AIM: To characterize the genome of an wild-type HAV isolate (DL3) in China.METHODS: A stool specimen was collected from hepatitis A patient from Dalian, China. HAV (DL3) was isolated and viral RNA was extracted. The g...AIM: To characterize the genome of an wild-type HAV isolate (DL3) in China.METHODS: A stool specimen was collected from hepatitis A patient from Dalian, China. HAV (DL3) was isolated and viral RNA was extracted. The genome of DL3 was amplified by reverse transcription and polymerase chain reaction (RT-PCR),followed by cloning into pGEM-T vector. The positive colonies were selected and sequenced. The full-length genome of DL3 was analyzed and compared with other wild-type HAV isolates.RESULTS: The genome of DL3 was 7 476 nucleotides (nt)in size, containing 732-nt 5'untranslated region (UTR), 6 681-nt open reading frame (ORF) which encoded a polyprotein of 2 227 amino acids (aa), and 63-nt 3'UTR. The base composition was 28.96 % A (2 165), 16.08 % C (1 202), 22.11% G(1 653)and 32.85% U (2 456). Genomic comparisons with wildtype HAV isolates revealed that DL3 had the highest identity of 97.5 % for nt (185 differences) with AH1, the lowest identity of 85.7 % (1 066 differences) with SLF88. The highest identity of 99.2 % for amino acid (18 differences)appeared among DL3, AH2 and FH3, and the lowest identity of 96.8 % (72 differences) between DL3 and SLF88. Based upon comparisons of the VP1/2A junction and the VP1 amino terminus, DL3 was classified as subgenotype IA. Phylogenetic analysis showed that DL3 was closest to the isolates in Japan.CONCLUSION: The sequence comparison and phylogenetic analysis revealed that DL3 is most similar to the isolates in Japan, suggesting the epidemiological link of hepatitis A happened in China and Japan.展开更多
The aim of this study was to explore the molecular basis for the attenuation of the Japanese encephalitis virus (JEV) vaccine strain SA14-14-2. The virulence of SA14 wild Japanese encephalitis virus (JEV) and its seve...The aim of this study was to explore the molecular basis for the attenuation of the Japanese encephalitis virus (JEV) vaccine strain SA14-14-2. The virulence of SA14 wild Japanese encephalitis virus (JEV) and its several attenuated viruses was tested by intracerebral (i.c.) or intraperitonial (i.p.) inoculation of 10-12 g mice. The stability of neuroattenuation was tested by one passage in suckling mouse brain. The E protein genes of those viruses were amplified by PCR, sequenced and compared. Three attenuated virus strains, SA14-14-2 vaccine virus, SA14-9-7 and SA14-5-3, did not exhibit lethal infections by i.c. or i.p. inoculation of 10-12 g mice and revert to the virulence. The other virus strain, SA14-12-1-7, showed no neuroinvasiveness by i.p. inoculation but residual neurovirulence by i.c. inoculation and reverted to high virulence after one brain passage. Comparison of the E protein gene sequences of the five virus strains indicated that there were differences of twelve nucleotides and eight amino acids between the parent strain SA14 and vaccine strain SA14-14-2, of which six amino acids (E-107, E-176, E-439, E-138, E-279, E-315) exhibited changes common to those of SA14-9-7 and SA14-5-3, three substitutions common to SA14-12-1-7. Two amino acid substitutions at the sites E177 (T→A) and E264 (Q→H) are unique to the SA14-14-2 vaccine virus. The results suggest that the mutations of E-107 (Leu→Phe), E-176 (He→Val), and E-439 (Lys→Arg) may contribute for the attenuation of neuroinvasiveness and partially for the attenuation of neurovirulence, the mutations of E-138, E-279, E-315 may not only critical to the neuroattenuation but also to its stability.展开更多
文摘AIM: To characterize the genome of an wild-type HAV isolate (DL3) in China.METHODS: A stool specimen was collected from hepatitis A patient from Dalian, China. HAV (DL3) was isolated and viral RNA was extracted. The genome of DL3 was amplified by reverse transcription and polymerase chain reaction (RT-PCR),followed by cloning into pGEM-T vector. The positive colonies were selected and sequenced. The full-length genome of DL3 was analyzed and compared with other wild-type HAV isolates.RESULTS: The genome of DL3 was 7 476 nucleotides (nt)in size, containing 732-nt 5'untranslated region (UTR), 6 681-nt open reading frame (ORF) which encoded a polyprotein of 2 227 amino acids (aa), and 63-nt 3'UTR. The base composition was 28.96 % A (2 165), 16.08 % C (1 202), 22.11% G(1 653)and 32.85% U (2 456). Genomic comparisons with wildtype HAV isolates revealed that DL3 had the highest identity of 97.5 % for nt (185 differences) with AH1, the lowest identity of 85.7 % (1 066 differences) with SLF88. The highest identity of 99.2 % for amino acid (18 differences)appeared among DL3, AH2 and FH3, and the lowest identity of 96.8 % (72 differences) between DL3 and SLF88. Based upon comparisons of the VP1/2A junction and the VP1 amino terminus, DL3 was classified as subgenotype IA. Phylogenetic analysis showed that DL3 was closest to the isolates in Japan.CONCLUSION: The sequence comparison and phylogenetic analysis revealed that DL3 is most similar to the isolates in Japan, suggesting the epidemiological link of hepatitis A happened in China and Japan.
文摘The aim of this study was to explore the molecular basis for the attenuation of the Japanese encephalitis virus (JEV) vaccine strain SA14-14-2. The virulence of SA14 wild Japanese encephalitis virus (JEV) and its several attenuated viruses was tested by intracerebral (i.c.) or intraperitonial (i.p.) inoculation of 10-12 g mice. The stability of neuroattenuation was tested by one passage in suckling mouse brain. The E protein genes of those viruses were amplified by PCR, sequenced and compared. Three attenuated virus strains, SA14-14-2 vaccine virus, SA14-9-7 and SA14-5-3, did not exhibit lethal infections by i.c. or i.p. inoculation of 10-12 g mice and revert to the virulence. The other virus strain, SA14-12-1-7, showed no neuroinvasiveness by i.p. inoculation but residual neurovirulence by i.c. inoculation and reverted to high virulence after one brain passage. Comparison of the E protein gene sequences of the five virus strains indicated that there were differences of twelve nucleotides and eight amino acids between the parent strain SA14 and vaccine strain SA14-14-2, of which six amino acids (E-107, E-176, E-439, E-138, E-279, E-315) exhibited changes common to those of SA14-9-7 and SA14-5-3, three substitutions common to SA14-12-1-7. Two amino acid substitutions at the sites E177 (T→A) and E264 (Q→H) are unique to the SA14-14-2 vaccine virus. The results suggest that the mutations of E-107 (Leu→Phe), E-176 (He→Val), and E-439 (Lys→Arg) may contribute for the attenuation of neuroinvasiveness and partially for the attenuation of neurovirulence, the mutations of E-138, E-279, E-315 may not only critical to the neuroattenuation but also to its stability.
