Abstract:
The Hantavirinae encompass a large number of species which are distributed worldwide. Although hantaviruses generally reside in murine hosts, they are also the causative agents of a number of human diseases including hantavirus pulmonary syndrome (HPS) and hemorrhagic fever with renal syndrome (HFRS). Tissue samples were obtained from hantavirus-infected rodents (e.g.,  Oligoryzomys microtis ) and subjected to reverse transcription-polymerase chain reaction (RT-PCR) analysis to amplify hantaviral-specific nucleic acids. A molecular clone encoding the complete nucleocapsid (N) protein of the Rio Mamore Virus (RMV) was obtained and used to express high-levels of protein. The availability of the RMV N protein and its incorporation into immunodiagnostic assays will facilitate the detection of hantavirus-specific antibodies.

Description:
This application is a continuation of Ser. No. 08/673,230 filed Jun. 27, 1996. 
    
    
     BACKGROUND OF THE INVENTION 
     1. Field of Art 
     Hantaviruses 
     Hantaviruses are a group of at least 19 diverse agents. They occur worldwide in rodent hosts and cause either (1) no known human disease; (2) hemorrhagic fever with renal syndrome, HFRS, or (3) Hantavirus pulmonary syndrome (HPS) when transmitted to man. The following is a compilation of currently recognized types; there is not universal agreement among workers in the field as to the degree to which each is clearly distinct from each other: 
     
       
         
               
               
               
               
               
               
             
           
               
                   
               
               
                 Virus 
                 Abbreviation 
                 Synonyms 
                 Host 
                 Distribution of Host 
                 Disease 
               
               
                   
               
             
             
               
                 Hantaan 
                 HTN 
                   
                 
                   A. agrarius 
                 
                 Central &amp; E Asia, Central &amp; E Europe 
                 HFRS 
               
               
                 Seoul 
                 SEO 
                 Baltimore rat 
                 
                   R. norvegicus, R. 
                 
                 Worldwide; commensal rat hosts 
                 HFRS 
               
               
                   
                   
                 virus; many others 
                 
                   rattus 
                 
               
               
                 Dobrava/Belgrade 
                 DOB 
                 BEL 
                 
                   A. flavicollis 
                 
                 Asia Minor Europe, Palestine 
                 HFRS 
               
               
                 Puumala 
                 PUU 
                   
                 
                   C. glareolus 
                 
                 Russia, Europe, Asia Minor 
                 HFRS 
               
               
                 Sin Nombre 
                 SN 
                 Four Corners; 
                 
                   P. maniculatus 
                 
                 Throughout US, W Canada 
                 HPS 
               
               
                   
                   
                 Muerto Canyon; 
               
               
                   
