Phylogenetic analysis reveals a new introduction of Yellow Fever virus in São Paulo State, Brazil, 2023.

Yellow Fever (YF) is a viral arbovirosis of Public Health importance. In Brazil, surveillance is focused mainly on detecting epizootic events of Platyrrhini. Herein, we compared the detection and phylogenetic analysis of YF virus in two neotropical primates (NTP), a Callithrix detected in the previous epidemic period (2016-2020), and a Callicebus nigrifons, showing a new introduction of YF in 2023. This paper illustrates the importance of joint actions of laboratory and field teams to ensure quick response to Public Health emergencies, such as the intensification of vaccination of susceptible human populations.

Yellow fever outbreak affecting Alouatta populations in southern Brazil (Rio Grande do Sul State), 2008-2009.

The natural transmission cycle of Yellow Fever (YF) involves tree hole breeding mosquitoes and a wide array of nonhuman primates (NHP), including monkeys and apes. Some Neotropical monkeys (howler monkeys, genus Alouatta) develop fatal YF virus (YFV) infections similar to those reported in humans, even with minimum exposure to the infection. Epizootics in wild primates may be indicating YFV circulation, and the surveillance of such outbreaks in wildlife is an important tool to help prevent human infection. In 2001, surveillance activities successfully identified YF-related death in a black-and-gold howler monkey (Alouatta caraya), Rio Grande do Sul State (RGS) in southern Brazil, and the YFV was isolated from a species of forest-dwelling mosquito (Haemagogus leucocelaenus). These findings led the State Secretariat of Health to initiate a monitoring program for YF and other 18 arboviral infections in Alouatta monkeys. The monitoring program included monkey captures, reporting of monkey casualties by municipalities, and subsequent investigations. If monkey carcasses were found in forests, samples were collected in a standardized manner and this practice resulted in increased reporting of outbreaks. In October 2008, a single howler monkey in a northwestern RGS municipality was confirmed to have died from YF. From October 2008 to June 2009, 2,013 monkey deaths were reported (830 A. caraya and 1,183 A. guariba clamitans). Viruses isolation in blood, viscera, and/or immunohistochemistry led to the detection of YF in 204 of 297 (69%) (154 A. g. clamitans and 50 A. caraya) dead Alouatta monkeys tested. The number of municipalities with confirmed YFV circulation in howlers increased from 2 to 67 and 21 confirmed human cases occurred. This surveillance system was successful in identifying the largest YF outbreak affecting wild NHP ever recorded.

Spatio-temporal dynamics of dengue-related deaths and associated factors.

Since the reintroduction of dengue viruses in 1987, Sao Paulo State (SP), Brazil, has experienced recurrent epidemics in a growing number of municipalities, each time with more cases and deaths. In the present study, we investigated the spatio-temporal dynamics of dengue-related deaths and associated factors in SP. This was an ecological study with spatial and temporal components, based on notified dengue-related deaths in the municipalities of SP between 2007 and 2017. A latent Gaussian Bayesian model with Poisson probability distribution was used to estimate the standardized mortality ratios (SMR) for dengue and relative risks (RR) for the socioeconomic, demographic, healthcare-related, and epidemiological factors considered. Epidemiological factors included the annual information on the number of circulating serotypes. A total of 1,019 dengue-related deaths (0.22 per 100,000 inhabitant-years) between 2007 and 2017 were confirmed in SP by laboratory testing. Mortality increased with age, peaking at 70 years or older (1.41 deaths per 100,000 inhabitant-years). Mortality was highest in 2015, and the highest SMR values were found in the North, Northwest, West, and coastal regions of SP. An increase of one circulating serotype, one standard deviation in the number of years with cases, and one standard deviation in the degree of urbanization were associated with increases of 75, 35, and 45% in the risk of death from dengue, respectively. The risk of death from dengue increased with age, and the distribution of deaths was heterogeneous in space and time. The positive relationship found between the number of dengue serotypes circulating and years with cases at the municipality/micro-region level indicates that this information can be used to identify risk areas, intensify surveillance and control measures, and organize healthcare to better respond to this disease.

Anti-DENV-NS1 monoclonal antibody for the differential histopathological diagnosis of hemorrhagic fever caused by dengue.

