Andrés Moreira-Soto

High Zika Virus Seroprevalence in Salvador, Northeastern Brazil Limits the Potential for Further Outbreaks

ABSTRACT During 2015 to 2016, Brazil reported more Zika virus (ZIKV) cases than any other country, yet population exposure remains unknown. Serological studies of ZIKV are hampered by cross-reactive immune responses against heterologous viruses. We conducted serosurveys for ZIKV, dengue virus (DENV), and Chikungunya virus (CHIKV) in 633 individuals prospectively sampled during 2015 to 2016, including microcephaly and non-microcephaly pregnancies, HIV-infected patients, tuberculosis patients, and university staff in Salvador in northeastern Brazil using enzyme-linked immunosorbent assays (ELISAs) and plaque reduction neutralization tests. Sera sampled retrospectively during 2013 to 2015 from 277 HIV-infected patients were used to assess the spread of ZIKV over time. Individuals were georeferenced, and sociodemographic indicators were compared between ZIKV-positive and -negative areas and areas with and without microcephaly cases. Epidemiological key parameters were modeled in a Bayesian framework. ZIKV seroprevalence increased rapidly during 2015 to 2016, reaching 63.3% by 2016 (95% confidence interval [CI], 59.4 to 66.8%), comparable to the seroprevalence of DENV (75.7%; CI, 69.4 to 81.1%) and higher than that of CHIKV (7.4%; CI, 5.6 to 9.8%). Of 19 microcephaly pregnancies, 94.7% showed ZIKV IgG antibodies, compared to 69.3% of 257 non-microcephaly pregnancies (P = 0.017). Analyses of sociodemographic data revealed a higher ZIKV burden in low socioeconomic status (SES) areas. High seroprevalence, combined with case data dynamics allowed estimates of the basic reproduction number R0 of 2.1 (CI, 1.8 to 2.5) at the onset of the outbreak and an effective reproductive number Reff of <1 in subsequent years. Our data corroborate ZIKV-associated congenital disease and an association of low SES and ZIKV infection and suggest that population immunity caused cessation of the outbreak. Similar studies from other areas will be required to determine the fate of the American ZIKV outbreak. IMPORTANCE The ongoing American Zika virus (ZIKV) outbreak involves millions of cases and has a major impact on maternal and child health. Knowledge of infection rates is crucial to project future epidemic patterns and determine the absolute risk of microcephaly upon maternal ZIKV infection during pregnancy. For unknown reasons, the vast majority of ZIKV-associated microcephaly cases are concentrated in northeastern Brazil. We analyzed different subpopulations from Salvador, a Brazilian metropolis representing one of the most affected areas during the American ZIKV outbreak. We demonstrate rapid spread of ZIKV in Salvador, Brazil, and infection rates exceeding 60%. We provide evidence for the link between ZIKV and microcephaly, report that ZIKV predominantly affects geographic areas with low socioeconomic status, and show that population immunity likely caused cessation of the outbreak. Our results enable stakeholders to identify target populations for vaccination and for trials on vaccine efficacy and allow refocusing of research efforts and intervention strategies. IMPORTANCE The ongoing American Zika virus (ZIKV) outbreak involves millions of cases and has a major impact on maternal and child health. Knowledge of infection rates is crucial to project future epidemic patterns and determine the absolute risk of microcephaly upon maternal ZIKV infection during pregnancy. For unknown reasons, the vast majority of ZIKV-associated microcephaly cases are concentrated in northeastern Brazil. We analyzed different subpopulations from Salvador, a Brazilian metropolis representing one of the most affected areas during the American ZIKV outbreak. We demonstrate rapid spread of ZIKV in Salvador, Brazil, and infection rates exceeding 60%. We provide evidence for the link between ZIKV and microcephaly, report that ZIKV predominantly affects geographic areas with low socioeconomic status, and show that population immunity likely caused cessation of the outbreak. Our results enable stakeholders to identify target populations for vaccination and for trials on vaccine efficacy and allow refocusing of research efforts and intervention strategies.