                   
                 Convict Creek 
               
               
                 Black Creek Canal 
                 BCC 
                   
                 
                   S. hispidus 
                 
                 SE US to Peru 
                 HPS 
               
               
                 Muleshoe 
                 MULE 
                   
                 
                   S. hispidus 
                 
                 W. Texas 
                 unknown 
               
               
                 Monongahela 
                 MON 
                   
                 
                   P. maniculatus 
                 
                 Appalachians, W. Va. to New York 
                 unknown 
               
               
                 New York 
                 NY 
                 SI-1 
                 
                   P. leucopus 
                 
                 NE US, SE Canada 
                 HPS 
               
               
                 Bayou 
                 BAY 
                   
                 
                   O. palustris 
                 
                 SE US, Kansas to New Jersey 
                 HPS 
               
               
                 Thottapalayam 
                 TPM 
                   
                 
                   S. murinus 
                 
                 Africa, India, SE Asia 
                 unknown 
               
               
                 Tula 
                 TUL 
                   
                 
                   M. arvalis 
                 
                 Russia, Europe, Asia Minor 
                 unknown 
               
               
                 Thai 
                 THAI 
                   
                 
                   B. indica 
                 
                 SE Asia, India 
                 unknown 
               
               
                 Prospect Hill 
                 PH 
                   
                 
                   M. pennsylvanicus 
                 
                 N, E US, Canada, Alaska 
                 unknown 
               
               
                 Bloodland Lake 
                 BLLL 
                 PVV 
                 
                   M. ochrogaster 
                 
                 Midwestern, E US, S Canada 
                 unknown 
               
               
                 Khabarovsk 
                 KBR 
                   
                 
                   M. fortis 
                 
                 E Russia 
                 unknown 
               
               
                 Isla Vista 
                 ILV 
                 CMMV 
                 
                   M. californicus 
                 
                 California, Oregon, Mexico 
                 unknown 
               
               
                 El Moro Canyon 
                 ELMC 
                 HMV-1 
                 
                   R. megalotis 
                 
                 W US, Mexico, SW Canada 
                 unknown 
               
               
                 Rio Segundo 
                 RIOS 
                 HMV-2 
                   R. mexicanus ? 
                 Mexico, Costa Rica, Ecuador 
                 unknown 
               
               
                 Rio Mamoré 
                 RM 
                   
                 Oligoryzomysmicrotis 
                 Bollvia, Brazil, Paraguay, Peru, 
                 unknown 
               
               
                   
                   
                   
                   
                 Argentina 
               
               
                   
               
             
          
         
       
     
     Since there are so many distinct species of Hantaviruses, there is no single test or single reagent that allows the diagnosis of all hantavirus infection. In each case, the best reagents for detection of antibodies to a given hantavirus are those which are based upon the hantavirus species that actually caused the infection. The nucleocapsid (N) protein is the portion of each hantavirus that is most strongly immunogenic, and the standard for diagnosis of Hantaviruses has increasingly been to rely upon the expression of homologous N protein in bacteria or other microbial expression system to generate high concentrations of recombinant-expressed antigen. Classical methods of viral antibody detection have depended upon the growth of the virus in culture, with use of the viral antigens from infected cultures in immunologic detection, but these methods are increasingly falling out of favor for a variety of technical and practical reasons. 
     2. Discussion of Related Art 
     Specific diagnostic tests are available for several previously-described Hantaviruses. For Hantaviruses in general, antibody tests are much preferred over direct detection of infectious viral particles, viral genomic RNA, or viral antigens because of the inherently superior stability, sensitivity, specificity, and ease of transfer of antibody assay technologies. The following modalities are in common use (C) or are under development or research use (D) for the following Hantaviruses: 
     
       
         
               
               
               
               
               
             
           
               
                   
               
               
                   
                 High density particle 
                   
                   
                   
               
               
                 Virus 
                 agglutination 
                 IFA* 
                 ELISA** 
                 Western blot 
               
               
                   
               
             
             
               
                 Hantaan 
                 C 
                 C 
                 C 
                 D 
               
               
                 Seoul 
                 C 
                 C 
                 C 
                 D 
               
               
                 Puumala 
                 C 
                 C 
                 C 
                 C/D 
               
               
                 Sln Nombre 
                   
                 D 
                 C 
                 C 
               
               
                 Dobrava 
                   
                 D 
                 D 
               
               
                   
               
               
                 *IFA, immunofluorescence assay  
               
               
                 **ELISA, enzyme-linked immunosorbent assay. Both native (cultured virus) and recombinant-expressed antigens are used.  
               
             
          
         
       
