Cloning and sequencing of Puumala virus Sotkamo strain S and M RNA segments: evidence for strain variation in hantaviruses and expression of the nucleocapsid protein

Abstract

The prototype Puumala virus (PV) Sotkamo strain small (S) and medium (M) RNA genome segments were amplified by the polymerase chain reaction (PCR), cloned and sequenced. The S segment is 1830 nucleotides long with an open reading frame coding for 433 amino acids. The identity to the PV H~illn~is strain was 83% at the nucleotide and 96% at the amino acid level. The M segment in the Sotkamo strain is 3616 nucleotides long and contains one open reading frame of 1148 amino acids with 83% nucleotide and 94% amino acid identity to the H/illn~is strain. Most amino acid changes were conservative and the five predicted glycosylation sites are identical. The amino acid identity to the prototype hantavirus, Hantaan virus, was 62 and 54% for S and M segments, respectively. The coding region of the S segment was further amplified by PCR, ligated to pEX vectors and expressed in Escherichia coli as a fl-galactosidase fusion protein and was seen to be specifically detected by nephropathia epidemica sera in immunoblotting.

Human recombinant Puumala virus antibodies: cross-reaction with other hantaviruses and use in diagnostics.

“…The data suggested a fine-tuned specificity of these human Fabs against the infective (homologous) agent and will provide most valuable reagents for characterization of the E.-M. Salonen and others E.-M. Salonen and others rapidly growing number of recognized hantaviruses. The present results also confirm that the human IgG response against N is mainly directed against the amino-terminal region of the protein (aa 1-61) (Vapalahti et al, 1992(Vapalahti et al, , 1995Elgh et al, 1996 ;Lundkvist et al, 1996).…”

“…The library was first panned against recombinant PUU N expressed by using the baculovirus system in Sf9 cells (bac-PUU-N) coated to microtitre plates (10 µg\ml in 0n01 M PBS, pH 7n4). The library was also panned against a β-octylglycoside (0n1 %) lysate of sucrose gradient purified PUU (Sotkamo strain) grown in Vero E6 cells (Schmaljohn et al, 1985 ;Vapalahti et al, 1992). The lysate was diluted 1 : 5 in PBS and coated to the wells of a microtitre plate.…”

Hantaviruses: genome structure, expression and evolution

“…L36929) ; BCC, Black Creek Canal virus (Ravkov et al, 1995;L39949); DOB, Dobrava virus (Avsic-Zupanc et al, 1995;L41916); ELMC, El Moro Canyon virus, strain RM-97 (Hjelle et al, 1994;U11427) ; HTN, Hantaan virus, strain 76118 (Schmaljohn et o/., 1986;M14626) ; ILV, Isla Vista virus, strain MC-SB-1 U31534) ; KBR, Khabarovsk virus, strain MF-43 (H6rling et al, 1996a; U35254) ' , NY, New York virus, strain RI-1 (Hjelle et al, 1995b; U09488) ' , PUU/Sotk, Puumala virus, strain Sotkamo (Vapalahti et al, 1992;X61035) ; PUU/Vind, Puumala virus, strain Vindeln/L20Cg/83 (H6rling et al, 1995; Z48586) ' , PUU/Bash, Puumala virus, strain Bashkiria/CG1820 (Stohwasser et al, 1990;M32750) ; PH, Prospect Hill virus, strain PH-1 (this paper', Z49098) ; SN/H1 O, Sin Nombre virus, strain H10 (Spriopoulou et al, 1994;L25784) ; SN/CC, Sin Nombre virus, strain Convict Creek 107 (Li et al, 1995;L33683) ; RIOS, Rio Segundo virus, strain RMx-Costa-1 (Hjelle et al, 1995a; U18100) ; SEO, Seoul virus, strain SR-11 (Arikawa et al, 1990', M34882) ; TUL/Tula, TUL, strain Tula/76Ma/87 (Plyusnin et al, 1994a; Z30941 ) ' , TUL/Mal, TUG strain Nlalacky/Nla32/94 (Sibold eta/., 1995', Z48235) ; TUL/Nlor, TUG strain Moravia/5286Ma/94 (Plyusnin et aL, 1995b;Z48573). The PHYLIP program package was used to make 500 bootstrap replicates of the sequence data (Seqboot).…”

Development of Novel Immunoglobulin G (IgG), IgA, and IgM Enzyme Immunoassays Based on Recombinant Puumala and Dobrava Hantavirus Nucleocapsid Proteins

“…Viral gene sequences. Expression plasmids contained N-coding sequences of one of the following hantavirus strains: DOBV-Aa, strain East Slovakia-862-Aa (48); HTNV, strain Fojnica (47); PUUV, strain Vranica/Hällnäs (38) or strain Sotkamo (51).…”