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.

Detection of herpesviruses in neotropical primates from São Paulo, Brazil.

Transmission of herpesvirus between humans and non-human primates represents a serious potential threat to human health and endangered species conservation. This study aimed to identify herpesvirus genomes in samples of neotropical primates (NTPs) in the state of São Paulo, Brazil. A total of 242 NTPs, including Callithrix sp., Alouatta sp., Sapajus sp., and Callicebus sp., were evaluated by pan-herpesvirus polymerase chain reaction (PCR) and sequencing. Sixty-two (25.6%) samples containing genome segments representative of members of the family Herpesviridae, including 16.1% for Callitrichine gammaherpesvirus 3, 6.1% for Human alphaherpesvirus 1, 2.1% for Alouatta macconnelli cytomegalovirus, and 0.83% for Cebus albifrons lymphocryptovirus 1. No co-infections were detected. The detection of herpesvirus genomes was significantly higher among adult animals (p = 0.033) and those kept under human care (p = 0.008671). These findings confirm the importance of monitoring the occurrence of herpesviruses in NTP populations in epizootic events.

Genetic characterization of the rare Bruconha virus (Bunyavirales: Orthobunyavirus) isolated in Vale do Ribeira (Atlantic Forest biome), Southeastern Brazil.

Brazil is a great source of arbovirus diversity, mainly in the Amazon region. However, other biomes, especially the Atlantic Forest, may also be a hotspot for emerging viruses, including Bunyaviruses (Negarnaviricota: Bunyavirales). For instance, Vale do Ribeira, located in the Southeastern region, has been widely studied for virus surveillance, where Flavivirus, Alphavirus and Bunyaviruses were isolated during the last decades, including Bruconha virus (BRCV), a member of Orthobunyavirus genus Group C, in 1976. Recently, a new isolate of BRCV named Span321532 was obtained from an adult sentinel mouse placed in Iguape city in 2011, and a full-length genome was generated with nucleotide differences ranging between 1.5%, 5.3% and 5% (L, M and S segments, respectively) from the prototype isolated 35 years earlier. In addition, each segment placed BRCV into different clusters, showing the high variety within Bunyavirales. Although no evidence for reassortants was detected, this finding reiterates the need for new surveillance and genomic studies in the area considering the high mutation rates of arbovirus, and also to identify the hosts capable of supporting the continuous circulation of Orthobunyavirus.

Serological evidence of arenavirus circulation in wild rodents from central-west, southeast, and south regions of Brazil, 2002-2006.

Viral hemorrhagic fevers caused by arenaviruses are severe zoonotic diseases. In reservoirs, the presence of antibodies may indicate viral circulation in a population of a specific region, and these data can be used as an indicator for further investigations by molecular techniques. The present study aimed to detect the presence of arenavirus antibodies in wild rodents captured from 1998 to 2008 during epidemiological surveillance activities. A retrospective analysis of 2243 wild rodent blood samples using a broad cross-reactive in-house developed enzyme-linked immunosorbent assay (ELISA) revealed a 0.44% (10/2243) positive rate in wild rodents, which included Necromys lasiurus (6/1012), Calomys callosus (2/94), and Akodon sp. (2/273) species. These rodents were captured between 2002 to 2006 in Campo Alegre de Goiás/GO, Bodoquena/MS, Nuporanga/SP, and Mogi das Cruzes/SP. Our findings suggest the sylvatic circulation of arenavirus among wild rodents in the southeast region of Brazil. However, future virological and molecular studies are necessary to confirm the viral presence in these regions.

Lack of molecular evidence of fecal-borne viruses in capybaras from São Paulo state, Brazil, 2018-2020: a minor public health issue.

