Dengue virus and its four serotypes (DENV-1 to DENV-4) infect 390 million people and are implicated in at least 25,000 deaths annually, with the largest disease burden in tropical and subtropical regions. We investigated the spatial dynamics of DENV-1, DENV-2 and DENV-3 in Brazil by applying a statistical framework to complete genome sequences. For all three serotypes, we estimated that the introduction of new lineages occurred within 7 to 10-year intervals. New lineages were most likely to be imported from the Caribbean region to the North and Northeast regions of Brazil, and then to disperse at a rate of approximately 0.5 km/day. Joint statistical analysis of evolutionary, epidemiological and ecological data indicates that aerial transportation of humans and/or vector mosquitoes, rather than Aedes aegypti infestation rates or geographical distances, determine dengue virus spread in Brazil.

Zika virus infection during pregnancy can cause congenital abnormities or fetal demise. The persistence of Zika virus in the male reproductive system poses a risk of sexual transmission. Here we demonstrate that live-attenuated Zika virus vaccine candidates containing deletions in the 3' untranslated region of the Zika virus genome (ZIKV-3'UTR-LAV) prevent viral transmission during pregnancy and testis damage in mice, as well as infection of nonhuman primates. After a single-dose vaccination, pregnant mice challenged with Zika virus at embryonic day 6 and evaluated at embryonic day 13 show markedly diminished levels of viral RNA in maternal, placental, and fetal tissues. Vaccinated male mice challenged with Zika virus were protected against testis infection, injury, and oligospermia. A single immunization of rhesus macaques elicited a rapid and robust antibody response, conferring complete protection upon challenge. Furthermore, the ZIKV-3'UTR-LAV vaccine candidates have a desirable safety profile. These results suggest that further development of ZIKV-3'UTR-LAV is warranted for humans.Zika virus infection can result in congenital disorders and cause disease in adults, and there is currently no approved vaccine. Here Shan et al. show that a single dose of a live-attenuated Zika vaccine prevents infection, testis damage and transmission to the fetus during pregnancy in different animal models.

Reporter virus neutralization test (RVNT) has been used as an alternative to the more laborious and time-demanding conventional PRNT assay for both DENV and ZIKV. However, few studies have investigated how these techniques would perform in epidemic areas with the circulation of multiple flavivirus. Here, we evaluate the performance of ZIKV and DENV Rluc RVNT and ZIKV mCh RVNT assays in comparison to the conventional PRNT assay against patient sera collected before and during ZIKV outbreak in Brazil. These samples were categorized into groups based on (1) acute and convalescent samples according to the time of disease, and (2) laboratorial diagnostic results (DENV and ZIKV RT-PCR and IgM-capture ELISA). Our results showed that DENV Rluc assay presented 100% and 78.3% sensitivity and specificity, respectively, with 93.3% accuracy, a similar performance to the traditional PRNT. ZIKV RVNT90, on the other hand, showed much better ZIKV antibody detection performance (around nine-fold higher) when compared to PRNT, with 88% clinical sensitivity. Specificity values were on average 76.8%. Even with these results, however, ZIKV RVNT90 alone was not able to reach a final diagnostic conclusion for secondary infection in human samples due to flavivirus cross reaction. As such, in regions where the flavivirus differential diagnosis represents a challenge, we suggest the establishment of a RVNT panel including other flaviviruses circulating in the region, associated with the other serological techniques such as IgM ELISA and the investigation of seroconversion, in order to help define an accurate diagnostic conclusion using serology.

Compared with other flaviviruses, Zika virus (ZIKV) is uniquely associated with congenital diseases in pregnant women. One recent study reported that (i) ZIKV has higher thermostability than dengue virus (DENV [a flavivirus closely related to ZIKV]), which might contribute to the disease outcome; (ii) the higher thermostability of ZIKV could arise from an extended loop structure in domain III of the viral envelope (E) protein and an extra hydrogen-bond interaction between E molecules (V. A. Kostyuchenko, E. X. Y. Lim, S. Zhang, G. Fibriansah, T.-S. Ng, J. S. G. Ooi, J. Shi, and S.-M. Lok, Nature 533:425-428, 2016, https://doi.org/10.1038/nature17994). Here we report the functional analysis of the structural information in the context of complete ZIKV and DENV-2 virions. Swapping the prM-E genes between ZIKV and DENV-2 switched the thermostability of the chimeric viruses, identifying the prM-E proteins as the major determinants for virion thermostability. Shortening the extended loop of the E protein by 1 amino acid was lethal for ZIKV assembly/release. Mutations (Q350I and T351V) that abolished the extra hydrogen-bond interaction between the E proteins did not reduce ZIKV thermostability, indicating that the extra interaction does not increase the thermostability. Interestingly, mutant T351V was attenuated in A129 mice defective in type I interferon receptors, even though the virus retained the wild-type thermostability. Furthermore, we found that a chimeric ZIKV with the DENV-2 prM-E and a chimeric DENV-2 with the ZIKV prM-E were highly attenuated in A129 mice; these chimeric viruses were highly immunogenic and protective against DENV-2 and ZIKV challenge, respectively. These results indicate the potential of these chimeric viruses for vaccine development.

Zika virus (ZIKV) infection of the mother during pregnancy causes devastating Zika congenital syndrome in the offspring. A ZIKV vaccine with optimal safety and immunogenicity for use in pregnant women is critically needed. Toward this goal, we have developed a single-dose live-attenuated vaccine candidate that infects cells with controlled, limited infection rounds. The vaccine contains a 9-amino-acid deletion in the viral capsid protein and replicates to titers of > 106 focus-forming units (FFU)/mL in cells expressing the full-length capsid protein. Immunization of A129 mice with one dose (105 FFU) did not produce viremia, but elicited protective immunity that completely prevented viremia, morbidity, and mortality after challenge with an epidemic ZIKV strain (106 PFU). A single-dose vaccination also fully prevented infection of pregnant mice and maternal-to-fetal transmission. Intracranial injection of the vaccine (104 FFU) to 1-day-old mice did not cause any disease or death, underscoring the safety of this vaccine candidate.