     
     Because none of the prototype Hantaviruses listed above occurs in rodents with distribution in South America, it is virtually certain that human Hantavirus disease in South America is due to novel virus(es) that will be detected in a less-than-optimal manner by tests that utilize antigens derived from prototype species. These virus(es) are almost certainly associated with indigenous rodents of the subfamily Sigmodontinae, family Muridae, because the clinical disease that has been noted in Brazilian, Argentinean, and Paraguayan patents is closely similar to those diseases caused by North American Hantaviruses of sigmodontine rodents. Detection of Hantavirus infection in South America has relied most heavily upon cross-reactivity between the prototypic sigmodontine rodent-borne Hantavirus Sin Nombre (Four Corners) virus (SNV) and the South American virus(es). 
     SUMMARY OF THE DISCLOSURE 
     The invention provides a molecular clone encoding and expressing the complete nucleotide protein of Rio Mamoré virus. The RMV N protein includes antigenically active domains useful in immunoassays for detecting South American Hantavirus infection, and in vaccines. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     FIG.  1 . Consists of FIGS. 1A through 1E. FIG. 1E is a dried polyacrylamide gel containing 4 different protein preparations. The first lane (“C”) is a crude lysate of  E. coli  JM101 cells after induction of expression from the pET23b vector that lacks a viral genetic insert. The next 3 lanes contain the pET23b-expressed, affinity-purified viral N proteins from Bayou (BAY), Rio Mamoré (RM) or Sin Nombre (SN)Hantaviruses. 4 western blot membranes containing the same purified proteins are at left. Panels (clockwise from upper left) were probed with serum of (FIG. 1A) an RMV-infected  Oligoryzomys microtus  mouse; (FIG. 1B) a BAYV-infected rice rat ( Oryzomys palustris ) from Texas; (FIG. 1C) an uninfected deer mouse ( Peromyscus maniculatus ); (FIG. 1D) an SNV-infected deer mouse ( Peromyscus maniculatus ) from Texas. Antibodies were detected with an alkaline phosphatase-conjugated goat anti  P. leucopus  reagent, followed by exposure to alkaline phosphatase substrate as described (Jenison et al., 1994). The dark bands in the BAY, RMV, and SNV lanes indicate the presence of antigenically-active N protein that reacts with rodent serum. All N proteins are about 55 kD in apparent molecular weight Differential reactivity is evident in each panel, in each case stronger reactivity is evident against the homologous viral antigen. 
     FIG.  2 . Consists of FIGS. 2A through 2E. The complete sequence of the S genomic segment (SEQ ID NO: 12) and associated N protein (SEQ ID NO: 13) of Rio Mamoré virus, specimen OM NK 13556. 
     FIG.  3 . Maximum-parsimony phylogenetic tree showing position of RMV (RM-OM556) relative to prototypical Hantaviruses BAY (Bayou), SNV (SN), Hantaan (HTN, Puumala (PUU), (Isla Vista (ILV), Tula (TUL), Bloodland Lake (BLLL), New York (N.Y.), El Moro Canyon (ELMC), Black Creek Canal (BCC), Rio Segundo (RIOS), Prospect Hill (PH) and Muleshoe (MULE). The tree is based upon the entire N gene sequences (1287-nt). 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     Medically-important Hantaviruses generally are associated with ecologically dominant murid rodent species. Oryzomine rodents were selected as potentially important vectors of human Hantavirus disease in South America because (1) they are abundant, widespread, and occur in high density; (2) they favor disturbed habitat such as houses and other human habitations; (3) was recently identified as a North American oryzomine rodent,  Oryzomys palustris  is the host for an etiologic agent of HPS (Bayou virus) in Louisiana and Texas. Accordingly, tissue specimens of candidate oryzomine rodent hosts were obtained from the Museum of Southwestern Biology (University of New Mexico) and from the Museum of Vertebrate Zoology (University of California, Berkeley). These included: 
     
       
         
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                   
               
               
                 Date Captured 
                 Jurisdiction 
                 Country 
                 Species 
                 tested 
                 positive 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1994 
                 Bollvar Province 
                 Ecuador 
                 
                   Microryzomys sp. 
                 