Capybara (Hydrochoerus hydrochaeris) is the world's largest rodent species distributed throughout South America. These animals are incredibly tolerant to anthropogenic environments and are occupying large urban centers. Capybaras are known to carry potentially zoonotic agents, including R. rickettsia, Leishmania spp., Leptospira spp., Trypanosoma spp., Salmonella spp., Toxoplasma gondii, and rabies virus. Focusing on the importance of monitoring potential sources of emerging zoonotic viruses and new viral reservoirs, the aim of the present study was to assess the presence of fecal-borne viruses in the feces of capybaras living in urban parks in São Paulo state, Brazil. A total of 337 fecal samples were collected between 2018 and 2020 and screened for the following: (i) Rotavirus group A (RVA) by ELISA; (ii) non-RVA species and Picobirnavirus (PBV) using PAGE; (iii) Human Bocaparvovirus (HBoV), Bufavirus (BuV), Tusavirus (TuV), and Cutavirus (CuV) qPCR; (iv) Human Enterovirus (EV), Norovirus GII (NoV), and Hantavirus by in houses RT-qPCR; (v) SARS-CoV-2 via commercial RT-qPCR kit assay; and (vi) Astrovirus (AstV) and Adenovirus (AdV) using conventional nested (RT)-PCRs. All fecal samples tested were negative for fecal-borne viruses. This study adds further evidence that the fecal-borne viruses is a minor public health issue in Brazilian capybaras, at least during the surveillance period and surveyed areas. Continuous monitoring of sylvatic animals is essential to prevent and control the emergence or re-emergence of newly discovered virus as well as viruses with known zoonotic potential.

Genetic characterization of the rare Bruconha virus (Bunyavirales: Orthobunyavirus) isolated in Vale do Ribeira (Atlantic Forest biome), Southeastern Brazil

ABSTRACT Brazil is a great source of arbovirus diversity, mainly in the Amazon region. However, other biomes, especially the Atlantic Forest, may also be a hotspot for emerging viruses, including Bunyaviruses (Negarnaviricota: Bunyavirales). For instance, Vale do Ribeira, located in the Southeastern region, has been widely studied for virus surveillance, where Flavivirus, Alphavirus and Bunyaviruses were isolated during the last decades, including Bruconha virus (BRCV), a member of Orthobunyavirus genus Group C, in 1976. Recently, a new isolate of BRCV named Span321532 was obtained from an adult sentinel mouse placed in Iguape city in 2011, and a full-length genome was generated with nucleotide differences ranging between 1.5%, 5.3% and 5% (L, M and S segments, respectively) from the prototype isolated 35 years earlier. In addition, each segment placed BRCV into different clusters, showing the high variety within Bunyavirales. Although no evidence for reassortants was detected, this finding reiterates the need for new surveillance and genomic studies in the area considering the high mutation rates of arbovirus, and also to identify the hosts capable of supporting the continuous circulation of Orthobunyavirus.

A fatal case of dengue hemorrhagic fever associated with dengue virus 4 (DENV-4) in Brazil: genomic and histopathological findings.

Dengue infection is the most prevalent arthropod-borne viral disease in subtropical and tropical regions, whose primary vector is Aedes aegypti mosquitoes. The mechanisms of dengue virus (DENV) pathogenesis are little understood because we have no good disease models. Only humans develop symptoms (dengue fever, DF, or dengue hemorrhagic fever, DHF) and research has been limited to studies involving patients. Samples from serum, brain, cerebellum, heart, lungs, liver, and kidneys from a 13-year-old male patient that died with hemorrhagic manifestations were sent for differential diagnosis at Adolfo Lutz, using both classical virological methods (RT-qPCR, virus isolation, ELISA, and hemagglutination inhibition test) and immunohistochemistry (IHQ). A DENV serotype 4 was detected by a DENV multiplex RT-qPCR, and the C6/36 cell supernatant was used for NGS using Minion. Lesions were described in the heart, liver, lung, and kidney with positive IHQ in endothelial cells of the brain, cerebellum, heart, and kidney, and also in hepatocytes and Kuppfer cells. A whole genome was obtained, revealing a DENV-4 genotype II, with no evidence of secondary dengue infection.

Análise de algoritmo diagnóstico de febre amarela em amostras de primatas não humanos encaminhadas ao Centro de Patologia do Instituto Adolfo Lutz / Analysis of a diagnostic algorithm for yellow fever in primate samples non-humans sent to the Pathology Center of Instituto Adolfo Lutz

