OBJECTIVETo investigate the change of viral titers under different conditions in cultured cells persistently-infected with different strains of Japanese encephalitis virus (JEV) and find out the factors that influence viral multiplication.

METHODSJEV JaGAr-01 and Nakayama wild strains were used to infect human hepatoma cell line KN73 respectively, and the persistent infection model was established. Viral titers were examined by plaque methods using BHK cells. Human nerve fibroblastoma cell line IMR-32 was infected with the strains of the virus that can cause persistent infection, and the thermal sensitivity of the viral strains was measured at 30 degrees C and 37 degrees C. KN73 cells persistently infected with JEV were infected with two JEV strains respectively, and viral superinfection was studied. To explore the replication of the persistently-infected viruses, KN73 and IMR-32 cells were infected with the viral strains.

RESULTSTwo persistently infected viral strains did not show any thermal difference. The results of superinfection were that the viral titers of JaGAr-01 strains were 1.3 and 8.8 percent of the control, respectively, and the viral titers of Nakayama strain were 80 and 1.7 percent of the control, respectively. JaGAr-01 wild strains, Nakayama wild strains and their persistently-infected strains infected KN73 and IMR-32 respectively. The replication of the persistently-infected strains was obviously lower than the wild strains in KN73 cells, however, in IMR-32 cells their replication was similar.

CONCLUSIONSThe two strains of JEV were not found to be temperature-mutant. It is possible that mutant viruses containing DI particles exist in JEV persistently-infected strains. In different cells there may be different host factors hindering the replication of the persistently-infected strains.

Animals ; Cell Line ; Encephalitis Virus, Japanese ; genetics ; physiology ; Encephalitis, Japanese ; virology ; Humans ; Virus Cultivation ; Virus Replication

Child ; Clinical Laboratory Techniques ; Humans ; Microbiological Techniques ; Viral Load ; Virus Cultivation ; Virus Diseases ; diagnosis

To select the adaptive strain of Dengue-III virus D9964 strain (China strain) in KMB17 cells, elucidate the biological characteristics and proliferation kinetics of adapted strain,and to lay the foundation for the development dengue inactivated vaccine and attenuated live vaccine. Dengue-III virus D9964 strain was firstly identified by amplification of the type-specific gene segment of dengue virus by RT-PCR, and the titer was determined. The virus was then subcultured in KMB17 cells with 4.0 MOI till completely adaptive to multiply in cell S. After subculturing in KMB17 cells for 10 consecutive passages, the adapted strain was screened, and purified through plaque. Virus titer of each passage was measured by microtitrimetry, and the antigenicity was detected by IFA. The purified virus RNA extraction of 3-8 day cultured from KMB17 cells, was performed to detect the proliferation kinetics of adapted strain. The results showed that after continuous subculture, dengue-III virus D9964 (China) strain could stably proliferate in KMB17 cells, a highly puried virus adapted strain was obtained through plaque purification. Purified strain maintained the good antigenicity with a highest replicating activity during the 5th-6th day.
Cell Line ; Dengue ; virology ; Dengue Virus ; chemistry ; genetics ; growth & development ; physiology ; Humans ; Kinetics ; Virus Cultivation ; Virus Replication

To investigate biological characteristics of the IVpi-189 progeny virus derived from the culture of influenza A virus as a live-attenuated vaccine candidate. Persistent infection of a cultured cell line with influenza A virus (MDCK-IVpi) was established by incubating continuously influenza virus-infected cells at a lower temperature. The infectious progeny virus derived from MDCK-IVpi cells at the 189rd subculture was designated as the IVpi-189 strain of influenza virus. The cytopathic effect induced by IVpi-189 virus was observed under different temperature conditions. The production of infectious progeny virus was examined at 38 and 32 degrees C by plaque titration of cell-associated and released virus. IVpi-189 virus showed cytopathic effect as strong as that of IVwt in infected cell line of MDCK at 32 degrees C. However, when culture temperature was raised to 38 degrees C, the cytopathic effect induced by IVpi-189 virus was delayed and less pronounced. Virus growth in IVpi-189 virus-infected cells at 38 degrees C was significantly reduced as compared with that of IVwt virus, although both viruses yielded nearly equivalent high titers of cell-associated and released virus at 32 degrees C. The reasons of the decreased proliferative ability of IVpi-189 virus at high culture temperature were unrelated with virus inactivation or the release of progeny virus, but associated with the decreased replication of infectious progeny virus in the infected cells. IVpi-189 virus derived from MDCK cells infected persistently with influenza A virus showed biological characteristics as a potential live-attenuated vaccine candidate.
Animals ; Cell Line ; Cytopathogenic Effect, Viral ; Dogs ; Humans ; Influenza A virus ; genetics ; physiology ; Temperature ; Virus Cultivation ; methods ; Virus Replication

OBJECTIVEReal-time RT-PCR, cell culture and embryonated eggs culture for influenza detection were compared by analyzing the data of influenza surveillance in Shenzhen in second half of 2009.

METHODS1092 clinical samples (throat swabs) collected during second half of 2009 were tested by real-time RT-PCR, cell culture and embryonated eggs culture, and the results were analyzed by statistical methods.

RESULTSThe positive rate were 54.21%, 27.11% and 16.21% using real-time RT-PCR, cell culture and embryonated eggs culture, and the sensitive were 100%, 50% and 29.9%. The lowest dilutions of virus detected by real-time RT-PCR were 10(-2) TCID50/ml.

