecular techniques to detect presence of Rickettsia. Results. Of a total of 425 PCR reactions, 270 were positive for Rickettsia and 155 negative. Among the positive samples, 86 PCR amplified for the gltA gene (55% of positives) and 41 of these also amplified the ompA gene. DNA of Rickettsiaamblyommii was found in horses ticks (Amblyomma cajennense, Dermacentor nitens), dogs ticks (Rhipicephalus sanguineus) and free living nymphs in the forest. Additionally, DNA of R. felis was found in fleas from dogs Ctenocephalides felis. Conclusions. The presence of R. amblyommii and R. felis was detected in ticks and fleas of domestic animals in villages near Cerro Chucanti; however no Rickettsia DNA was found in ectoparasites of non-domestic wildlife.