O Centro de Patologia do Instituto Adolfo Lutz (CPA-IAL) é credenciado pelo Ministério da Saúde como laboratório de referência macrorregional para a vigilância epidemiológica de febre amarela (FA) em seres humanos e primatas não humanos (PNH) do Brasil, atuando por meio de análise histopatológica e imuno-histoquímica (IHQ). Até o ano de 2018, ambos os exames eram aplicados a todas as amostras de PNH recebidas para a pesquisa de FA. Em 2019, implantou-se um algoritmo diagnóstico baseado na triagem pelas características histopatológicas observadas no tecido hepático, possibilitando a racionalização do uso da IHQ. Objetivo: Avaliar a aplicação do algoritmo diagnóstico comparado ao período que antecedeu sua implantação. Métodos: Estudo retrospectivo de relatórios anatomopatológicos de PNH emitidos, entre 2018 e 2019, no CPA-IAL para determinação de índices de performance diagnóstica do exame histopatológico na vigilância epidemiológica de febre amarela, avaliação da sensibilidade do exame imuno-histoquímico para amostras com autólise de moderada a avançada e comparação da mediana de tempo decorrido para emissão dos relatórios em cada período. Resultados: Não houve diferença estatisticamente significante na performance da detecção de FA por histologia e IHQ entre os períodos pré e pós algoritmo; houve importante redução na quantidade de exames IHQ solicitados e no tempo de liberação dos relatórios (p<0,0001). Conclusões: O algoritmo resultou em desempenho semelhante, redução do tempo de liberação oportuno para a vigilância epidemiológica do agravo e da quantidade de reações IHQ realizadas, portanto, apresentando-se adequado para o diagnóstico de febre amarela em PNH no CPA-IAL.

Concurrent yellow fever and pulmonary aspergillosis due to Aspergillus fumigatus in a free-ranging howler monkey (Alouatta sp).

Herein, we describe a unique case of concomitant angioinvasive pulmonary aspergillosis due to Aspergillus fumigatus and yellow fever in a free-ranging howler monkey (Alouatta sp). Lung samples were negative for influenza viruses A and B.

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.

Norovirus strains in patients with acute gastroenteritis in rural and low-income urban areas in northern Brazil.

From 2010-2016, a total of 251 stool samples were screened for norovirus using next-generation sequencing (NGS) followed by phylogenetic analysis to investigate the genotypic diversity of noroviruses in rural and low-income urban areas in northern Brazil. Norovirus infection was detected in 19.9% (50/251) of the samples. Eight different genotypes were identified: GII.4_Sydney[P31] (64%, 32/50), GII.6[P7] (14%, 7/50), GII.17[P17] (6%, 3/50), GII.1[P33] (6%, 3/50), GII.3[P16] (4%, 2/50), GII.2[P16] (2%, 1/50), GII.2[P2] (2%, 1/50), and GII.4_New Orleans[P4] (2%, 1/50). Distinct GII.6[P7] variants were recognized, indicating the presence of different co-circulating strains. Elucidating norovirus genetic diversity will improve our understanding of their potential health burden, in particular for the GII.4_Sydney[P31] variant.

Enteric viruses circulating in undiagnosed central nervous system infections at tertiary hospital in São José do Rio Preto, São Paulo, Brazil.

Enterovirus (EV) is commonly associated with central nervous system (CNS) syndromes. Recently, gastroenteric viruses, including rotavirus (RVA), human astrovirus (HAstV), and norovirus (NoV), have also been associated with CNS neurological disorders. The aim of the present study was to investigate the presence of EV, RVA, HAst, and NoV associated to CNS infections with undiagnosed etiology in Northwest region of São Paulo State, Brazil, and to conduct the molecular characterization of the positive samples detected. A total of 288 cerebrospinal fluid samples collected from July to December 2017 were tested for EV and NoV by quantitative real-time polymerase chain reaction (RT-qPCR), HAstV by conventional RT-PCR, and RVA by enzyme-linked immunosorbent assay. Positive-EV samples were inoculated in cells lines, amplified by RT-PCR and sequenced. RVA, NoV, and HAstV were not detected. EV infection was detected in 5.5% (16/288), and five samples successful genotyped: echovirus 3 (E3) (1/5), coxsackie virus A6 (CVA6) (1/5), and coxsackie virus B4 (CVB4) (3/5). Meningitis was the main syndrome observed (12/16; 75%). CVA6, CVB4, and E3 were identified associated with aseptic meningitis. Reports of CVA6 associated with aseptic meningitis are rare, E3 had not been previously reported in Brazil, and epidemiological data on CVB4 in the country is virtually unknown. The present investigation illustrates the circulation of diverse EV types in a small regional sample set and in a short period of time, highlighting the importance of an active EV surveillance system in CNS infections. Enhanced understanding of undiagnosed CNS infections will assist in public health and health care planning.

Zika virus serological diagnosis: commercial tests and monoclonal antibodies as tools.