CONCLUSIONThe sensitive of real-time RT-PCR was higher than culture and the specificity was also very high. It was more suitable for emergency detect. The sensitive of cell culture for H3N2 subtype was higher, and sensitive of embryonated eggs culture for type B was higher.

Animals ; Chick Embryo ; Humans ; Orthomyxoviridae ; isolation & purification ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Virus Cultivation ; methods

Bombyx mori bidensovirus (BmBDV) has been identified as causing chronic densonucleosis in Bombyx mori specifically. The replication mechanism of BmBDV remains unknown. Its genome comprises two single stands DNA (VD1 and VD2). In order to rescue infectious virions in vitro, we obtained the total viral DNA extracted from the BmBDV-infected larvae midguts, subsequently cloned the full-length sequence of BmBDV genome fragments by PCR and constructed recombinant plasmids pMD18T-VD1 and pUC-VD2. The linear genome fragments were obtained by digesting recombinant plasmids with corresponding restriction enzymes, and then collectively transfected BmN cells by the method of liposome-embedding. We determined the replication of the virus gene by PCR with the template of demethylated total DNA extracted from the post-transfect BmN cells. Meanwhile, we collected the total proteins from the post-transfect BmN cells and the larvae midgut of feeding the post-transfect BmN cells to perform Western blotting analysis, and detected the expression of viral genes. Here we firstly confirm that infectious virions can be rescued in BmN cells by linear co-transfect method.
Animals ; Bombyx ; DNA, Viral ; Densovirus ; growth & development ; Larva ; Transfection ; Virion ; Virus Cultivation

Persistent baculovirus infection is observed frequently in insect populations. Persistent infection can be transformed to a replicative and infective state caused by stress factors and plays an important role in regulating the size of insect population and in epizoology of baculoviruses. The aim of this study is to establish a persistently baculovirus-infected cell system to explore the molecular mechanisms of baculoviral persistence. Spodoptera exigua nucleopolyhedrovirus (SeMNPV) was serially undiluted passaged in Se301 cells to reduce virulence. Upon infection of Se301 cells with the SeMNPV up to passage 8, a few cells survived even if most of cells died due to virus infection. The surviving cells were passaged and designated as P8-Se301 cell strain. P8-Se301 cells had a population doubling time of 58-65 hours and grew slower than Se301 cells. Light microscopy and electron microscopy observation showed symptom of baculovirus infection, such as virogenic stroma, viral particles and occlusion bodies, in some of P8-Se301 cells. End-point dilution assay and infectious center assay showed that 4.14% +/- 0.99% cells continually released infectious progeny virus which replicated slower than SeMNPV in Se301 cells. The result indicated that P8-Se301 cells show a typical character trait of baculovirus persistent infection.
Animals ; Cells, Cultured ; Nucleopolyhedrovirus ; growth & development ; physiology ; Spodoptera ; virology ; Virus Cultivation ; methods

Organoid is an in vitro multicellular form mimicking in vivo organ. Its similarity to human organ including cellular organization, molecular expression patterns, as well as genetic signatures enables to study the characteristics of infectious agents and host-pathogen interaction. For the features of organoid, this system also can be potentially used to cultivate currently uncultivable viruses of vaccine candidates. This paper will briefly describe problems in the current culture system for virus production and the possibility of organoid as culture system for viral vaccine and their current limitations that should be solved to meet the goal.
Host-Pathogen Interactions ; Humans ; In Vitro Techniques ; Organoids* ; Viral Vaccines ; Virus Cultivation

High-yield H3N2 subtype swine influenza virus for large-scale vaccine production in cell culture was generated by reverse genetics. The rescued H3N2 (rH3N2) candidate virus contained hemagglutinin (HA) and neuraminidase (NA) genes derived from a field isolate A/Swine/Henan/S4/01 (H3N2), PB2 gene from A/PR/8/34, and the other five internal genes from A/Goose/Dalian/3/01 (H9N2). The rH3N2 virus titer in MDCK cell culture were measured by hemagglutination assay and the maximum virus titre of 1:512 hemagglutination unit was obtained after infection of MDCK cell for 60 h. The results of the present study indicated that rH3N2 virus was suitable for growth in MDCK cell culture and is feasible to be used for the production of cell grown influenza vaccine.
Animals ; Cell Line ; Dogs ; Hemagglutination Tests ; Influenza A Virus, H3N2 Subtype ; classification ; genetics ; growth & development ; Influenza Vaccines ; Plasmids ; Virus Cultivation

Surface enhanced Raman spectroscopy technology (SERS), using gold nanoparticles as a base, was developed for rapid and sensitive detection of virus strains. SERS can be used as a rapid and reliable method to distinguish the titers of viral replication. In the present study, we characterized H1N1 subtypes of influenza A virus strains in different conditions of pH or temperatures, while we analyzed data from SERS technology using gold nanoparticles as a base and cell cultures were employed to further confirm the data from virus strains. Origin8.0 was used to collect Raman spectra, smooth and homogenize data, and to contrast spectra. Our results indicated that the peaks of different virus strains in optimal environmental conditions (T=37 ℃/pH=7.2) reached ≥3 000. This criterion was verified by subsequent virological method. The present data indicate that the established SERS protocol can be used as a rapid and reliable method to distinguish the replication rate of virus, which can be further used in clinical samples.
Gold ; Hydrogen-Ion Concentration ; Influenza A Virus, H1N1 Subtype ; growth & development ; Nanoparticles ; Spectrum Analysis, Raman ; Temperature ; Virus Cultivation ; methods