Dengue is the most prevalent arboviral disease in humans in tropical and subtropical regions, especially in urban areas, and can cause major epidemics. Although a self-limiting illness, it may sometimes have serious hemorrhagic manifestations, and the outcome of dengue hemorrhagic fever has similar clinical manifestations as in other infections, which could result in death. Therefore, autopsy procedures are required under certain circumstances such as in hemorrhagic fevers, sometimes to confirm or to clarify the diagnosis that may have epidemiological consequences. Normally, the Immunohistochemistry Laboratory of the Pathology Center of Adolfo Lutz Institute receives autopsy samples from different hospitals in Sao Paulo State to confirm a previous diagnosis, especially hemorrhagic fever of infectious etiology. For this diagnosis, we have been using a mouse polyclonal antibody to dengue virus that often does not provide a clear conclusion, because of background staining or no relevant immunostaining, which hampers the histopathological analysis. Accordingly, in the present study, anti-DENV-NS1 monoclonal antibody (4H2) was tested to determine its accuracy in immunohistochemical analysis. Twenty-four autopsy cases of hemorrhagic febrile syndrome showing histopathological alterations compatible with dengue disease were studied: twenty cases were confirmed by RT-PCR for DENV-2 and in four by RT-PCR for yellow fever virus. Samples from autopsied cases of deaths caused by other infectious diseases (two meningitis C and two severe acute respiratory syndrome caused by influenza A H1N1) were included as negative control cases. Positive immunostaining for DENV-NS1 was detected in 16/20 (80%) liver samples and 11/15 (73%) spleen samples from autopsied hemorrhagic dengue patients, whereas the polyclonal antibody detected DENV antigens in 12/20 (60%) liver and in 6/15 (40%) spleen samples from the same cases. Positive results were not obtained with liver biopsy samples from yellow fever or Neisseria meningitides and Flu-A cases. 4H2 mAb recognizes the native protein of the four DENV serotypes in infected cells and did not cross-react with native ZIKV- or CHKV-infected cells by immunohistochemical assay, so it is a useful tool for differential histopathological conclusion of acute febrile hemorrhagic deaths.

Guapiaçu virus, a new insect-specific flavivirus isolated from two species of Aedes mosquitoes from Brazil.

Classical insect-flaviviruses (cISFVs) and dual host-related insect-specific flavivirus (dISFV) are within the major group of insect-specific flavivirus. Remarkably dISFV are evolutionarily related to some of the pathogenic flavivirus, such as Zika and dengue viruses. The Evolutionary relatedness of dISFV to flavivirus allowed us to investigate the evolutionary principle of host adaptation. Additionally, dISFV can be used for the development of flavivirus vaccines and to explore underlying principles of mammalian pathogenicity. Here we describe the genetic characterization of a novel putative dISFV, termed Guapiaçu virus (GUAPV). Distinct strains of GUAPV were isolated from pools of Aedes terrens and Aedes scapularis mosquitoes. Additionally, we also detected viral GUAPV RNA in a plasma sample of an individual febrile from the Amazon region (North of Brazil). Although GUAPV did not replicate in tested mammalian cells, 3'UTR secondary structures duplication and codon usage index were similar to pathogenic flavivirus.

Detection of Wild-type Yellow Fever Virus in Breast Milk.

Yellow fever is an endemic disease in tropical areas in America and Africa. We report a case where the wild-type yellow fever virus was detected in a breast milk sample of a 33-year-old woman, from a rural area in the municipality of São Paulo, thus highlighting a potential risk for transmission of yellow fever virus through breast-feeding.

Genomic evidence of yellow fever virus in Aedes scapularis, southeastern Brazil, 2016.

The southeastern region of Brazil has recently experienced the largest yellow fever disease outbreak in decades. Since July 2016 epizootic events were reported in São Paulo state's north region, where 787 Culicidae were captured as part of public health surveillance efforts and tested using real-time quantitative PCR. One Aedes scapularis pool collected in November 2016 in an agriculture area in Urupês city tested positive for YFV-RNA. Using a validated multiplex PCR approach we were able to recover a complete virus genome sequence from this pool. Phylogenetic analysis of the novel strain and publicly available data indicates that the belongs to the South American genotype 1 clade circulating in Sao Paulo state and is basal to the recent outbreak clade in southeast Brazil. Our findings highlight the need of additional studies, including vector competence studies, to disentangle the role of Aedes scapularis in yellow fever transmission in the Americas.

The role of pregnant women with rash in the Zika virus sentinel surveillance.

