Investigation of the role of healthy dogs as potential carriers of rabies virus.

To investigate whether healthy animals are potential carriers of rabies virus in China, 153 domestic dogs were collected from a rabies enzootic area, Anlong county in Guizhou Province, and monitored for 6 months. Initially, findings of rabies virus antigen in the saliva of 15 dogs by an enzyme-linked immunosorbent assay (ELISA) test suggested they might be carriers. These 15 dogs were kept under observation for 6 months. None of the dogs showed any clinical signs of rabies during the observation period. Moreover, using the ELISA test alone, detection of rabies virus antigen in saliva of some animals was not consistent during the observation period. However, none of the saliva samples collected either at the time of acquisition or during the observation period was found to be positive for rabies virus RNA by reverse transcriptase-polymerase chain reaction (RT-PCR). Furthermore, neither viral antigen nor viral RNA was detected in the brain samples collected at the time of euthanasia. These results do not provide support for the contention that healthy dogs act as carriers in rabies. Caution is urged when preliminary and nondefinitive tests, such as ELISA, are used to infer clinical status related to rabies.

Safety and immunogenicity from a phase I trial of inactivated severe acute respiratory syndrome coronavirus vaccine.

BACKGROUND: Emergence of severe acute respiratory syndrome (SARS) from the winter of 2002 to the spring of 2003 has caused a serious threat to public health. METHODS: To evaluate the safety and immunogenicity of the inactivated SARS coronavirus (SARS-CoV) vaccine, 36 subjects received two doses of 16 SARS-CoV units (SU) or 32 SU inactivated SARS-CoV vaccine, or placebo control. RESULTS: On day 42, the seroconversion reached 100% for both vaccine groups. On day 56, 100% of participants in the group receiving 16 SU and 91.1% in the group receiving 32 SU had seroconverted. The geometric mean titre of neutralizing antibody peaked 2 weeks after the second vaccination, but decreased 4 weeks later. CONCLUSION: The inactivated vaccine was safe and well tolerated and can elicit SARS-CoV-specific neutralizing antibodies.

A molecular epidemiological study targeting the glycoprotein gene of rabies virus isolates from China.

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.

[An immunofluorescence assay for the detection of SARS associated coronavirus antibody based on recombinant nucleocapsid antigen and its application].

OBJECTIVE: To establish a new technique for SARS-CoV antibody test to detect infection of severer acute respiratory syndrome (SARS). METHODS: Nucleocapsid gene was obtained by reverse transcription and polymerase chain reaction from a SARS patient and inserted into the vector pFastBacHTa expressing baculovirus. Insect Sf9 cells were transfected with the recombinant baculovirus expressing SARS nucleocapsid antigen and then cultured, fixed by acetone so as to make SARS-specific antigen. Immunofluorescence assay (IFA) technique and plaque reduction neutralization test (PRNT) were used to detect 7 samples of sera of 4 newly diagnosed SARS patients collected in different days, 48 samples of convalescent sera of SARS patients, 24 serum samples of healthy person undergoing physical examination, and 40 serum samples from non-SARS patients with fever by double blind test. RESULTS: The recombinant SARS-specific antigen reacted only with SARS positive sera but not with normal sera. Double blind test showed that 45 of the 46 PRNT positive sera were IFA positive with an accordance rate of 97.8%. 7 samples of sera from 4 SARS patients in acute progressive stage in Guangdong province were all IFA positive. SARS antibody could be detected since the sixth day after onset, and the titer increased from 1:40 to 1:600 on the ninth day. CONCLUSION: Immunofluorescence assay is highly specific and sensitive in detection of SARS. This reagent is safe and easy to prepare.

[Analysis of full-length gene sequence of rabies vaccine virus aG strain].

To sequence and analyze the full-length gene sequence of rabies vaccine virus aG strain. The full-length gene sequence of aG strain was amplified by RT-PCR by 8 fragments,each PCR product was cloned into vector pGEM-T respectively, sequenced and assemblied; The 5' leader sequence was sequenced with method of 5' RACE. The homology between aG and other rabies vaccine virus was analyzed by using DNAstar and Mega4. 0 software. aG strain was 11 925nt(GenBank accession number: JN234411) in length and belonged to the genotype I . The Bioinformatics revealed that the homology showed disparation form different rabies vaccine virus. the full-length gene sequence of rabies vaccine virus aG strain provided a support for perfecting the standard for quality control of virus strains for production of rabies vaccine for human use in China.

[Studies on the biological and genetic characteristics of a highly neurovirulent Japanese encephalitis virus strain SA4].