Differential Infection Patterns and Recent Evolutionary Origins of Equine Hepaciviruses in Donkeys

ABSTRACT The hepatitis C virus (HCV) is a major human pathogen. Genetically related viruses in animals suggest a zoonotic origin of HCV. The closest relative of HCV is found in horses (termed equine hepacivirus [EqHV]). However, low EqHV genetic diversity implies relatively recent acquisition of EqHV by horses, making a derivation of HCV from EqHV unlikely. To unravel the EqHV evolutionary history within equid sister species, we analyzed 829 donkeys and 53 mules sampled in nine European, Asian, African, and American countries by molecular and serologic tools for EqHV infection. Antibodies were found in 278 animals (31.5%), and viral RNA was found in 3 animals (0.3%), all of which were simultaneously seropositive. A low RNA prevalence in spite of high seroprevalence suggests a predominance of acute infection, a possible difference from the mostly chronic hepacivirus infection pattern seen in horses and humans. Limitation of transmission due to short courses of infection may explain the existence of entirely seronegative groups of animals. Donkey and horse EqHV strains were paraphyletic and 97.5 to 98.2% identical in their translated polyprotein sequences, making virus/host cospeciation unlikely. Evolutionary reconstructions supported host switches of EqHV between horses and donkeys without the involvement of adaptive evolution. Global admixture of donkey and horse hepaciviruses was compatible with anthropogenic alterations of EqHV ecology. In summary, our findings do not support EqHV as the origin of the significantly more diversified HCV. Identification of a host system with predominantly acute hepacivirus infection may enable new insights into the chronic infection pattern associated with HCV. IMPORTANCE The evolutionary origins of the human hepatitis C virus (HCV) are unclear. The closest animal-associated relative of HCV occurs in horses (equine hepacivirus [EqHV]). The low EqHV genetic diversity implies a relatively recent acquisition of EqHV by horses, limiting the time span for potential horse-to-human infections in the past. Horses are genetically related to donkeys, and EqHV may have cospeciated with these host species. Here, we investigated a large panel of donkeys from various countries using serologic and molecular tools. We found EqHV to be globally widespread in donkeys and identify potential differences in EqHV infection patterns, with donkeys potentially showing enhanced EqHV clearance compared to horses. We provide strong evidence against EqHV cospeciation and for its capability to switch hosts among equines. Differential hepacivirus infection patterns in horses and donkeys may enable new insights into the chronic infection pattern associated with HCV.

Lineage-Specific Real-Time RT-PCR for Yellow Fever Virus Outbreak Surveillance, Brazil

The current yellow fever outbreak in Brazil prompted widespread yellow fever virus (YFV) vaccination campaigns, imposing a responsibility to distinguish between vaccine- and wild-type YFV-associated disease. We developed novel multiplex real-time reverse transcription PCRs that differentiate between vaccine and American wild-type YFV. We validated these highly specific and sensitive assays in an outbreak setting.

Exposure of dogs to spotted fever group rickettsiae in urban sites associated with human rickettsioses in Costa Rica.

The zoonotic transmission cycles of Rickettsia rickettsii and other spotted fever group (SFG) rickettsiae in Latin America have usually been associated with rural or sylvatic environments, although domestic dogs can be implicated in more populated settings. In this study, exposure of dogs to SFG rickettsiae in the Greater Metropolitan Area of Costa Rica was investigated. Dogs from sites associated with human cases and from dog shelters were evaluated by indirect immunofluorescence assay (IFA) using antigen of SFG rickettsiae. Rickettsia spp. were detected in ectoparasites by polymerase chain reaction (PCR). A total 18.5% (31/168) of dogs associated with human cases and 6.8% (11/161) of dogs in shelters had IgG end titers≥64 to Rickettsia spp. The odds of being seropositive were greater in dogs from areas associated with human cases when compared to shelters (OR: 3.2; 95% C.I: 1.5-5.6). Rhipicephalus sanguineus sensu lato (s. l.) was present in all sites associated with human cases. Rickettsia felis URRWXCal2 and R. felis-like RF2125 were detected in Ctenocephalides felis, and Rickettsia sp. IbR/CRC in Ixodes boliviensis. Results demonstrate that dogs from the main urban center of Costa Rica have been exposed to SFG rickettsiae, especially in areas with known human infection. Both human and animal health sectors must be aware of possible rickettsial diseases in urban areas, where dogs may also serve as sentinels for human infection.

