Marcos Lázaro Moreli

Hantavirus Pulmonary Syndrome, Central Plateau, Southeastern, and Southern Brazil

This syndrome is an increasing health problem because of human encroachment into habitats of rodent reservoirs.

Role of mixed Th1 and Th2 serum cytokines on pathogenesis and prognosis of hantavirus pulmonary syndrome

The hantavirus pulmonary syndrome (HPS) is an emerging syndrome in the Americas. The disease results from intense immune activation and changes in vascular permeability. The aim of this study was to determine the profile of serum cytokines in HPS patients looking for correlation with the clinical parameters, severity and outcome of illness. Studying 21 HPS patients, we found that IL-6 may have an important role in the pathogenesis of HPS, being associated with fatal outcome. Our results also support a mixed Th1/Th2 immune response during the course of HPS and that the magnitude of Th1 response effector cytokines is correlated to HPS severity. The decreased levels of TGF-beta observed in HPS patients suggest that immunoregulatory activity could be damaged in these patients.

Analysis of oropouche virus L protein amino acid sequence showed the presence of an additional conserved region that could harbour an important role for the polymerase activity.

Summary. We described here the complete nucleotide sequence of the L RNA segment of Oropouche virus (genus Orthobunyavirus, family Bunyaviridae). We found the L RNA segment is 6846 nucleotides long and encodes a putative RNA polymerase of 2250 amino acids. Phylogenetic analysis showed that ORO virus cluster to the Orthobunyavirus genus confirming the serological classification. It also showed that Bunyamwera and California viruses, from the Orthobunyavirus genus, are more closely related to each other than to ORO virus. Sequence comparisons performed between the L proteins of 15 bunyaviruses and the PB1 proteins of 3 influenza viruses revealed that ORO L protein contains the 3 regions characteristic of arenaviruses and bunyaviruses. These comparisons also showed the existence of an additional fourth conserved region in the L protein of bunyaviruses that contains at least two active sites.

Detection of Brazilian hantavirus by reverse transcription polymerase chain reaction amplification of N gene in patients with hantavirus cardiopulmonary syndrome

We report a nested reverse transcription-polymerase chain reaction (RT-PCR) assay for hantavirus using primers selected to match high homology regions of hantavirus genomes detected from the whole blood of hantavirus cardiopulmonary syndrome (HCPS) patients from Brazil, also including the N gene nucleotide sequence of Araraquara virus. Hantavirus genomes were detected in eight out of nine blood samples from the HCPS patients by RT-PCR (88.9% positivity) and in all 9 blood samples (100% positivity) by nested-PCR. The eight amplicons obtained by RT-PCR (P1, P3-P9), including one obtained by nested-PCR (P-2) and not obtained by RT-PCR, were sequenced and showed high homology (94.8% to 99.1%) with the N gene of Araraquara hantavirus. Although the serologic method ELISA is the most appropriate test for HCPS diagnosis, the use of nested RT-PCR for hantavirus in Brazil would contribute to the diagnosis of acute hantavirus disease detecting viral genomes in patient specimens as well as initial genomic characterization of circulating hantaviruses.

Expression of a hantavirus N protein and its efficacy as antigen in immune assays

Hantavirus cardiopulmonary syndrome (HCPS) has been recognized as an important public heath problem. Five hantaviruses associated with HCPS are currently known in Brazil: Juquitiba, Araraquara, Laguna Negra-like, Castelo dos Sonhos, and Anajatuba viruses. The laboratory diagnosis of HCPS is routinely carried out by the detection of anti-hantavirus IgM and/or IgG antibodies. The present study describes the expression of the N protein of a hantavirus detected in the blood sample of an HCPS patient. The entire S segment of the virus was amplified and found to be 1858 nucleotides long, with an open reading frame of 1287 nucleotides that encodes a protein of 429 amino acids. The nucleotide sequence described here showed a high identity with the N protein gene of Araraquara virus. The entire N protein was expressed using the vector pET200D and the Escherichia coli BL21 strain. The expression of the recombinant protein was confirmed by the detection of a 52-kDa protein by Western blot using a pool of human sera obtained from HCPS patients, and by specific IgG detection in five serum samples of HCPS patients tested by ELISA. These results suggest that the recombinant N protein could be used as an antigen for the serological screening of hantavirus infection.

Hantaviruses in São Paulo State, Brazil.