                 1 
                 0 
               
               
                 1994 
                 Bollvar Province 
                 Ecuador 
                 
                   Oryzomys albigularis 
                 
                 12 
                 0 
               
               
                 1991 
                 Amazonas State 
                 Brazil 
                 
                   Oryzomys capito 
                 
                 20 
                 0 
               
               
                 1991-1992 
                 Acre and Amazonas States 
                 Brazil 
                 
                   Oryzomys yunganus 
                 
                 29 
                 0 
               
               
                 1991 
                 Amazonas State 
                 Brazil 
                 
                   Oligoryzomys microtis 
                 
                 32 
                 0 
               
               
                 1991 
                 Acre and Amazonas State 
                 Brazil 
                 
                   Oecomys bicolor 
                 
                 6 
                 0 
               
               
                 1991 
                 Acre and Amazonas States 
                 Brazil 
                 
                   Oecomys roberti 
                 
                 13 
                 0 
               
               
                 1977-79 
                 Amazonas Dept. 
                 Peru 
                 
                   Oryzomys capito 
                 
                 14 
                 0 
               
               
                 1977-79 
                 Amazonas Dept. 
                 Peru 
                 
                   Nectomys squamlpes 
                 
                 2 
                 0 
               
               
                 1977-79 
                 Amazonas Dept. 
                 Peru 
                 
                   Oecomys bicolor 
                 
                 15 
                 0 
               
               
                 1985 
                 Beni Dept. 
                 Bollvia 
                 
                   Oligoryzomys microtis 
                 
                 10 
                 3 
               
               
                 1985, 1992 
                 La Paz Dept. 
                 Bollvia 
                 
                   Oligoryzomys microtis 
                 
                 12 
                 2 
               
               
                 1984-85 
                 Santa Cruz Dept 
                 Bollvia 
                 
                   Oligoryzomys microtis 
                 
                 13 
                 0 
               
               
                 1984, 1991 
                 Santa Cruz and Tarija Depts. 
                 Bollvia 
                 
                   Oligoryzomys flavescens 
                 
                 9 
                 0 
               
               
                 1990-91 
                 Santa Cruz, Chuquisaca, and Tarija 
                 Etquitsia 
                 
                   Oligoryzomys chacoensis 
                 
                 5 
                 0 
               
               
                   
                   
                   
                 Total 
                 192 
                 5 
               
               
                   
               
             
          
         
       
     