Specific detection of dengue and Zika virus antibodies using envelope proteins with mutations in the conserved fusion loop

Detection of antibodies is widely used for the diagnosis of infections with arthropod-borne flaviviruses including dengue (DENV) and Zika virus (ZIKV). Due to the emergence of ZIKV in areas endemic for DENV, massive co-circulation is observed and methods to specifically diagnose these infections and differentiate them from each other are mandatory. However, serological assays for flaviviruses in general, and for DENV and ZIKV in particular, are compromised by the high degree of similarities in their proteins which can lead to cross-reacting antibodies and false-positive test results. Cross-reacting flavivirus antibodies mainly target the highly conserved fusion loop (FL) domain in the viral envelope (E-) protein, and we and others have shown previously that recombinant E-proteins bearing FL-mutations strongly reduce cross-reactivity. Here we investigate whether such mutant E-proteins can be used to specifically detect antibodies against DENV and ZIKV in an ELISA-format. IgM antibodies against DENV and ZIKV virus were detected with 100% and 94.2% specificity and 90.7% and 87.5% sensitivity, respectively. For IgG the mutant E-proteins showed cross-reactivity, which was overcome by pre-incubation of the sera with the heterologous antigen. This resulted in specificities of 97.1% and 97.9% and in sensitivities of 100% and 100% for the DENV and ZIKV antigens, respectively. Our results suggest that E-proteins bearing mutations in the FL-domain have a high potential for the development of serological DENV and ZIKV tests with high specificity. Emerging Microbes & Infections (2017) 6, e99; doi:10.1038/emi.2017.87; published online 8 November 2017

Zoonotic Potential of Emerging Paramyxoviruses

The risk of spillover of enzootic paramyxoviruses and the susceptibility of recipient human and domestic animal populations are defined by a broad collection of ecological and molecular factors that interact in ways that are not yet fully understood. Nipah and Hendra viruses were the first highly lethal zoonotic paramyxoviruses discovered in modern times, but other paramyxoviruses from multiple genera are present in bats and other reservoirs that have unknown potential to spillover into humans. We outline our current understanding of paramyxovirus reservoir hosts and the ecological factors that may drive spillover, and we explore the molecular barriers to spillover that emergent paramyxoviruses may encounter. By outlining what is known about enzootic paramyxovirus receptor usage, mechanisms of innate immune evasion, and other host-specific interactions, we highlight the breadth of unexplored avenues that may be important in understanding paramyxovirus emergence.

Neotropical primary bat cell lines show restricted dengue virus replication

Dengue is the most widespread arboviral disease affecting humans. Bats are recognized carriers of emerging viral zoonoses and have been proposed as dengue reservoirs, since RNA/NS1 and/or antiviral antibodies have been detected. Yet, experimental inoculation of Artibeus bats failed to show virus replication. This conflicting results prevent drawing further conclusions of whether bats sustain dengue infection. To test bat cellular permissivity to dengue infection, we established primary bat embryonic cells from diverse organs and tissues of Artibeus jamaicensis, Molossus sinaloae, and Desmodus rotundus. We observed a limited serotype-, organ-, and bat species- specific dengue susceptibility. Only some Molossus-derived primary cells sustained poorly initial Dengue serotype-1 replication, though it was latter absent. To elucidate if Molossus bats may play a role in dengue replication, ecological or in vivo experiments must be performed. Taken together our results show that Dengue did not replicate efficiently in cell lines derived from Neotropical bat species.

