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

Abstract

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. In 1993, a new hantavirus disease, hantavirus cardiopulmonary syndrome (HCPS), was described in the Americas associated with wild rodents of the subfamily Sigmodontinae (1).The hantavirus genus of the Bunyaviridae family encompasses a large number of rodent-borne viruses that have a worldwide distribution. These viruses are transmitted mainly by inhalation of aerosols of infected rodent excreta. Hantaviruses are enveloped viruses with three single-stranded segments of negative-sense RNA genome, defined as small (S), medium (M), and large (L). The RNA segments are covered by the nucleocapsid (N) protein forming individual L, M and S nucleocapsids (2). The S segment encodes the N protein, the M segment encodes a glycoprotein precursor that is processed into the enveloped G1 and G2 glycoproteins, and the L segment encodes the viral RNA polymerase. The N protein is synthesized early after infection and is the most abundant viral protein. The encapsidation of viral RNA by N protein subunits is recognized as a central event in the virus replication cycle (3). It is believed that N protein protects newly synthesized viral RNA from nuclease degradation. In addition, the N protein of the ribonucleoprotein core interacts directly with the cytoplasmic tail of the G1 protein of the virus to initiate virion assembly. It has been shown that N protein of some hantaviruses interacts with host cell pro-

Diagnosis of hantavirus infection in humans and rodents in Ribeirão Preto, State of São Paulo, Brazil

“…These include RT-PCR for hantavirus genome detection in clinical samples and ELISA using N recombinant protein from ARAV for use as the antigen in serological diagnostic tests for such infections [6][7][8] . These diagnostic techniques have been useful for diagnosing HPCS among patients in this region 13 .…”

“…Serological tests Human serum was tested by means of IgM-ELISA and IgG-ELISA using a recombinant nucleocapsid protein from ARAV as the antigen, as previously reported by Figueiredo et al 6,7 . The rodent blood was also tested by means of IgG-ELISA using the same technique, but changing to a mixture that included anti-Peromyscus and anti-mouse peroxidase conjugates.…”

Serosurvey of hantavirus infection in humans in the border region between Brazil and Argentina

“…Today, over 1,200 cases of hantavirus pulmonary syndrome have occurred in Brazil since 1993, fatalities were reported at a rate of 39%, according to geographic and ethnic differences 14 . Five hantaviruses associated with HPS are currently known in Brazil: the Juquitiba, Araraquara, Laguna Negra-like, Castelo dos Sonhos and Anajatuba viruses 15 .…”

“…The first death from hantavirus in the state was reported in 2001 and 48 deaths have been reported up to the present, making this the state with the second highest death rate 16 . HPS is an emerging public health problem in Brazil due to the overlap of urban and agricultural areas, livestock and the disorderly growth of human population in areas where imbalance occurs, which increases human contact with several species of rodents that are reservoirs of hantavirus 15 . This study was conducted to investigate the presence of antibodies against hantavirus in the far western region of the State of Santa Catarina and the southern border of Brazil and northern Argentina and correlate this presence through interviews to elucidate the medical history and morbidity of the participants.…”

Prevalence of serum antibodies to hantavirus in a rural population from the southern state of Santa Catarina, Brazil

“…Sera were tested using an indirect IgG ELISA using the N recombinant (rN) protein of the Araraquara hantavirus (ARAV) as the antigen. This technique has been previously described by Figueiredo et al 7 . Briefly, polystyrene microtiter plates (Greiner Bio-one, Germany) were coated overnight in a wet chamber at 4°C with 2μg/mL of ARAV rN protein (lines A to D) or with negative…”