INTRODUCTION: The state of São Paulo has been monitoring cases of microcephaly and pregnant women presenting with acute rash, through CeVeSP. METHODS: This was a descriptive study focusing on pregnant women with rash and the outcome of their pregnancy, based on the notifications through the CeVeSP. RESULTS: During 2016, 2,209 cases of pregnant women with rash were reported and investigated. Of these, 36.6% were confirmed. Of the pregnant women who tested positive for ZIKV, 6.4% did not have a favorable outcome. CONCLUSIONS: Our results allowed the characterization of pregnant women exposed to ZIKV and the outcome of pregnancy.

Epizootics due to Yellow Fever Virus in São Paulo State, Brazil: viral dissemination to new areas (2016-2017).

Beginning in late 2016 Brazil faced the worst outbreak of Yellow Fever in recent decades, mainly located in southeastern rural regions of the country. In the present study we characterize the Yellow Fever Virus (YFV) associated with this outbreak in São Paulo State, Brazil. Blood or tissues collected from 430 dead monkeys and 1030 pools containing a total of 5,518 mosquitoes were tested for YFV by quantitative RT-PCR, immunohistochemistry (IHC) and indirect immunofluorescence. A total of 67 monkeys were YFV-positive and 3 pools yielded YFV following culture in a C6/36 cell line. Analysis of five nearly full length genomes of YFV from collected samples was consistent with evidence that the virus associated with the São Paulo outbreak originated in Minas Gerais. The phylogenetic analysis also showed that strains involved in the 2016-2017 outbreak in distinct Brazilian states (i.e., Minas Gerais, Rio de Janeiro, Espirito Santo) intermingled in maximum-likelihood and Bayesian trees. Conversely, the strains detected in São Paulo formed a monophyletic cluster, suggesting that they were local-adapted. The finding of YFV by RT-PCR in five Callithrix monkeys who were all YFV-negative by histopathology or immunohistochemistry suggests that this YFV lineage circulating in Sao Paulo is associated with different outcomes in Callithrix when compared to other monkeys.

Natural Plasmodium infections in Brazilian wild monkeys: reservoirs for human infections?

Four hundred and forty-eight samples of total blood from wild monkeys living in areas where human autochthonous malaria cases have been reported were screened for the presence of Plasmodium using microscopy and PCR analysis. Samples came from the following distinct ecological areas of Brazil: Atlantic forest (N=140), semideciduous Atlantic forest (N=257) and Cerrado (a savannah-like habitat) (N=51). Thick and thin blood smears of each specimen were examined and Plasmodium infection was screened by multiplex polymerase chain reaction (multiplex PCR). The frequency of Plasmodium infections detected by PCR in Alouatta guariba clamitans in the São Paulo Atlantic forest was 11.3% or 8/71 (5.6% for Plasmodium malariae and 5.6% for Plasmodium vivax) and one specimen was positive for Plasmodium falciparum (1.4%); Callithrix sp. (N=30) and Cebus apella (N=39) specimens were negative by PCR tests. Microscopy analysis was negative for all specimens from the Atlantic forest. The positivity rate for Alouatta caraya from semideciduous Atlantic forest was 6.8% (16/235) in the PCR tests (5.5, 0.8 and 0.4% for P. malariae, P. falciparum and P. vivax, respectively), while C. apella specimens were negative. Parasitological examination of the samples using thick smears revealed Plasmodium sp. infections in only seven specimens, which had few parasites (3.0%). Monkeys from the Cerrado (a savannah-like habitat) (42 specimens of A. caraya, 5 of Callithrix jacchus and 4 of C. apella) were negative in both tests. The parasitological prevalence of P. vivax and P. malariae in wild monkeys from Atlantic forest and semideciduous Atlantic forest and the finding of a positive result for P. falciparum in Alouatta from both types of forest support the hypothesis that monkeys belonging to this genus could be a potential reservoir. Furthermore, these findings raise the question of the relationship between simian and autochthonous human malaria in extra-Amazonian regions.

Circulation of Chikungunya virus East-Central-South African genotype during the 2020­21 outbreak in São Paulo State, Brazil