To the Editor: Hantavirus pulmonary syndrome (HPS) is an emerging health problem in Brazil. This syndrome was first reported in 1993 in three persons living in a rural area of Juquitiba County; two of them died of acute respiratory failure (1). Although Juquitiba County is part of the metropolitan area of greater Sao Paulo City, patients lived in a recently deforested region. From 1993 through 2002, approximately 200 HPS cases were reported in Brazil, with a 40% case-fatality ratio (Ministry of Health of Brazil, Report on Hantavirus cases 1993–2002, unpub. data). The wild rodent Bolomys laziurus is believed to be the most important hantavirus reservoir in the State of Sao Paulo, based on high levels of specific antibodies observed in serum from captured specimens (L.E. Pereira, Adolpho Lutz Institute, pers. comm., 2001). The economy of the inland region of Ribeirao Preto in the State of Sao Paulo, with its 3.5 million inhabitants, is based on the sugar cane agroindustry. The region has been almost completely deforested, with important consequences to the environment and wild rodent ecology. Twenty HPS cases were reported in Ribeirao Preto in the last 5 years, with a 60% case-fatality ratio. Review of medical records showed that a prodromic fever occurred in all 14 case-patients studied; dyspnea, cough, hypotension, and tachycardia occurred in about two thirds of patients; and hemorrhagic phenomena (hematuria, melena, and hypermenorrhea) in about one third. Thrombocytopenia was observed in all the patients, elevated hematocrit in about three fourths, and leukocytosis with neutrophilia and a left shift in the differential count in about two thirds. Serum creatinine levels were also increased (average level 2 mg/dL). Chest radiographs showed diffuse alveolar flocculant infiltrates in most cases (2,3). Laboratory diagnosis of HPS was made by serologic testing (enzyme-linked immunosorbent assay [ELISA]) in 18 cases and by reverse transcription–polymerase chain reaction (RT-PCR) in 11 cases; for 7 cases, both techniques were used. We performed a nucleotide sequence analysis of the N gene of hantavirus (residues 236–477) obtained from the blood of 11 of the 20 patients. This analysis showed that the infections were caused by Araraquara virus, a previously known hantavirus that had been detected by RT-PCR in the serum of an HPS patient living in a nearby county (4). Thus, Araraquara virus is the causative agent of a severe form of HPS, with a high death rate. This high death rate could also be related to the lack of adequate initial therapy provided by clinicians who probably did not immediately suspect HPS and may have not recommended hospitalization in intensive-care units. In addition, some hospitalized patients were in shock when first seen and were rehydrated with massive quantities of fluids, which may have aggravated pulmonary edema and contributed to death. The occurrence of 10% of the Brazilian HPS reported cases in Ribeirao Preto indicates that this region is suitable for studying the epidemiology of hantavirus infections. A serologic survey conducted in the region in 1999, which included 567 primary-care patients from Ribeirao Preto, Guariba, and Jardinopolis Counties, found that 7 (1.23%) of them had immunoglobulin (Ig) G antibodies to Sin Nombre virus by ELISA and that 5 of those lived in Jardinopolis (population 30,000), a county where a fatal case of HPS occurred in 1999 (5). Thus, Jardinopolis County was chosen for a population-based survey. In May 2001, we obtained personal information and collected fingerprick blood samples from 818 participants, 15–70 years of age, living in urban and rural areas of the county. IgG antibodies to the N recombinant protein of Andes virus were detected by ELISA in the blood samples of 14.3% of the participants (5). Even though all HPS cases in Ribeirao Preto were associated with rural activity and rodent exposure, these serologic data suggest that hantavirus infections are common in Jardinopolis County, independent of sex, profession, or history of contact with rodents. None of the 14.3% participants with IgG antibodies to hantavirus had a history of HPS-like disease, and the ELISA test showed cross-reactions with most of the South American hantaviruses, including Araraquara. Persons living in the urban area had higher levels of antibodies to hantavirus than those from rural areas. In Ribeirao Preto, the physical boundaries of cities have expanded to incorporate other areas, encroaching upon rural areas with many popular subsidized housing complexes. Work-related and recreational rural activities in that region are also frequent, which makes it difficult to interpret these data. These results suggest that in this region of southeast Brazil, hantaviruses may be causing undiagnosed asymptomatic or clinically minor infections in addition to typical HPS. This finding envokes important questions. Is more than one hantavirus circulating in this region, causing mostly benign infections? Is Araraquara virus widespread, causing mostly inapparent infections and only rarely causing HPS? Would HPS be associated with some predisposing condition in the infected person? If more than one hantavirus is circulating in the region, could urban rodents be reservoirs? Further studies are necessary to better understand the epidemiology and clinical signs and symptoms of hantavirus infection in the region of Ribeirao Preto. Such studies should emphasize determining the reservoirs, the modes of virus transmission to people, and the possible distinct clinical forms of hantavirus infections.