Zika virus (ZIKV), an emerging arthropod-borne virus (arbovirus) of the Flaviviridae family, is a current issue worldwide, particularly because of the congenital and neurological syndromes associated with infection by this virus. As the initial clinical symptoms of all diseases caused by this group are very similar, clinical diagnosis is difficult. Furthermore, laboratory diagnostic efforts have failed to identify specific and accurate tests for each virus of the Flaviviridae family due to the cross-reactivity of these viruses in serum samples. This situation has resulted in underreporting of the diseases caused by flaviviruses. However, many companies developed commercial diagnostic tests after the recent ZIKV outbreak. Moreover, health regulatory agencies have approved different commercial tests to extend the monitoring of ZIKV infections. Considering that a specific and sensitive diagnostic method for estimating risk and evaluating ZIKV propagation is still needed, this review aims to provide an update of the main commercially approved serological diagnostics test by the US Food and Drug Administration (FDA) and Brazilian National Health Surveillance Agency (ANVISA). Additionally, we present the technologies used for monoclonal antibody production as a tool for the development of diagnostic tests and applications of these antibodies in detecting ZIKV infections worldwide.

Detection of Zika and dengue viruses in wild-caught mosquitoes collected during field surveillance in an environmental protection area in São Paulo, Brazil.

Species of the genus Flavivirus are widespread in Brazil and are a major public health concern. The country's largest city, São Paulo, is in a highly urbanized area with a few forest fragments which are commonly used for recreation. These can be considered to present a potential risk of flavivirus transmission to humans as they are home simultaneously to vertebrate hosts and mosquitoes that are potential flavivirus vectors. The aim of this study was to conduct flavivirus surveillance in field-collected mosquitoes in the Capivari-Monos Environmental Protection Area (EPA) and identify the flavivirus species by sequence analysis in flavivirus IFA-positive pools. Monthly mosquito collections were carried out from March 2016 to April 2017 with CO2-baited CDC light traps. Specimens were identified morphologically and grouped in pools of up to 10 individuals according to their taxonomic category. A total of 260 pools of non-engorged females were inoculated into C6/36 cell culture, and the cell suspensions were analyzed by indirect immunofluorescence assay (IFA) after the incubation period. IFA-positive pools were tested by qRT-PCR with genus-specific primers targeting the flavivirus NS5 gene to confirm IFA-positive results and sequenced to identify the species. Anopheles cruzii (19.5%) and Wyeomyia confusa (15.3%) were the most frequent vector species collected. IFA was positive for flaviviruses in 2.3% (6/260) of the sample pools. This was confirmed by qRT-PCR in five pools (83.3%). All five flavivirus-positive pools were successfully sequenced and the species identified. DENV serotype 2 (DENV-2) was detected in Culex spp. and Culex vaxus pools, while ZIKV was identified in An. cruzii, Limatus durhamii and Wy. confusa pools. To the best of our knowledge, detection of flavivirus species of medical importance has never previously been reported in these species of wild-caught mosquitoes. The finding of DENV-2 and ZIKV circulating in wild mosquitoes suggests the existence of an enzootic cycle in the area. In-depth studies of DENV-2 and ZIKV, including investigation of mosquito infection, vector competence and infection in sylvatic hosts, are needed to shed light on the transmission dynamics of these important viruses and the potential risk of future outbreaks of DENV-2 and ZIKV infections in the region.

Possible non-sylvatic transmission of yellow fever between non-human primates in São Paulo city, Brazil, 2017-2018.

Yellow Fever (YF) is a severe disease caused by Yellow Fever Virus (YFV), endemic in some parts of Africa and America. In Brazil, YFV is maintained by a sylvatic transmission cycle involving non-human primates (NHP) and forest canopy-dwelling mosquitoes, mainly Haemagogus-spp and Sabethes-spp. Beginning in 2016, Brazil faced one of the largest Yellow Fever (YF) outbreaks in recent decades, mainly in the southeastern region. In São Paulo city, YFV was detected in October 2017 in Aloutta monkeys in an Atlantic Forest area. From 542 NHP, a total of 162 NHP were YFV positive by RT-qPCR and/or immunohistochemistry, being 22 Callithrix-spp. most from urban areas. Entomological collections executed did not detect the presence of strictly sylvatic mosquitoes. Three mosquito pools were positive for YFV, 2 Haemagogus leucocelaenus, and 1 Aedes scapularis. In summary, YFV in the São Paulo urban area was detected mainly in resident marmosets, and synanthropic mosquitoes were likely involved in viral transmission.

