Ricardo Luiz Moro de Sousa

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.

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.

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.

Detection and Quantification of Antibodies to Newcastle Disease Virus in Ostrich and Rhea Sera Using a Liquid Phase Blocking Enzyme-Linked Immunosorbent Assay

ABSTRACT A liquid phase blocking ELISA (LPB-ELISA) was adapted for the detection and quantification of antibodies to Newcastle disease virus. Sera from vaccinated and unvaccinated commercial flocks of ostriches (Struthio camelus) and rheas (Rhea americana) were tested. The purified and nonpurified virus used as the antigen and the capture and detector antibodies were prepared and standardized for this purpose. The hemagglutination-inhibition (HI) test was regarded as the reference method. The cutoff point for the LPB-ELISA was determined by a two-graph receiver operating characteristic analysis. The LPB-ELISA titers regressed significantly (P < 0.0001) on the HI titers with a high correlation coefficient (r = 0.875). The two tests showed good agreement (κ = 0.82; P < 0.0001), relative sensitivity (90.91%) and specificity (91.18%), and accuracy (91.02%), suggesting that they are interchangeable.

Inducible nitric oxide synthase-deficient mice show exacerbated inflammatory process and high production of both Th1 and Th2 cytokines during paracoccidioidomycosis

Paracoccidioidomycosis, the major systemic mycosis in Latin America, is caused by fungus Paracoccidioides brasiliensis. To analyze the influence of inducible nitric oxide synthase (iNOS) in this disease, iNOS-deficient (iNOS(-/-)) and wild-type (WT) mice were infected intravenously with P. brasiliensis 18 isolate. We found that, unlike WT mice, iNOS(-/-) mice did not control fungal proliferation, and began to succumb to infection by day 50 after inoculation of yeast cells. Typical inflammatory granulomas were found in WT mice, while, iNOS(-/-) mice presented incipient granulomas with intense inflammatory process and necrosis. Additionally, splenocytes from iNOS(-/-) mice did not produce nitric oxide, however, their proliferative response to Con-A was impaired, just like infected WT mice. Moreover, infected iNOS(-/-) mice presented a mixed pattern of immune response, releasing high levels of both Th1 (IL-12, IFN-gamma and TNF-alpha) and Th2 (IL-4 and IL-10) cytokines. These data suggest that the enzyme iNOS is a resistance factor during paracoccidioidomycosis by controlling fungal proliferation, by influencing cytokines production, and by appeasing the development of a high inflammatory response and consequently formation of necrosis. However, iNOS-derived nitric oxide seems not being the unique factor responsible for immunosuppression observed in infections caused by P. brasiliensis.

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.

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.

First detection and molecular characterization of Nebovirus in Brazil.

Nebovirus is a new genus of viruses belonging to the Caliciviridae family recently characterized in cattle, and is associated with gastrointestinal disorders, such as diarrhoea, anorexia and intestinal lesions particularly in calves. The aim of this study was to investigate the prevalence of neboviruses in Brazilian cattle and analyse phylogenetically the virus strains detected. A prevalence of 4·8% of neboviruses in faecal samples from 62 head of cattle from different Brazilian states was detected. All positive animals were aged 96·0% nt (100% aa) sequence identity between the virus sequences in this study and >88·8% nt (>94·4% aa) identity with Newbury1/UK. Our results indicate, for the first time, the occurrence of neboviruses in Brazil as well as in South America, and the first Newbury1-like nebovirus found outside the UK.

Carryover of aflatoxins from feed to lambari fish (Astyanax altiparanae) tissues

ABSTRACT The aim of this study was to verify the carryover of aflatoxin B1 from feed to lambari fish. Aflatoxins (AF) were incorporated into feed, checking the levels by HPLC. Treatments were: Control, feed without toxin; A, feed + 10 µg AFB1 kg–1; B, feed + 20 µg AFB1 kg–1; and C, feed + 50 µg AFB1 kg–1. Juveniles of lambari fish were placed in 12 aquariums at a density of 50 fish/m2. Fish were fed twice a day with extruded feed, at 5% of animal biomass. The unit sample was constituted by a pool of 10 fish. AFs B1, B2, G1, G2 and M1 were quantified by HPLC in fish muscle and liver after 30, 60, 90 and 120 days of experiment. There was accumulation of AFs is fish liver and muscle, mainly after 90 days. Fish from treatment C had higher levels of AFB1 in muscle when compared with the others, and AFB1 in muscle at 120 days was similar to the levels in feed. Therefore, when lambari fish is exposed on a daily and long-term basis to AFs in feed, the regulation limits for AFs in animal feed do not guarantee safety for consumers.

Serosurvey of selected avian pathogens in brazilian commercial Rheas (Rhea americana) and Ostriches (Struthio camelus)

Ratite farming of has expanded worldwide. Due to the intensive farming methods used by ratite producers, preventive medicine practices should be established. In this context, the surveillance and control of some avian pathogens are essential for the success of the ratite industry; however, little is known on the health status of ratites in Brazil. Therefore, the prevalence of antibodies against Newcastle Disease virus, Chlamydophila psittaci, Mycoplasma gallisepticum, Mycoplasma synoviae, and Salmonella Pullorum were evaluated in 100 serum samples collected from commercial ostriches and in 80 serum samples from commercial rheas reared in Brazil. All sampled animals were clinically healthy. The results showed that all ostriches and rheas were serologically negative to Newcastle disease virus, Chlamydophila psittaci, Mycoplasma gallisepticum, and Mycoplasma synoviae. Positive antibody responses against Salmonella Pullorum antigen were not detected in ostrich sera, but were detected in two rhea serum samples. These results can be considered as a warning as to the presence of Salmonella spp. in ratite farms. Therefore, the implementation of good health management and surveillance programs in ratite farms may contribute to improve not only animal production, but also public health conditions.