Virus isolation and genotype identification of human respiratory syncytial virus in Guizhou Province, China

To investigate the genetic variation and molecular epidemiology characteristics of Human Respiratory Syncytial Virus (HRSV) in Guizhou Province, nasopharyngeal aspirates were collected from patients with acute respiratory infection (ARI) in Guizhou Provincial People's Hospital, from December 2017 to March 2018, and inoculated to Hep-2 cells to isolate HRSV. Cells that showed cytopathic effect (CPE) were then confirmed by indirect immunofluorescence assay and reverse transcription. The sequence of the PCR products was determined for HRSV isolates, and the genetic variation was analyzed. Out of 196 nasopharyngeal aspirate samples, HRSV were isolated in 39. The second hypervariable region at the 3' terminal of glycoprotein gene (HVR2) sequence analysis showed that subgroup A was dominant. Seventy-nine percent of the isolates belonged to subgroup A, ON1 genotype, and 21 % belonged to subgroup B, BA9 genotype, which indicates that the dominant HRSV circulating in Guizhou Province was subgroup A, genotype ON1, co-circulating with a less prevalent subgroup B, genotype BA9.

Keywords:
Human respiratory syncytial virus; Virus isolation; RT-PCR; HVR2; Phylogenetic analysis; Molecular epidemiology

Authorship

Ting Zhao

Guizhou University, Medical College, Guiyang, ChinaGuizhou UniversityChinaGuiyang, ChinaGuizhou University, Medical College, Guiyang, China

Guizhou University, Guizhou Provincial People's Hospital, Department of Central Laboratory, GuiyangChinaGuizhou UniversityChinaGuiyang, ChinaGuizhou University, Guizhou Provincial People's Hospital, Department of Central Laboratory, GuiyangChina

http://orcid.org/0000-0002-1326-9610

Zhixu Ye ^**Corresponding authors. E-mail addresses: yezhixu126@126.com (Z. Ye), yufuxun@126.com (F. Yu).

Guizhou University, Guizhou Provincial People's Hospital, Department of Pediatrics, Guiyang, ChinaGuizhou UniversityChinaGuiyang, ChinaGuizhou University, Guizhou Provincial People's Hospital, Department of Pediatrics, Guiyang, China

Guizhou Provincial People's Hospital, NHC Key Laboratory of Pulmonary Immunological Diseases, Guiyang, ChinaGuizhou Provincial People's HospitalChinaGuiyang, ChinaGuizhou Provincial People's Hospital, NHC Key Laboratory of Pulmonary Immunological Diseases, Guiyang, China

Binlin Wang

People's Hospital of QianNan, Department of Clinical Labotatory, Guizhou Province, ChinaPeople's Hospital of QianNanChinaChinaPeople's Hospital of QianNan, Department of Clinical Labotatory, Guizhou Province, China

Yuxia Cui

Yingjie Nie

Bin Yang

Kun Chen

Hua Zhang

Guizhou University, Guizhou Provincial People's Hospital, Department of Clinical Laboratory, Guiyang, ChinaGuizhou UniversityChinaGuiyang, ChinaGuizhou University, Guizhou Provincial People's Hospital, Department of Clinical Laboratory, Guiyang, China

Fangfang Hu

Fuxun Yu ^**Corresponding authors. E-mail addresses: yezhixu126@126.com (Z. Ye), yufuxun@126.com (F. Yu).

*Corresponding authors. E-mail addresses: yezhixu126@126.com (Z. Ye), yufuxun@126.com (F. Yu).

Author contributions

FY, YC, ZY and YN designed the study. TZ, BW, WH, LL, ZL, BY, QX, KC, FH, HZ and FY performed the experiments. TZ, ZY and FY analyzed the data. TZ and FY wrote the paper. All authors read and approved the final manuscript.

Conflict of interest

The authors declare no conflicts of interest.

SCIMAGO INSTITUTIONS RANKINGS

Guizhou University, Medical College, Guiyang, ChinaGuizhou UniversityChinaGuiyang, ChinaGuizhou University, Medical College, Guiyang, China

Figures

Figures (5)

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Fig. 1
Cytopathic effect and indirect immunofluoresence test of HRSV isolates on Hep-2 cells. A, Mock Hep-2 cell; B, Isolate GZA17-01 infected Hep-2 cells; C, Indirect immunofluoresence test of mock Hep-2 cell; D, Indirect immunofluoresence test of isolate GZA17-01 infected Hep-2 cell.

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Fig. 2
Comparison of homology between the Guizhou HRSV subgroup A isolates 3' end of G gene nucleotide and amino acid sequences with that of the prototype strain A2 in colored heat map. A, comparison of nucleotide sequences homology of subgroup A; B, homology comparison of amino acid sequences of subgroup A. Red indicates the maximum identity, blue indicates the minimum identity, white indicates the middle value, green indicates the maximum divergence, red indicates the minimum divergence, yellow indicates the middle value.