Virus-host interactions determine an infection outcome. The Asian lineage of Zika virus (ZIKV), responsible for the recent epidemics, has fixed a mutation in the NS1 gene after 2012 that enhances mosquito infection. Here we report that the same mutation confers NS1 to inhibit interferon-β induction. This mutation enables NS1 binding to TBK1 and reduces TBK1 phosphorylation. Engineering the mutation into a pre-epidemic ZIKV strain debilitates the virus for interferon-β induction; reversing the mutation in an epidemic ZIKV strain invigorates the virus for interferon-β induction; these mutational effects are lost in IRF3-knockout cells. Additionally, ZIKV NS2A, NS2B, NS4A, NS4B, and NS5 can also suppress interferon-β production through targeting distinct components of the RIG-I pathway; however, for these proteins, no antagonistic difference is observed among various ZIKV strains. Our results support the mechanism that ZIKV has accumulated mutation(s) that increases the ability to evade immune response and potentiates infection and epidemics.

The Asian lineage of Zika virus (ZIKV) is responsible for the recent epidemics in the Americas and severe disease, whereas the African lineage of ZIKV has not been reported to cause epidemics or severe disease. We constructed a cDNA infectious clone (IC) of an African ZIKV strain, which, together with our previously developed Asian ZIKV strain IC, allowed us to engineer chimeric viruses by swapping the structural and non-structural genes between the two lineages. Recombinant parental and chimeric viruses were analyzed in A129 and newborn CD1 mouse models. In the A129 mice, the African strain developed higher viremia, organ viral loading, and mortality rate. In CD1 mice, the African strain exhibited a higher neurovirulence than the Asian strain. A chimeric virus containing the structural genes from the African strain is more virulent than the Asian strain, whereas a chimeric virus containing the non-structural genes from the African strain exhibited a virulence comparable to the Asian strain. These results suggest that (i) African strain is more virulent than Asian strain and (ii) viral structural genes primarily determine the virulence difference between the two lineages in mouse models. Other factors may contribute to the discrepancy between the mouse and epidemic results.

We associated Laguna Negra virus with hantavirus pulmonary syndrome in Mato Grosso State, Brazil, and a previously unidentified potential host, the Calomys callidus rodent. Genetic testing revealed homologous sequencing in specimens from 20 humans and 8 mice. Further epidemiologic studies may lead to control of HPS in Mato Grosso State.

To study the dynamics of wild rodent populations and identify potential hosts for hantavirus, we conducted an eco-epidemiologic study in Campo Novo do Parecis, Mato Grosso State, Brazil. We detected and genetically characterized Castelo dos Sonhos virus found in a species of pygmy rice rat (Oligoryzomys utiaritensis).

We describe 2 bat-transmitted outbreaks in remote, rural areas of Portel and Viseu Municipalities, Pará State, northern Brazil. Central nervous system specimens were taken after patients' deaths and underwent immunofluorescent assay and histopathologic examination for rabies antigens; also, specimens were injected intracerebrally into suckling mice in an attempt to isolate the virus. Strains obtained were antigenically and genetically characterized. Twenty-one persons died due to paralytic rabies in the 2 municipalities. Ten rabies virus strains were isolated from human specimens; 2 other cases were diagnosed by histopathologic examination. Isolates were antigenically characterized as Desmodus rotundus variant 3 (AgV3). DNA sequencing of 6 strains showed that they were genetically close to D. rotundus-related strains isolated in Brazil. The genetic results were similar to those obtained by using monoclonal antibodies and support the conclusion that the isolates studied belong to the same rabies cycle, the virus variants found in the vampire bat D. rotundus.

Zika virus (ZIKV) infection causes devastating congenital abnormities and Guillain-Barré syndrome. The ZIKV envelope (E) protein is responsible for viral entry and represents a major determinant for viral pathogenesis. Like other flaviviruses, the ZIKV E protein is glycosylated at amino acid N154. To study the function of E glycosylation, we generated a recombinant N154Q ZIKV that lacks the E glycosylation and analyzed the mutant virus in mammalian and mosquito hosts. In mouse models, the mutant was attenuated, as evidenced by lower viremia, decreased weight loss, and no mortality; however, knockout of E glycosylation did not significantly affect neurovirulence. Mice immunized with the mutant virus developed a robust neutralizing antibody response and were completely protected from wild-type ZIKV challenge. In mosquitoes, the mutant virus exhibited diminished oral infectivity for the Aedes aegypti vector. Collectively, the results demonstrate that E glycosylation is critical for ZIKV infection of mammalian and mosquito hosts.

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.

The emergence of Zika virus (ZIKV) and its association with congenital malformations has prompted the rapid development of vaccines. Although efficacy with multiple viral vaccine platforms has been established in animals, no study has addressed protection during pregnancy. We tested in mice two vaccine platforms, a lipid nanoparticle-encapsulated modified mRNA vaccine encoding ZIKV prM and E genes and a live-attenuated ZIKV strain encoding an NS1 protein without glycosylation, for their ability to protect against transmission to the fetus. Vaccinated dams challenged with a heterologous ZIKV strain at embryo day 6 (E6) and evaluated at E13 showed markedly diminished levels of viral RNA in maternal, placental, and fetal tissues, which resulted in protection against placental damage and fetal demise. As modified mRNA and live-attenuated vaccine platforms can restrict in utero transmission of ZIKV in mice, their further development in humans to prevent congenital ZIKV syndrome is warranted.