Chikungunya virus (CHIKV), a mosquito-borne alphavirus, has traditionally circulated in Africa and Asia, causing human febrile illness accompanied by severe, chronic joint pain. The Asian and East-Central-South African (ECSA) genotypes were introduced in Brazil in 2014, in Bahia State and Northeast region. CHIKV virus rapidly spread, causing epidemics in urban areas, and the ECSA genotype was detected also in other regions. In the last five years, more than 700,000 cases of Chikungunya fever were reported in Brazil. Nevertheless, there is limited information about the genomic epidemiology of CHIKV from surveillance studies. In São Paulo (SP), the most populous Brazilian state, until 2015 only imported cases were identified. In 2020­21, an outbreak was detected in the coastal region of SP, accounting for 98% of confirmations in SP. To better understand the disease dynamics in SP, we generated 53 nearcomplete genomes of CHIKV isolates from Baixada Santista, obtained from clinical samples. Our results demonstrate that SP-CHIKV clade belongs to a distinct clade from those previously found in Brazil, supporting the local circulation of a specific lineage in the period. None of the SP-CHIKV carry the substitutions A226V (E1 protein) and L210Q (E2 protein) associated with increased transmission in Aedes albopictus mosquitoes. The results confirm that the ECSA lineage continues to spread across the country and reinforce that genomic data can provide information about virus genetic diversity and transmission dynamics, assisting the establishment of a more effective surveillance framework. (AU)

First Complete Genome Sequence of Zika Virus (Flaviviridae, Flavivirus) from an Autochthonous Transmission in Brazil.

We report here the genome sequence of Zika virus, strain ZikaSPH2015, containing all structural and nonstructural proteins flanked by the 5' and 3' untranslated region. It was isolated in São Paulo state, Brazil, in 2015, from a patient who received a blood transfusion from an asymptomatic donor at the time of donation.

Zika virus in the Americas: Early epidemiological and genetic findings.

Brazil has experienced an unprecedented epidemic of Zika virus (ZIKV), with ~30,000 cases reported to date. ZIKV was first detected in Brazil in May 2015, and cases of microcephaly potentially associated with ZIKV infection were identified in November 2015. We performed next-generation sequencing to generate seven Brazilian ZIKV genomes sampled from four self-limited cases, one blood donor, one fatal adult case, and one newborn with microcephaly and congenital malformations. Results of phylogenetic and molecular clock analyses show a single introduction of ZIKV into the Americas, which we estimated to have occurred between May and December 2013, more than 12 months before the detection of ZIKV in Brazil. The estimated date of origin coincides with an increase in air passengers to Brazil from ZIKV-endemic areas, as well as with reported outbreaks in the Pacific Islands. ZIKV genomes from Brazil are phylogenetically interspersed with those from other South American and Caribbean countries. Mapping mutations onto existing structural models revealed the context of viral amino acid changes present in the outbreak lineage; however, no shared amino acid changes were found among the three currently available virus genomes from microcephaly cases. Municipality-level incidence data indicate that reports of suspected microcephaly in Brazil best correlate with ZIKV incidence around week 17 of pregnancy, although this correlation does not demonstrate causation. Our genetic description and analysis of ZIKV isolates in Brazil provide a baseline for future studies of the evolution and molecular epidemiology of this emerging virus in the Americas.

The role of pregnant women with rash in the Zika virus sentinel surveillance

INTRODUCTION: The state of São Paulo has been monitoring cases of microcephaly and pregnant women presenting with acute rash, through CeVeSP. METHODS: This was a descriptive study focusing on pregnant women with rash and the outcome of their pregnancy, based on the notifications through the CeVeSP. RESULTS: During 2016, 2,209 cases of pregnant women with rash were reported and investigated. Of these, 36.6% were confirmed. Of the pregnant women who tested positive for ZIKV, 6.4% did not have a favorable outcome. CONCLUSIONS Our results allowed the characterization of pregnant women exposed to ZIKV and the outcome of pregnancy.

The role of pregnant women with rash in the Zika virus sentinel surveillance

Abstract INTRODUCTION: The state of São Paulo has been monitoring cases of microcephaly and pregnant women presenting with acute rash, through CeVeSP. METHODS: This was a descriptive study focusing on pregnant women with rash and the outcome of their pregnancy, based on the notifications through the CeVeSP. RESULTS: During 2016, 2,209 cases of pregnant women with rash were reported and investigated. Of these, 36.6% were confirmed. Of the pregnant women who tested positive for ZIKV, 6.4% did not have a favorable outcome. CONCLUSIONS Our results allowed the characterization of pregnant women exposed to ZIKV and the outcome of pregnancy.

Yellow fever epizootics in non-human primates, São Paulo state, Brazil, 2008-2009.