Applying a pan-flavivirus RT-qPCR assay in Brazilian public health surveillance.

The aim of this study was to improve flavivirus field monitoring in Brazil using a reliable probe-based RT-qPCR assay. Standard flavivirus strains were employed to evaluate the performance of the assay, and its applicability was evaluated using 235 stored pools of Culicidae samples collected between 1993 and 1997 and in 2016. Flavivirus species were identified by sequencing. Sixteen (6.8%) samples tested positive: Ilheus virus, Iguape virus, and Saint Louis encephalitis virus were identified in historical specimens from 1993-1994, while insect-specific flaviviruses were detected in the samples from 2016. This approach was demonstrated to be accurate for flavivirus detection and characterization, and it can be successfully applied for vector surveillance and for monitoring and discovery of insect specific flaviviruses.

Detection and characterization of Ilheus and Iguape virus genomes in historical mosquito samples from Southern Brazil.

In Brazil, flaviviruses have caused massive outbreaks. Surveillance programs designed to monitor virus activity in vectors provides a system for mapping disease distribution and for identifying specific vector species for targeted control. The present study aimed to describe the detection, whole genome characterization and phylogenetic analysis of Ilheus virus (ILHV) and Iguape virus (IGUV) strains obtained from historical mosquito's samples. Twelve isolates of pooled mosquito specimens (inoculated in neonate mouse brain) collected in the state of São Paulo, Brazil, in 1993, 1994 and 1997 were investigated. Viral RNA was extracted and analyzed by qRT-PCR using Flavivirus genus-specific primers. Positive samples were sequenced and underwent phylogenetic analyses. Flavivirus was detected in 50% of the specimens. Positive samples were successfully Sanger sequenced. Three Anopholes cruzii pools collected in 1994 were positive for IGUV. One Culex sp. pool, one Anopheles triannulatus pool, and one Coquillettidia juxtamansonia pool, collected in 1994, were positive for ILHV. Metagenomic sequencing successfully characterize one ILHV and four IGUV full genomes, and revealed a high degree of homology between the Brazilian ILHV and IGUV strains and isolates available in GenBank. Phylogenetic analysis of partial ILHV NS5 gene revealed three distinct lineages (clades), an indication of genetic heterogeneity in strains circulating in Brazil. Nucleotide insertions and a high-level of nucleotide diversity were observed in the NS1 protein and capsid region of IGUV strains, respectively. Detection of ILHV and IGUV in mosquitoes from Southeastern Brazil confirms the historical circulation of these viruses in this area. Furthermore, this first evidence of ILHV in Anopheles triannulatus suggests the potential importance of Anopheles mosquitoes in the IGUV transmission cycle. Genomic and phylogenetic analysis of these viruses provided insights into their diversity and evolution, which are important for the emergence patterns of flaviviruses and their evolutionary trends in Brazil, an endemic country for several arbovirus. in In-depth studies of ILHV and IGUV including vector competence and molecular studies are needed to shed light on their epidemiology and potential risk of future emergence.

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.

Zika virus serological diagnosis: commercial tests and monoclonal antibodies as tools

Zika virus (ZIKV), an emerging arthropod-borne virus (arbovirus) of the Flaviviridae family, is a current issue worldwide, particularly because of the congenital and neurological syndromes associated with infection by this virus. As the initial clinical symptoms of all diseases caused by this group are very similar, clinical diagnosis is difficult. Furthermore, laboratory diagnostic efforts have failed to identify specific and accurate tests for each virus of the Flaviviridae family due to the cross-reactivity of these viruses in serum samples. This situation has resulted in underreporting of the diseases caused by flaviviruses. However, many companies developed commercial diagnostic tests after the recent ZIKV outbreak. Moreover, health regulatory agencies have approved different commercial tests to extend the monitoring of ZIKV infections. Considering that a specific and sensitive diagnostic method for estimating risk and evaluating ZIKV propagation is still needed, this review aims to provide an update of the main commercially approved serological diagnostics test by the US Food and Drug Administration (FDA) and Brazilian National Health Surveillance Agency (ANVISA). Additionally, we present the technologies used for monoclonal antibody production as a tool for the development of diagnostic tests and applications of these antibodies in detecting ZIKV infections worldwide.(AU)

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.