RESUMO
BACKGROUND: Large-scale vaccine production requires downstream processing that focuses on robustness, efficiency, and cost-effectiveness. METHODS: To assess the robustness of the current vaccine production process, three batches of COVID-19 Omicron BA.1 strain hydrolytic concentrated solutions were selected. Four gel filtration chromatography media (Chromstar 6FF, Singarose FF, Bestarose 6B, and Focurose 6FF) and four ion exchange chromatography media (Maxtar Q, Q Singarose, Diamond Q, and Q Focurose) were used to evaluate their impact on vaccine purification. The quality of the vaccine was assessed by analyzing total protein content, antigen content, residual Vero cell DNA, residual Vero cell protein, and residual bovine serum albumin (BSA). Antigen recovery rate and specific activity were also calculated. Statistical analysis was conducted to evaluate process robustness and the purification effects of the chromatography media. RESULTS: The statistical analysis revealed no significant differences in antigen recovery (p = 0.10), Vero HCP residue (p = 0.59), Vero DNA residue (p = 0.28), and BSA residue (p = 0.97) among the three batches of hydrolytic concentrated solutions processed according to the current method. However, a significant difference (p < 0.001) was observed in antigen content. CONCLUSIONS: The study demonstrated the remarkable robustness of the current downstream process for producing WIBP-CorV vaccines. This process can adapt to different batches of hydrolytic concentrated solutions and various chromatography media. The research is crucial for the production of inactivated SARS-CoV-2 vaccines and provides a potential template for purifying other viruses.
RESUMO
The complete genomic sequence of a rabies virus isolate WH11, isolated from brain tissue of a rabid donkey in China, was determined and compared with other rabies viruses. This is the first Chinese street strain which was isolated from donkey and the entire length and organization of the virus was similar to that of other rabies viruses. Multiple alignments of amino acid sequences of the nucleoprotein, phosphoprotein, matrix protein, glycoprotein, and large protein of WH11 with those of other rabies viruses were undertaken to examine the conservative degree of functional regions. Phylogenetic analysis using the complete genomic sequence of WH11 determined that this isolate is most closely related with rabies viruses previously isolated in China and the attenuated Chinese vaccine strain CTN181.
Assuntos
Equidae/virologia , Genoma Viral , Vírus da Raiva/genética , Raiva/veterinária , Sequência de Aminoácidos , Animais , Antígenos Virais/genética , Encéfalo/virologia , China , Sequência Conservada , Glicoproteínas/genética , Chaperonas Moleculares , Dados de Sequência Molecular , Proteínas do Nucleocapsídeo/genética , Fosfoproteínas/genética , Filogenia , RNA Viral/genética , Raiva/virologia , Vírus da Raiva/isolamento & purificação , Alinhamento de Sequência , Proteínas do Envelope Viral/genética , Proteínas Estruturais Virais/genéticaRESUMO
To investigate the evolutionary dynamics of rabies virus (RABV) in China, we collected and sequenced 55 isolates sampled from 14 Chinese provinces over the last 40 years and performed a coalescent-based analysis of the G gene. This revealed that the RABV currently circulating in China is composed of three main groups. Bayesian coalescent analysis estimated the date of the most recent common ancestor for the current RABV Chinese strains to be 1412 (with a 95 % confidence interval of 1006-1736). The estimated mean substitution rate for the G gene sequences (3.961x10(-4) substitutions per site per year) was in accordance with previous reports for RABV.
Assuntos
Evolução Molecular , Vírus da Raiva/classificação , Vírus da Raiva/genética , Raiva/virologia , Animais , Antígenos Virais/genética , China , Análise por Conglomerados , Genótipo , Glicoproteínas/genética , Humanos , Dados de Sequência Molecular , Filogenia , Mutação Puntual , Vírus da Raiva/isolamento & purificação , Análise de Sequência de DNA , Homologia de Sequência , Proteínas do Envelope Viral/genéticaRESUMO
Attenuated recombinant rabies vector could be an ideal system for delivery of contrast agent gene for Magnetic Resonance Imaging (MRI) because of its neurotropic nature. In this study, the gene of a biomolecular contrast agent, ferritin, was successfully cloned into two rabies virus vectors, vaccine-based pCTN and street strain-based pNH. Recombinant virus granules were obtained and proved to express ferritin by RT-PCR after transfection of CTN-ferritin and NH-ferritin vector systems in BHK-21 cells. The recovered rabies virus-rCTN-ferritin was of similar ability to rNH-ferritin, which suggests the possibility of application of this safe and effective rabies vector system in delivery of diagnostic or therapeutic genes into the brain.