Zoonotic Potential of Emerging Paramyxoviruses: Knowns and Unknowns

The risk of spillover of enzootic paramyxoviruses and the susceptibility of recipient human and domestic animal populations are defined by a broad collection of ecological and molecular factors that interact in ways that are not yet fully understood. Nipah and Hendra viruses were the first highly lethal zoonotic paramyxoviruses discovered in modern times, but other paramyxoviruses from multiple genera are present in bats and other reservoirs that have unknown potential to spillover into humans. We outline our current understanding of paramyxovirus reservoir hosts and the ecological factors that may drive spillover, and we explore the molecular barriers to spillover that emergent paramyxoviruses may encounter. By outlining what is known about enzootic paramyxovirus receptor usage, mechanisms of innate immune evasion, and other host-specific interactions, we highlight the breadth of unexplored avenues that may be important in understanding paramyxovirus emergence.

Neotropical bats that co-habit with humans function as dead-end hosts for dengue virus

Several studies have shown Dengue Virus (DENV) nucleic acids and/or antibodies present in Neotropical wildlife including bats, suggesting that some bat species may be susceptible to DENV infection. Here we aim to elucidate the role of house-roosting bats in the DENV transmission cycle. Bats were sampled in households located in high and low dengue incidence regions during rainy and dry seasons in Costa Rica. We captured 318 bats from 12 different species in 29 households. Necropsies were performed in 205 bats to analyze virus presence in heart, lung, spleen, liver, intestine, kidney, and brain tissue. Histopathology studies from all organs showed no significant findings of disease or infection. Sera were analyzed by PRNT90 for a seroprevalence of 21.2% (51/241), and by PCR for 8.8% (28/318) positive bats for DENV RNA. From these 28 bats, 11 intestine samples were analyzed by RT-PCR. Two intestines were DENV RNA positive for the same dengue serotype detected in blood. Viral isolation from all positive organs or blood was unsuccessful. Additionally, viral load analyses in positive blood samples by qRT-PCR showed virus concentrations under the minimal dose required for mosquito infection. Simultaneously, 651 mosquitoes were collected using EVS-CO2 traps and analyzed for DENV and feeding preferences (bat cytochrome b). Only three mosquitoes were found DENV positive and none was positive for bat cytochrome b. Our results suggest an accidental presence of DENV in bats probably caused from oral ingestion of infected mosquitoes. Phylogenetic analyses suggest also a spillover event from humans to bats. Therefore, we conclude that bats in these urban environments do not sustain DENV amplification, they do not have a role as reservoirs, but function as epidemiological dead end hosts for this virus.

Examining the Role of Transmission of Chelonid Alphaherpesvirus 5

Marine turtle fibropapillomatosis (FP) is a devastating neoplastic disease characterized by single or multiple cutaneous and visceral fibrovascular tumors. Chelonid alphaherpesvirus 5 (ChHV5) has been identified as the most likely etiologic agent. From 2010 to 2013, the presence of ChHV5 DNA was determined in apparently normal skin, tumors and swab samples (ocular, nasal and cloacal) collected from 114 olive ridley (Lepidochelys olivacea) and 101 green (Chelonia mydas) turtles, with and without FP tumors, on the Pacific coasts of Costa Rica and Nicaragua. For nesting olive ridley turtles from Costa Rica without FP, 13.5% were found to be positive for ChHV5 DNA in at least one sample, while in Nicaragua, all olive ridley turtles had FP tumors, and 77.5% tested positive for ChHV5 DNA. For green turtles without FP, 19.8% were found to be positive for ChHV5 DNA in at least one of the samples. In turtles without FP tumors, ChHV5 DNA was detected more readily in skin biopsies than swabs. Juvenile green turtles caught at the foraging site had a higher prevalence of ChHV5 DNA than adults. The presence of ChHV5 DNA in swabs suggests a possible route of viral transmission through viral secretion and excretion via corporal fluids.