     Briefly, the supernatants of these tissue (kidney, heart, and/or liver) samples were screened for Hantavirus antibodies using a recombinant western blot assay for antibodies reactive with SNV (see PCT/US94/09416). The antigen comprised the recombinant-expressed TrpE-SNV N fusion protein, which was transferred to a nitrocellulose membrane as described in PCT/US94109416 (Jenison S, Yamada T, Morris C, Anderson B, Torrez-Martinez N, Keller N, Hjelle B., “Characterization of human antibody responses to Four Corners Hantavirus infections among patients with hantavirus pulmonary syndrome.”  J Virol  1994; 68:3000-6). The membrane strips were rocked overnight at 4° in a bath containing 5% dry milk in phosphate-buffered saline and a 1:200 dilution of tissue sample supernatant (as source of antibodies). Bound antibodies were detected with a secondary antibody consisting of alkaline phosphatase-conjugated goat anti- Peromyscus leucopus  IgG. 
     Of the above specimens, only 5 (all  Oligowyzomys microtis  from Bolivia, collected in 1985) were positive for Hantavirus antibodies. These tissue samples were used to prepare RNA, and the RNA was then subjected to reverse transcription-polymerase chain reaction (RT-PCR) analysis to identify the virus and to prepare recombinant antigens through expression of the PCR-amplified DNA in molecular clones. 
     The availability of a molecular clone encoding the complete N protein of Rio Mamoré Virus (RMV) makes it possible to detect Hantavirus infection in South American people and rodents with homologous antigens for the first time. The high-level expression of the N protein allows incorporation into a variety of antibody testing formats to produce the most efficient and accurate diagnostic tests. The RMV N protein is antigenically active in western blot and ELISA formats. Additional conventional formats such as immunofluorescence assay, particle agglutination and radioimmunoprecipitation assays are contemplated. 
     Serologic (antibody) tests to screen for, or confirm, the presence of antibodies to RMVN protein, whether in humans, rodents, or other animals are described herein. The western blot and ELISA assays have been reduced to practice, and other methods are readily adapted from these procedures (given the purified antigen) by trivial manipulations. Although many different configurations are within the scope of the invention, an ELISA system in which a microtiter well is first coated with goat IgG directed against human IgM is of particular interest. The well is then treated with the serum of a patient with suspected RMV infection, washed, then treated with purified recombinant antigen. After washing, a biotin-labeled rabbit antibody directed against the recombinant antigen of RMV (see below) is applied. Finally a streptavidin-conjugated alkaline phosphatase is used to detect the bound biotin. A chromogenic alkaline phosphatase substrate is used to detect bound alkaline phosphatase. 
     Even in the event that laboratory is able to grow RMV in culture, and develop an ELISA based upon the native antigens of the culture-adapted virus, rDNA-derived antigens will continue to be useful diagnostic tools. In many cases rDNA antigens provide valuable supplementary information to that provided by ELISAs using cultured virus as antigen; in some cases, rDNA antigens have superior sensitivity or superior ability to differentiate among antibodies directed against different related viruses, or ability to diagnose infection by viruses for which there is no method of culture. At present, virtually all North American HPS virus infections are diagnosed with recombinant antigen-based systems. 
     I. Experimental Methods 
     Design of PCR Primers. 
     Reaction Mixes and Conditions: Reverse Transcription and “First Round” PCR. 
     The initial reaction mixes (for reverse transcription and subsequent PCR thermal cycling) were as follows. All mixtures contained 10 pmol of each primer; 1.7 mM 2-mercaptoethanol; 1.5 mM MgCl 2 ; 10 mM Tris-HCl (pH 8.3); 50 mM KCl; 200 uM each of dATP, dTTP, dGTP, and dCTP; 10 units of AMV reverse transcriptase (Boehringer-Mannheim), and 2.5 units of AmpliTaq™ DNA polymerase (Perkin-Elmer), in a final volume of 100 ul. After addition of all reagents to a 0.6 ml Eppendorf tube, the tubes were overlaid with 3 drops of mineral oil (Perkin-Elmer), and placed in a thermal cycler. Each tube was warmed to 42° C. for 1 h, then subjected to temperature cycling of 94°-40°-72° for 1 minute, 1 minute, and 3 minutes per cycle for 8 cycles, then 94-45-72° (1 minute, 1 minute, and 3 minutes) for 29 more cycles. 
     “Second Round” PCR. 
     After the initial amplification described above, all of the samples were subjected to some form of “nesting” PCR reaction, in which the amplified product was further amplified by using primers internal to those used in the first round of amplification. Fifty pmol of each “second round” primer was used; reaction ingredients included 3 ul of the first-round PCR product, and the same ingredients as those in the first round (except no 2-mercaptoethanol or reverse transcriptase was added): 
     The “second round” PCR product was prepared by thermal cycling at 94-40-72 (1 minute, 1 minute, and 3 minutes, respectively) for 8 cycles, followed by 29 more cycles at 94-42-72 (1 minute, 1 minute, and 3 minutes, respectively). The reaction was then subjected to an elongation step of 70° for 10 minutes. The DNA product was then loaded on an 1.2%-1.6% agarose gel, electrophoresed for 1 h at 80V, and the band of the appropriate molecular weight was then excised with a razor blade. The DNA was extracted from the gel with a glass-milk resin (Qiaex resin, Qiagen Inc.) after melting the gel in a sodium iodide solution. After washing the resin (according to the instructions for Qiaex resin), the PCR product was taken up in 10 ul of (10 mM Tris-HCl, pH 8/1 mM EDTA), and 5-10 ul was ligated to the pCRII vector according to the manufacturer&#39;s instructions (Invitrogen Corp.). One ul of the 10 ul ligation mix was used to transform  E. coli  cells according to the manufacturer&#39;s instructions (Invitrogen), and the transformed cells plated onto LB media containing 50 ug/mi ampicillin and 0.005% X-Gal; plates were incubated at 37° overnight. Clear colonies were selected from the plate the following morning and expanded in 4 ml of LB media containing 50 ug/ml ampicillin. 
     All PCR primers were designed either as consensus primers (conserved portions of other Hantavirus S segments that were predicted to be conserved in RMV) or by directly reading the sequences of RMV clones and designing primers from those sequences. 
     Primers (S genomic segment) successfully used in amplifying RMV cDNAs: 
     