Assuntos
Ferritinas/biossíntese , Expressão Gênica , Vetores Genéticos , Vírus da Raiva/genética , Transdução Genética , Animais , Linhagem Celular , Meios de Contraste/metabolismo , Cricetinae , Ferritinas/genética , Humanos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genéticaRESUMO
In this study, the complete genomic sequence of a rabies virus isolate HN10, recovered from brain tissue of a rabid patient in China, was determined. This is the first Chinese street isolate that has been fully characterized. The overall organization of this virus is typical of that observed for all other rabies viruses. Alignments of amino acid sequences of the phosphoprotein, glycoprotein and large protein of HN10 with those of other rabies viruses were used to examine the extent of conservation of known functional regions. Phylogenetic analysis using either the complete or partial genomic sequence of HN10 determined that this isolate is most closely associated with viruses previously shown to circulate in Guangxi and Hunan provinces. In addition, of all vaccine strains used for comparison, the attenuated Chinese vaccine strain CTN181 is most closely related to HN10.
Assuntos
Genoma Viral , Vírus da Raiva/genética , Raiva/virologia , Sequência de Aminoácidos , Sequência de Bases , Encéfalo/virologia , China , Humanos , Lactente , Masculino , Dados de Sequência Molecular , Filogenia , RNA Viral/análise , RNA Viral/genética , Vírus da Raiva/isolamento & purificação , Alinhamento de Sequência , Proteínas Virais/análiseRESUMO
A group of 31 rabies viruses (RABVs), recovered primarily from dogs, one deer and one human case, were collected from various areas in China between 1989 and 2006. Complete G gene sequences determined for these isolates indicated identities of nucleotide and amino acid sequences of >or=87% and 93.8%, respectively. Phylogenetic analysis of these and some additional Chinese isolates clearly supported the placement of all Chinese viruses in Lyssavirus genotype 1 and divided all Chinese isolates between four distinct groups (I-IV). Several variants identified within the most commonly encountered group I were distributed according to their geographical origins. A comparison of representative Chinese viruses with other isolates retrieved world-wide indicated a close evolutionary relationship between China group I and II viruses and those of Indonesia while China group III viruses formed an outlying branch to variants from Malaysia and Thailand. China group IV viruses were closely related to several vaccine strains. The predicted glycoprotein sequences of these RABVs variants are presented and discussed with respect to the utility of the anti-rabies biologicals currently employed in China.
Assuntos
Antígenos Virais/genética , Glicoproteínas/genética , Vírus da Raiva/classificação , Vírus da Raiva/genética , Raiva/virologia , Proteínas do Envelope Viral/genética , Sequência de Aminoácidos , Animais , Antígenos Virais/química , Sequência de Bases , China , Cervos , Cães , Evolução Molecular , Feminino , Genótipo , Glicoproteínas/química , Glicosilação , Humanos , Dados de Sequência Molecular , Filogenia , Raiva/veterinária , Vírus da Raiva/isolamento & purificação , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Proteínas do Envelope Viral/químicaRESUMO
The CTN rabies virus was isolated from a human in China in 1953 and subsequently attenuated by multiple passaging to a vaccine strain now approved by the WHO. In this study, we describe the development of a reverse genetics system for the CTN rabies virus strain. The recombinant full-length genomic cDNA was flanked by a hammerhead ribozyme (HamRz) and the hepatitis delta virus ribozyme (HdvRz) while the non-coding G-L region was replaced with a green fluorescent protein (GFP) gene. A set of helper plasmids encoding nucleoprotein (N), phosphoprotein (P), and large protein (L) were constructed and co-transfected with recombinant full-length genome plasmid into BHK-21 cells. Recombinant virus was successfully recovered from cloned cDNA under control of the CMV promoter driven by RNA polymerase II. The recombinant virus, CTN-GFP, stably expressed GFP as detected by fluorescence microscopy. A group of 1-day-old suckling mice was challenged with the CTN-GFP strain by intracerebral inoculation, resulting in 100% morbidity and GFP expression was detected in brain tissues. The recombinant virus CTN-GFP strain recovered from cloned cDNA will be useful as a viral vector to express other foreign genes.
Assuntos
Engenharia Genética/métodos , Genética Microbiana/métodos , Vacina Antirrábica/genética , Vírus da Raiva/genética , Animais , Linhagem Celular , China , Cricetinae , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Vírus Delta da Hepatite/enzimologia , Vírus Delta da Hepatite/genética , Humanos , Camundongos , Plasmídeos , RNA Catalítico/genética , RNA Catalítico/metabolismo , Raiva/virologia , Vacina Antirrábica/imunologia , Vírus da Raiva/imunologia , Análise de SobrevidaRESUMO
To construct a expression plasmid containing the full-length cDNA of rabies virus, four overlapped fragments covering full length cDNA of rabies virus street stain HN10 were cloned into pVAX1 sequentially in the genome except for the G-L noncoding region which was replaced with GFP gene. The plasmid containing the full-length viral cDNA was flanked by hammerhead ribozyme (HamRz) and hepatitis delta ribozyme (HdvRz) sequences and arranged under the control of the cytomegalovirus (CMV) promoter. The constructed plasmid could be directly used for the following procedure of producing the recombinant rabies virus street HN10.