Table 1

The primers of plasmid constraction and fragment length"

Fig. 1

Construction diagram of pTH-B1 plasmid A, B, C, D indicate the fragments range to viral cDNA; N, P, M, F, M2, SH, G, L represent viral genes; T7: T7 RNA polymerase transcriptional promoter; HdvRz&T7ter. Hepatitis delta virus ribozyme and T7 RNA polymerase transcriptional terminator; Mlu Ⅰ, Xho Ⅰ, Kpn Ⅰ, Not Ⅰ. The specific restriction enzyme site contained in the plasmid, the number in parentheses is the position of the enzyme restriction site in the pTH-B1 plasmid; . Genetic marker"

Fig. 2

Construction diagram of pTH-B1EGFP plasmid M, F. The viral genes of M and F; NGS. The transcriptional initiation sequence of N gene; NGE. The transcriptional termination sequence of N gene; Pme Ⅰ. Cleavage site; EGFP. Enhanced green fluorescent protein gene"

Fig. 3

Construction diagram of two auxiliary plasmids and mini-genome plasmid A. Auxiliary plasmid pCI-NPM2.1 (CMV. Human cytomegalovirus RNA transcription promoter; SV40 polyA. RNA transcriptional termination and polyadenylation signal sequence); B. Helper plasmid pCI-L; C. Mini-genome plasmid (Trailer. 5′untranslated regulatory sequence of the genome of strain B1; Leader. 3′untranslated regulatory sequence of the genome of strain B1; GE. Transcriptional termination sequence of L gene; GS. Transcriptional initiation sequence of N gene)"

Table 2

Detection primers and fragment length"

Fig. 4

The amplification results of B1 genome In Fig. A, 1. 3′RACE amplification band; 2. 5′RACE amplification band; M. DL5000 bp DNA marker. In Fig.B, 1-5. 1-5 amplification band; M. DL 5000 bp DNA marker"

Fig. 5

The aa comparison result of G protein at carboxyl terminal "." show the same aa; "-" show the missing aa; the red box shows the different aa site"

Fig. 6

The comparison of different intergenic region of subtype B aMPV strains A. The intergenic region of M-F gene; B. The intergenic region of M2-SH gene; C. The intergenic region of SH-G gene; D. The intergenic region of G-L gene; E. The 5′untranslated region of viral genome; "." shows the same base; "-" shows the missing base; the red boxes show the different nucleic acids"

Fig. 7

The identification results of genome plasmid A. The pTH-B1 plasmid (1. XhoⅠ-NotⅠ; 2. MluⅠ-KpnⅠ; 3. MluⅠ-NotⅠ; M. DL15 kb DNA marker); B. The pTH-B1EGFP plasmid (1. PmeⅠ-NotⅠ; 2. PmeⅠ-MluⅠ; M. DL15 kb DNA marker); C. pTH-B1EGFP plasmid (1-2. PCR detection; M. DL5 000 bp DNA marker)"

Fig. 8

Identification of auxiliary plasmids and mini-genome plasmid A. The pCI-NPM2.1 plasmid (1. PspOMⅠ-EcoR Ⅴ-Kpn I; M. DL15 kb DNA marker); B. The pCI-L plasmid (1. KpnⅠ; M. DL15 kb DNA marker); C. The pTH-miniG plasmid (1. HindⅢ-SalⅠ; M. DL5000 bp DNA marker)"

Fig. 9

Fluorescence observation of the cells transfected with microgenomic plasmid and auxiliary plasmids(200×) A. Transfected cells of pTH-miniG, pCI-NPM2.1 and pCI-L; B. Transfected cells of pTH-miniG; C. Transfected cells of pCI-NPM2.1 and pCI-L; D. Non-transfection cells"

Fig. 10

The observation result of cells infected by rescued strains at120 h post-infection(100×) A. Wild-type B1 strain; B. Rescued rB1 strain; C. Resued rB1-EGFP strain; D. Mock; Arrows indicate syncytium formation"

Fig. 11

Fluorescence observation of the Vero cells infected by rB1-EGFP strain at 72 h post-infection (100×)"

Fig. 12

Identification of rescued strains A. Detection of N gene (1. B1 strain; 2. rB1 strain; 3. rB1-EGFP strain; M. DL2000 bp DNA marker; 4. Negative control; 5. Positive control); B. Detection of Genetic marker (1. SalⅠ enzyme-digested product of B1 strain; 2. SalⅠ enzyme-digested product of rB1 strain; 3. SalⅠ enzyme-digested product of rB1-EGFP strain; M. DL2000 bp DNA marker); C. Sequencing analysis of rB1 genetic marker"

Fig. 13

Indirect immunofluorescence assay(IFA)of rescued strain (200×)"

Fig. 14

Western blot identification of rescue strains M. Page ruler; 1. B1 strain; 2. rB1 strain; 3. rB1-EGFP strain; 4. Negative control"

Fig. 15

Growth curve of the rescued strains (n=3)"