Síndrome pulmonar e cardiovascular por hantavírus: aspectos clínicos de uma doença emergente no sudeste brasileiro

Pulmonary and cardiovascular syndrome due to hantavirus is a disease caused by inhalation of aerosols from the excreta of wild rodents contaminated by viruses of the Bunyaviridae family. We studied the clinical and laboratory manifestations of 70 cases that occurred in the region of Ribeirão Preto, SP, Brazil, between 1998 and 2007. The frequency of symptoms was as follows: dyspnea (87%), fever (81%), coughing (44%), headache (34%), tachycardia (81%), low arterial blood pressure (56%), metabolic acidosis (57%), lymphocytopenia (51%), hematocrit > 45% (70%), leukocytosis with left deviation (67%), creatinine (51%) and urea (42%). Mortality (54.3%) occurred mainly on the fourth day. Respiratory insufficiency, low arterial blood pressure and shock occurred after 24 to 48 hours. High hematocrit and decreased platelet levels were signs strongly suggestive of the disease. The diagnostic hypothesis of atypical pneumonia was associated with a good prognosis (p = 0.0136). Fluid infusion greater than 2,000 ml and arterial hypotension were associated with a poor prognosis (p = 0.0286 and p = 0.0453).

Association of −308G/A polymorphism in the tumor necrosis factor-α gene promoter with susceptibility to development of hantavirus cardiopulmonary syndrome in the Ribeirão Preto region, Brazil

Activation of the immune response in hantavirus cardiopulmonary syndrome (HCPS) leads to a high TNF production, probably contributing to the disease. The polymorphic TNF2 allele (TNF −308G/A) has been associated with increased cytokine production. We investigated the association of the TNF2 allele with the outcome of hantavirus infection in Brazilian patients. A total of 122 hantavirus-exposed individuals (26 presenting HCPS and 96 only hantavirus seroconversion) were studied. The TNF2 allele was more frequently found in HCPS patients than in individuals with positive serology for hantavirus but without a history of HCPS illness, suggesting that the TNF2 allele could represent a risk factor for developing HCPS.

Brazilian Flavivirus phylogeny based on NS5

In this work, a comprehensive phylogenetic study based on 600 base pair nucleotide and on putative 200 amino acid sequences of NS5 was carried out in order to establish genetic relationships among 15 strains of 10 Brazilian flaviviruses: Bussuquara, Cacipacore, dengue type 1, 2 and 4, Iguape, Ilheus, Rocio, Saint Louis encephalitis (SLE), and yellow fever. Phylogenetic trees were created by neighbor-joining and maximum parsimony methods. These trees showed Brazilian flaviviruses grouped into three main branches: yellow fever branch, dengue branch subdivided in types 1, 2 and 4 branches, and Japanese encephalitis virus (JEV) complex branch including SLE virus strains, Cacipacore, Iguape, Rocio, Ilheus and Bussuquara. Viruses transmitted by Aedes mosquitoes, such as dengue and urban yellow fever, that are also the only Flavivirus causing hemorrhagic fevers in Brazil, were grouped in the same cluster. Encephalitis associated viruses, transmitted by Culex mosquitoes such as JEV complex branch including SLE virus strains, Cacipacore, Iguape, Rocio, Ilheus and Bussuquara were also grouped in the same clade.

Natural Host Relationships and Genetic Diversity of Rodent-Associated Hantaviruses in Southeastern Brazil

Objective: Hantaviruses are rodent-borne RNA viruses that have caused hantavirus cardiopulmonary syndrome in several Brazilian regions. In the present study, geographical distribution, seroprevalence, natural host range, and phylogenetic relations of rodent-associated hantaviruses collected from seven counties of Southeastern Brazil were evaluated. Methods: ELISA, RT-PCR and phylogenetic analysis were used in this study. Results: Antibodies to hantavirus were detected in Bolomys lasiurus, Akodon sp. and Oligoryzomys sp., performing an overall seroprevalence of 5.17%. All seropositive rodents were associated with grasslands or woods surrounded by sugar cane fields. Phylogenetic analysis of partial S- and M-segment sequences showed that viral sequences isolated from B. lasiurus specimens clustered with Araraquara virus. However, a sequence from Akodon sp. shared 100% similarity with Argentinian/Chilean viruses based on the partial S-segment amino acid sequence. Conclusion: These results indicate that there are associations between rodent reservoirs and hantaviruses in some regions of Southeastern Brazil, and suggest the existence of additional hantavirus genetic diversity and host ecology in these areas.