       
         
               
               
               
               
             
           
               
                   
               
               
                 Primer number (primer 
                   
                   
                   
               
               
                 type) 
                 Coordinate (S segment) 
                 Sequence (5′-3′) 
               
               
                   
               
             
             
               
                 1 (C*) 
                 1 sense 
                 TAG TAG TAG ACT CCT TGA GAA GCT AC 
                 (SEQ ID NO:1) 
               
               
                   
               
               
                 2 (RMV) 
                 25 sense 
                 ACT ACT GCA TAT GCT GGT ATG AG 
                 (SEQ ID NO:2) 
               
               
                   
               
               
                 3 (RMV) 
                 1688 antisense 
                 TCT ATG ACT TAA CAC TAT ATG GAT C 
                 (SEQ ID NO:3) 
               
               
                   
               
               
                 4 (RMV) 
                 43 sense 
                 T AAG CTT ATG AGC MC CTC CAA GAA GTA CAA GA 
                 (SEQ ID NO:4) 
               
               
                   
               
               
                 5 (RMV) 
                 1326 antisense 
                 C CTC GAG CAA TTT CAA TGG CTC TTG GTT TGA 
                 (SEQ ID NO:5) 
               
               
                   
               
               
                 6 (RMV) 
                 997 sense 
                 CTT TAT GTT GCA GG(AT) GT(TA) CCT GA 
                 (SEQ ID NO:6) 
               
               
                   
               
               
                 7 (RMV) 
                 1039 sense 
                 ATC CTG CAG GA(CT) ATG (CA)GA AAT AC 
                 (SEQ ID NO:7) 
               
               
                   
               
               
                 8 © 
                 184 sense 
                 CGG GCA GCT GTG TCT GCA TTG GA 
                 (SEQ ID NO:8) 
               
               
                   
               
               
                 9 © 
                 626 antisense 
                 GG TGT GAT TTC ATC TGC (C/T) TT CAT 
                 (SEQ ID NO:9) 
               
               
                   
               
               
                 10 © 
                 1085 antisense 
                 CC TAC AGA CTT TGA TGC CAT (GAT) AT 
                 (SEQ ID NO:10) 
               
               
                   
               
               
                 11 © 
                 1975 antisense 
                 TAG TAG TAT ACT CCT TGA AAA GCA A 
                 (SEQ ID NO:11) 
               
               
                   
               
               
                 *(C)= consensus hantavirus primer; (RMV) = primer designed from empirically-determined sequences of RMV sample OM NK 13556. Bolded sequences in primers 4 and 5 represent restriction endonuclease sites that were introduced as the primers were designed. All coordinates refer the location of the 5′-most residue of the primer; each primer is written in 5′-3′ direction.  
               
