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.

Síndrome pulmonar e cardiovascular por Hantavirus: aspectos epidemiológicos, clínicos, do diagnóstico laboratorial e do tratamento / Hantavirus pulmonary and cardiovascular syndrome: epidemiology, clinical presentation, laboratory diagnosis and treatment

The Hantavirus pulmonary and cardiovascular syndrome (HPCVS) is an emerging disease in Brazil. In this study, eight confirmed cases of HPCVS were studied. All the patients presented fever and dyspnea as well as thrombocytopenia and hypoxemia. Tachycardia, malaise, hypotension and lung rales occurred in 75 to 87.5% of the cases. Hemoconcentration, blood cell count increased and immature neutrophils, and high levels of creatinine were observed in 75 to 87.5%. Intravenous liquid infusion, the use of drugs for increasing systemic vascular resistance and inotropism, and mechanic ventilation were used for the patients. Mechanical ventilation and volume administration should be started precociously, preferable in intensive care units employing recommended universal and respiratory precautions. Careful volume administration should be limited if signs of pulmonary edema develop. Mortality (50%) is high and probably related to the severity of the disease as well as to a delayed attending of the patients for intensive management. It is important to report hantaviruses and HPCVS to the Brazilian medical community considering that many cases could be undiagnosed.

A síndrome pulmonar e cardiovascular por Hantavirus (SPCVH), é doença emergente com descrição crescente de casos no Brasil. Neste trabalho, estudou-se 8 casos confirmados da doença. Todos apresentaram febre e dispnéia. Taquicardia, astenia, hipotensão e estertoração pulmonar ocorreram em 75 a 87,5% dos casos. Plaquetopenia e hipoxemia ocorreram em 100% dos casos, hemoconcentração, leucocitose com desvio à esquerda e elevação de uréia e creatinina séricas em 75 a 87,5%. Assistência respiratória, hidratação endovenosa e utilização de aminas vasoativas foram as medidas utilizadas nos pacientes. Ressalta-se que o suporte ventilatório e cardiovascular deve ser precocemente instituído, preferencialmente em unidades de terapia intensiva, com precauções universais e respiratórias de isolamento. Deve-se ter cuidados com infusão excessiva de líquidos para não agravar o edema pulmonar. A mortalidade observada, de 50%, é elevada, deveu-se à gravidade da doença e ao comparecimento tardio para tratamento intensivo. Deve-se informar sobre a SPCVH aos profissionais de saúde, considerando que casos de SPCVH, provavelmente, vêm passando desapercebidos.

Cytomegalovirus infection in renal transplant recipients diagnosed by nested-PCR

A prospective study of cytomegalovirus (CMV) infection was carried out on 34 renal transplant recipients managed at a General Hospital in Ribeirão Preto, SP, Brazil. Serologic tests showed that all patients were infected with CMV before renal transplantation. Two nested-PCR techniques with primers that recognize sequences of the glycoprotein B (gB) and H (gH) genes were used for CMV detection in blood and urine samples during the post-transplantation period. CMV was detected more frequently in blood samples than in urine samples (P<0.001). Thirty-three patients had CMV detected at least once in blood and/or urine samples. Seven of these patients (21.2 percent) were diagnosed as having symptomatic CMV infection and showed a worse clinical outcome, with a higher death rate (P = 0.03). No association between CMV viremia and graft rejection was observed. Nested-PCR was not useful to identify patients at risk for symptomatic CMV infection since only 21.2 percent of the patients with CMV infection were symptomatic

Detection of cytomegalovirus infections by PCR in renal transplant patients

Cytomegalovirus (CMV) is the single most important infectious agent affecting recipients of organ transplants. To evaluate the incidence and the clinical importance of CMV infection in renal transplants in Brazil, 37 patients submitted to renal allograft transplants were tested periodically for the presence of cytomegalovirus DNA in urine using the polymerase chain reaction (PCR), and for the presence of IgM and IgG antibodies against CMV by enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescence (IIF). The PCR-amplified products were detected by gel electrophoresis and confirmed by dot-blot hybridization with oligonucleotide probes. Thirty-two of the 37 patients (86.4 percent) were positive by at least one of the three methods. In six patients, PCR was the only test which detected the probable CMV infection. Ten patients had a positive result by PCR before transplantation. In general, the diagnosis was achieved earlier by PCR than by serologic tests. Active infection occurred more frequently during the first four months after transplantation. Sixteen of the 32 patients (50 percent) with active CMV infection presented clinical symptoms consistent with CMV infection. Five patients without evidence of active CMV infection by the three tests had only minor clinical manifestations during follow-up. Our results indicate that PCR is a highly sensitive procedure for the early detection of CMV infection and that CMV infection in renal transplant patients is a frequent problem in Brazil

Diagnóstico de infecção congênita e perinatal por citomegalovírus utilizando a reação em cadeia da polimerase / Diagnosis of congenital and perinatal infection by cytomegalovirus using polymerase chain reaction

Aplicou-se uma reação em cadeia da polimerase (PCR) no diagnóstico de infecção congênita e perinatal por citomegalovirus, comparando-a com a técnica de isolamento viral em cultura celular. Foram processadas 305 amostras de urina de crianças de 0 a 6 meses, por ambas as técnicas. Utilizou-se na PCR os primers que amplificam parte do gene codificador do principal antígeno precoce imediato de CMV. Detectou-se virúria em 47 amostras por PCR e comparando os resultados com aqueles obtidos pelo isolamento viral, observou-se copositividade de 89,6% e conegatividade de 98,5%. Estas amostras positivas tiveram o resultado confirmado por PCR utilizando outros primers que amplificam regiões dos genes codificadores das glicoproteínas B e H de CMV. O diagnóstico de infecção congênita e perinatal por CMV pela PCR mostrou sensibilidade comparável à do isolamento viral e o uso de vários primers conferiu alta especificidade ao teste.

The practical application of a polymerase chain reaction (PCR) amplification for the diagnosis of congenital and perinatal cytomegalovirus (CMV) infections was evaluated. Three hundred five urine samples were tested by PCR and conventional virus isolation in cell culture. Viruria was detected in 47 urine samples by PCR using a primer pair which amplifies part of the major immediate-early (MIE) CMV genome. The PCR compared to virus isolation showed 89.6% sensitivity, 98.5% specificity and 91.5% positive predictive value. PCR with primer pairs amplifying parts of the glycoprotein B and glycoprotein H genes of CMV were used for confirmation of the positivity of the 47 urine samples. We concluded that this CMV PCR assay in urine has a suitable sensitivity for the diagnosis of congenital and perinatal infections and its specificity is highly increased by use of more than one pair of primers among the ones we used.

Donated organs as a source of cytomegalovirus (CMV) in renal transplant patients

Two patients receiving the same cadaver kidney graft were investigated prospectively for cytomegalovirus (CMV) infection using the polymerase chain reaction (PCR) and serologic tests (ELISA and IFI). The data indicate that a strain of CMV was probably transmitted from the same donor to both kidney recipients including one who was seropositive for CMV