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Fig. 3
Comparison of homology between Guizhou HRSV subgroup B isolates 3' end of G gene nucleotide and amino acid sequences with that of the prototype strain CH18537 in colored heat map. A, comparison of nucleotide sequences homology of subgroup B; B, homology comparison of amino acid sequences of subgroup B. Red indicates the maximum identity, blue indicates the minimum identity, white indicates the middle value, green indicates the maximum divergence, red indicates the minimum divergence, yellow indicates the middle value.

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Fig. 4
Phylogenetic trees of nucleotide sequences from the second variable region of the G gene of HRSV subgroup A (A) and subgroup B (B) isolates. Trees were generated by using the Neighbor-joining methods. Bootstrap values greater than 70 % are shown at the branch notes. The value represents the bootstrap probability. The genotypes obtained by isolates in this study are indicated by brackets on the right side. Reference sequences describing each genotype (ON1, NA1 to NA4, GA1 to GA7, SAA1, GB1 to GB4, SAB1 to SAB3, BA1 to BA10) were obtained from the Genbank database and included in the tree. Prototype strains (strain A2 for subgroup A and strain CH18537 for subgroup B) were used as the outgroup sequences.

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Fig. 5
Deduced amino acid alignment of HVR2 from Guizhou HRSV isolates shown relative to the sequence of prototype strains. A, deduced amino acid alignment of HVR2 from Guizhou subgroup A isolates shown relative to the sequence of strain A2 (acc. nº M11486); B, deduced amino acid alignment of HVR2 from Guizhou subgroup B isolates shown relative to the sequence of strain CH18537 (Number acc. nº JX198143). Different amino acids are shown in different colors, identical residues are indicated by dots, dashes designate gaps corresponding to nucleotide insertions and asterisks indicate stop codons.

Fig. 1 Cytopathic effect and indirect immunofluoresence test of HRSV isolates on Hep-2 cells. A, Mock Hep-2 cell; B, Isolate GZA17-01 infected Hep-2 cells; C, Indirect immunofluoresence test of mock Hep-2 cell; D, Indirect immunofluoresence test of isolate GZA17-01 infected Hep-2 cell.

Fig. 2 Comparison of homology between the Guizhou HRSV subgroup A isolates 3' end of G gene nucleotide and amino acid sequences with that of the prototype strain A2 in colored heat map. A, comparison of nucleotide sequences homology of subgroup A; B, homology comparison of amino acid sequences of subgroup A. Red indicates the maximum identity, blue indicates the minimum identity, white indicates the middle value, green indicates the maximum divergence, red indicates the minimum divergence, yellow indicates the middle value.

Fig. 3 Comparison of homology between Guizhou HRSV subgroup B isolates 3' end of G gene nucleotide and amino acid sequences with that of the prototype strain CH18537 in colored heat map. A, comparison of nucleotide sequences homology of subgroup B; B, homology comparison of amino acid sequences of subgroup B. Red indicates the maximum identity, blue indicates the minimum identity, white indicates the middle value, green indicates the maximum divergence, red indicates the minimum divergence, yellow indicates the middle value.

Fig. 4 Phylogenetic trees of nucleotide sequences from the second variable region of the G gene of HRSV subgroup A (A) and subgroup B (B) isolates. Trees were generated by using the Neighbor-joining methods. Bootstrap values greater than 70 % are shown at the branch notes. The value represents the bootstrap probability. The genotypes obtained by isolates in this study are indicated by brackets on the right side. Reference sequences describing each genotype (ON1, NA1 to NA4, GA1 to GA7, SAA1, GB1 to GB4, SAB1 to SAB3, BA1 to BA10) were obtained from the Genbank database and included in the tree. Prototype strains (strain A2 for subgroup A and strain CH18537 for subgroup B) were used as the outgroup sequences.

Fig. 5 Deduced amino acid alignment of HVR2 from Guizhou HRSV isolates shown relative to the sequence of prototype strains. A, deduced amino acid alignment of HVR2 from Guizhou subgroup A isolates shown relative to the sequence of strain A2 (acc. nº M11486); B, deduced amino acid alignment of HVR2 from Guizhou subgroup B isolates shown relative to the sequence of strain CH18537 (Number acc. nº JX198143). Different amino acids are shown in different colors, identical residues are indicated by dots, dashes designate gaps corresponding to nucleotide insertions and asterisks indicate stop codons.

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Virus isolation and genotype identification of human respiratory syncytial virus in Guizhou Province, China

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[1] *Corresponding authors. E-mail addresses: yezhixu126@126.com (Z. Ye), yufuxun@126.com (F. Yu).