             
          
         
       
     
     The following primer combinations were used in cloning the RMV S genome, in the indicated order: 
     1. Primers 1 and 10, nested with primers 8 and 9, yielding a 442-nt product. Tissue RNAs from five seropositive  O. microtus  were used in this round, and all were positive. Since specimen OM NK 13556 (from Beni Department, Bolivia) produced the strongest amplification signal, it was used exclusively in further amplification reactions, and represents the prototype RMV S segment sequence. 
     2. Primers 1 and 10, heminested with primers 8 and 10, yielding a 901-nt product The sequence of this product was used to synthesize primers 6 and 7. 
     3. Primers 6 and 11, heminested with primers 7 and 11, yielding a 939-nt product, spanning the 3′ end of the gene. 
     4. Primers 1 and 10, heminested with primers 1 and 9, yielding a 626-nt product that spanned the 5′ end of the gene. 
     5. The intact N gene open reading frame was then cloned by amplifying with primers 2 and 3, followed by nesting with primers 4 and 5, yielding a 1287-nt product. 
     Plasmid DNA was prepared from the cultures according to standard methods, and then digested with various restriction enzymes to verify that the correct insert had been obtained. Clones that appeared to have the correct restriction enzyme digestion pattern were subjected to DNA sequencing (according to the manufacturer of the Sequenase™ sequencing system, US Biochemicals) to verify that DNA with characteristics appropriate for a novel Hantavirus had been amplified and cloned. The DNA was examined for strong homology to (but not identity to) previously-described Hantaviruses SNV and Bayou virus, and relative conservation of the protein product that would be predicted from the nucleotide sequence. 
     II. Expression of the RMV N Protein 
     The complete N gene open reading frame was excised from the cloning vector pCRII, and subcloned at the Hind III and Xho I restriction sites (which were incorporated into the PCR primers 2 and 3). The gene was transferred into the pET 23b vector (Novagen Inc.) at the Hind III and Xho I sites and expanded in  E. coli  strain BL21, wherein the gene is not expressed. This vector places the N gene in fusion with a small leader genetic element derived from the T7 bacteriophage. Correct clones were identified by restriction endonuclease digestion and DNA sequencing. The clone was then transfected into the expression-competent cell, BL21 (DE3), where N gene expression was induced with IPTG according to the manufacturers instructions. 
     Induction with IPTG resulted in the high-level production of a band at approximately 55,000 Daltons apparent molecularweight by SDS-polyacrylamide gel electrophoresis. A band of identical size was produced with a control induction that results in the expression of a T7-Bayou Hantavirus N protein, and a somewhat larger band was produced by expression of the Sin Nombre virus N protein as 2 Trp E fusion partner (the larger size of the SNV N protein is explained by the larger size of the Trp E fusion partner relative to the T7 fusion partner). The proteins on the SDS gel were transferred to a nitrocellulose membrane by western blotting (Jenison S, Yamada T, Morris C, Anderson B, Torrez-Martinez N, Keller N, Hjelle B. Characterization of human antibody responses to Four Comers Hantavirus infections among patients with Hantavirus pulmonary syndrome.  J Virol  1994; 68:3000-6). To verify that the RMV N protein produced in this manner is antigenically active, the western blot was probed with serum from a patient with SNV-induced HPS, and all three N proteins (RMV, BAYV, and SNV) were strongly reactive. By contrast, a lane in which the pET 23b vector alone was used in induction experiments yielded no immunoreactive bands, and a serum from a patient without HPS also produced no. bands. 
     The use of the pET 23b expression system is designed to allow the ready purification of recombinant antigens for preparation of ELISAs and “slot-blot” format assays such as the RIBA™ (Chiron Corporation). These assays require that the antigen be extremely pure, because they are subject to false reactivities that can be associated with even small amounts of contaminating  E coli  antigens. The purification of the recombinant proteins is facilitated by the presence in the pET 23b of a genetic segment that encodes a “histidine tag”, ie, a series of 6 histidine residues in tandem. This peptide tag has high affinity for certain metal ions (Cesium, Nickel) that can be incorporated into an affinity column. The Novagen Corporation sells such columns as kits for purification of recombinant proteins. These columns were purchased and found to work very well for purification of the RMV N protein. The purified N protein was placed on western blots and in microtiter wells, and probed with serum from animals and humans with and without Hantavirus infection (FIG.  1 ). Strong immunoreactivity of the RMV N protein was evident in these studies, indicating that it is useful in detection of Hantavirus antibodies. 