Since 2000, the expansion of Sylvatic Yellow Fever (YF) has been observed in the southeast of Brazil, being detected in areas considered silent for decades. Epizootics in non-human primates (NHPs) are considered sentinel events for the detection of human cases. It is important to report epizootic events that could have impact on the conservation status of susceptible species. We describe the epizootics in NHPs, notified in state of São Paulo, Brazil, between September 2008 to August 2009. Ninety-one epizootic events, involving 147 animals, were reported in 36 counties. Samples were obtained from 65 animals (44.2%). Most of the epizootics (46.6%) were reported between March and April, the same period during which human cases of YF occurred in the state. Biological samples were collected from animals found dead and were sent to Instituto Adolfo Lutz, in São Paulo. Two samples, collected in two counties without an indication for YF vaccination, were positive for the virus. Another 48 animals were associated with YF by clinical-epidemiological linkage with laboratory confirmed cases. Because the disease in human and NHPs occurred in the same period, the detection of the virus in NHPs did not work as sentinel, but aided in the delineation of new areas of risk.

First isolation of Dengue 4 in the state of São Paulo, Brazil, 2011.

We report the first isolation of Dengue virus 4 (DENV-4) in the state of São Paulo, from two patients - one living in São José do Rio Preto and the other one in Paulo de Faria, both cities located in the Northwest region of the state. The virus isolations were accomplished in the clone C6/36 Aedes albopictus cell line, followed by indirect immunofluorescence assays, performed with type-specific monoclonal antibodies that showed positive reactions for DENV-4. The results were confirmed by Nested RT-PCR and Real-Time RT-PCR assays. The introduction of DENV-4 in a country that already has to deal with the transmission of three other serotypes increases the possibility of the occurrence of more severe cases of the disease. The importance of early detection of dengue cases, before the virus spreads and major outbreaks occur, should be emphasized.

First complete Genome Sequence of Zika Virus (Flaviviridae, Flavivirus) from an Autochthonous Transmission in Brazil

We report here the genome sequence of Zika virus, strain ZikaSPH2015, containing all structural and nonstructural proteins flanked by the 5' and 3' untranslated region. It was isolated in São Paulo state, Brazil, in 2015, from a patient who received a blood transfusion from an asymptomatic donor at the time of donation.

Evaluation of dengue NS1 antigen detection for diagnosis in public health laboratories, São Paulo State, 2009.

The present work evaluated the diagnostic accuracy of detection of Dengue NS1 antigen employing two NS1 assays, an immunochromatographic assay and ELISA, in the diagnostic routine of Public Health laboratories. The results obtained with NS1 assay were compared with virus isolation and, in a subpopulation of cases, they were compared with the IgM-ELISA results obtained with convalescent samples. A total of 2,321 sera samples were analyzed by one of two NS1 techniques from March to October 2009. The samples were divided into five groups: groups I, II and III included samples tested by NS1 and virus isolation, and groups IV and V included patients with a first sample tested by NS1 and a second sample tested by IgM-ELISA. Sensitivity, specificity, positive and negative predictive values, Kappa Index and Kappa Concordance were calculated. The results showed that NS1 testing in groups I, II and III had high sensitivity (98.0%, 99.5% and 99.3%), and predictive values and Kappa index between 0.9 - 1.0. Groups IV and V only had Kappa Concordance calculated, since the samples were analyzed according to the presence of NS1 antigen or IgM antibody. Concordance of 92.1% was observed when comparing the results of NS1-negative samples with IgM-ELISA. Based on the findings, it is possible to suggest that the tests for NS1 detection may be important tools for monitoring the introduction and spread of Dengue serotypes.

Isolation of yellow fever virus (YFV) from naturally infected Haemagogus (Conopostegus) leucocelaenus (diptera, cukicudae) in São Paulo State, Brazil, 2009.

After detecting the death of Howlers monkeys (genus Alouatta) and isolation of yellow fever virus (YFV) in Buri county, São Paulo, Brazil, an entomological research study in the field was started. A YFV strain was isolated from newborn Swiss mice and cultured cells of Aedes albopictus - C6/36, from a pool of six Haemagogus (Conopostegus) leucocelaenus (Hg. leucocelaenus) mosquitoes (Dyar & Shannon) collected at the study site. Virus RNA fragment was amplified by RT-PCR and sequenced. The MCC Tree generated showed that the isolated strain is related to the South American I genotype, in a monophyletic clade containing isolates from recent 2008-2010 epidemics and epizootics in Brazil. Statistical analysis commonly used were calculated to characterize the sample in relation to diversity and dominance and indicated a pattern of dominance of one or a few species. Hg. leucocelaenus was found infected in Rio Grande do Sul State as well. In São Paulo State, this is the first detection of YFV in Hg. leucocelaenus.