     III. Preparation of Recombinant Antigens. II. Use in Development of Antiviral Antibodies 
     After expressing recombinant proteins in  E coli  or baculovirus systems from our rDNA RMV clones, the proteins are purified and used to immunize rabbit, mica and/or other animals by injection. The resultant antibodies (both polyclonal and monoclonal) are useful in diagnosis, treatment, or prophylaxis of RMV infection in the following ways. (1) As a method for amplification of any specific signal in an ELISA system (such as that described above) designed to detect antiviral antibodies in humans or animals; (2) as a reagent for detecting viral antigens, either in tissue samples or peripheral blood of humans or animals with possible RMV infection, or in cell cultures; (3) as potential sources of passive immunizations of humans with exposure to, or disease caused by, RMV. Passive antibodies are routinely used as prophylaxis after exposure to viruses such as rabies and hepatitis B virus. 
     IV. Preparation of Recombinant Antigens. III. Use in Development of Vaccines 
     Molecular clones encoding a majority of the antigenic domains of RMV are potentially important vaccination reagents. While many possible approaches can be foreseen as a means of exploiting these reagents, the following are particularly contemplated. (1) The RMV N antigen is expressed in cultured cells under the control of a vaccinia or other heterologous virus&#39; replication machinery, and used to prepare live or killed-virus vaccinia antigens. (2) The RMV DNA is used as a substrate for “naked DNA vaccines”, i.e., immunization by injection of purified RMV DNA intramuscularly into humans or animals. (3) Purified RMV N protein, expressed in baculovirus, yeast, or  E. coli , is injected into humans or animals in combination with a customary pharmacological carrier to allow development of an immune response and protective immunity. 
     V. Development of a PCR Diagnosis for RMV 
     Alternative reverse transcriptase-PCR-based systems for diagnosis of RMV in humans and animals may be used. While one system has been described herein, we expect that the following goals for improved diagnostic PCR may be met by using a system of this design. (1) A PCR system with increased sensitivity, made possible by choosing primer pairs designed solely from RMV sequences, as opposed to primers designed on the basis of conservation of sequences of related viruses. (2) A system with greater potential for decontamination, made possible by choosing a larger PCR target (such as 300 nt). Such a modification should make the system less prone to false positive tests. With the sequence of the entire RMV S genome in hand, many primer sets for the virus are possible. 
     PCR can be conducted quantitatively. It is expected that successful treatment of RMV infection will result in a rapid drop in the level of circulating virus during the course of a patient&#39;s treatment. We intend to explore that possibility, with the intent of developing a routine method for monitoring the efficacy of various therapies for HPS. 
     VI. Use of RMV Clones for in Situ Hybridization 
     The availability of the RMV S genome sequence allows the preparation of specific probes for detection of RMV RNA in cell culture, in human and animal tissues, and in human blood cells. Specific probes can be made to either strand of the virus. Plus strand probes, corresponding to the mRNA of the virus, should be useful in detecting RMV (a minus-strand virus), wherever it occurs. Minus strand probes should be useful in detecting the antigenomic strand and mRNA of RMV, which would occur only in tissues in which the virus is replicating. In situ hybridization has great potential as a research tool for understanding the replication of the virus, and some potential as a diagnostic, prognostic, or therapeutic tool if it proves able to detect replicating and non-replicating RMV in patient blood specimens, human or animal tissues, or viral cultures. This method is developed by expressing radio labeled RNA from DNA clones of the RMV in vitro, and using the RNA probes as reagents for in situ hybridization studies of infected tissues. 
     In the following claims, the claimed nucleotide or peptide sequences which are substantially equivalent in structure and function.