The biological and genetic characteristics of a highly neurovirulent JE virus strain SA4 were studied. Mice were inoculated intracerebrally with strain SA4 and SA14, and observed for 14 days, respectively. On different days, mice brains were harvested for titrations of the virus content in the brains. Full-length genome of SA4 was sequenced and compared with SA14 as well as other JE virus strains in the world. The results indicated that the mice inoculated by SA4 induced sickness and death more rapidly (24 hours faster) than those induced by the SA14. The virus titers in the brains of mice infected with SA4 were 0.5-1.0 lg PFU/mL higher than that infected with SA14. The sequence comparison indicated that the nucleotide and amino acid homology between SA4 and the other 21 JE strains were 84.6%-99.0% and 95.2%-99.7% respectively. Comparison with strain SA14 revealed that there were 17 amino acid differences between the two strains, of which 5 were in the E protein region. The results demonstrate that strain SA4 is a highly neurovirulent strain. The substitutions of the 17 amino acids in the SA4 strain can be the molecular basis for the biological characteristics of high neurovirulence.

[Comparison of viremia formation between guinea-pigs infected with wild and attenuated (SA14-14-2) Japanese encephalitis viruses].

OBJECTIVE: To study the viremia formation in guinea-pigs infected with wild type and attenuated Japanese encephalitis virus (JEV). METHODS: Guniea pigs were inoculated intraperitoneally with different wild JEV strains and the attenuated vaccine strain and its parent virulent strain. Viremia was detected on different days following virus inoculation. RESULTS: All the guinea-pigs inoculated with the wild JEV strains induced different levels of viremia (1.00-3.40 Lg pfu) on the 1st and 3rd day post inoculation. Using a virus titer of 10(4) pfu for inoculation, the animals inoculated with the SA14 parent strain induced relatively high viremia (10(2.4)-10(3.4) pfu), however no viremia coulds be detected on any tested days. CONCLUSION: The degree of viremia in guinea pigs can be used as a new method to evaluate the attenuation of JEV.

[Analysis of full-length gene sequence of a rabies vaccine strain CTN-1 for human use in China].

CTN-1 is one of the rabies vaccine strains for human use in China, but there has been no report on the full-length gene sequence of CTN-1. In this study, the full-length gene of CTN-1 was amplified by RT-PCR, each PCR product was cloned into T vector and then sequenced, assemblied and compared with other vaccine strains as well as the wild Chinese rabies isolates. The phylogenetic tree of G gene was constructed and the genetic homology was analyzed. The results revealed that CTN-1 was 11 925nt (GenBank accession number: FJ959397)in length and belonged to the genotype I. The full-length nucleotide homologies among CTN-1 and other rabies virus strains were between 81.5%-93.4%, of which the lowest 81.5% was between CTN-1 strain and bat isolate SHBRV, and the highest 93.4% was between CTN-1 and Chinese isolate HN10. The phylogenetic analysis revealed that the majority of Chinese isolates could be grouped into the same clade with the CTN-1 strain, but aG and some vaccine strains from abroad such as Flury, PM, PV, ERA, RC-HL and a few Chinese strains were grouped in another clade. Comparsion of the G protein genes also showed that the homologies among CTN-1 and most of the Chinese isolates were higher than that of the other vaccine strains to those Chinese strains. Therefore, it suggests that the CTN-1 strain is more suitable and rational to be used for the production of rabies inactivated vaccine in China than the others.

[Completed sequences analysis on the Chinese attenuated yellow fever 17D vaccine strain and the WHO standard yellow fever 17D vaccine strain].

OBJECTIVE: To compare the molecular characteristics of the Chinese attenuated yellow fever 17D vaccine strain and the WHO reference yellow fever 17D vaccine strain. METHODS: The primers were designed according to the published nucleotide sequences of YFV 17D strains in GenBank. Total RNA of was extracted by the Trizol and reverse transcripted. The each fragments of the YFV genome were amplified by PCR and sequenced subsequently. The fragments of the 5' and 3' end of the two strains were cloned into the pGEM T-easy vector and then sequenced. RESULTS: The nucleotide acid and amino acid sequences of the homology to both strains were 99% with each other. No obvious nulceotide changes were found in the sequences of the entire genome of each 17D strains. Moreover, there was no obvious changes in the E protein genes. But the E173 of YF17D Tiantan, associted with the virulence, had mutantions. And the two live attenuated yellow fever 17D vaccine strains fell to the same lineage by the phylogenetic analysis. CONCLUSION: The results indicated that the two attenuated yellow fever 17D vaccine viruses accumulates mutations at a very low frequency and the genomes were relative stable.

[Study on the phenotypic characteristics of Japanese encephalitis virus strains isolated from different years].

In order to reveal the phenotypic characteristics of 17 JE virus strains isolated from different years, plaque sizes, mice neurovirulence and mice neuroinvasiveness of the isolates were studied and compared. BHK21 cell monolayers were used for testing the plaque sizes. The virus neurovirulence was tested in 9-11g mice inoculated intracerebrally and the virus neuroinvasiveness was tested in 9-11g and 14-16g by subcutaneous inoculation. Results showed that all the viruses produced clear plaques on the BHK21 cell monolayers with different sizes and all the virus strains appeared high neurovirulence in the mice with higher than lg8. 0/0.03 mL virus titers, while no apparent difference among them. The neuroinvasiveness (subcutaneous virulence) tested in the 9-11g mice had shown a little difference, but when tested in the 12-14 g mice,the difference was apparent. The results demonstrated that JEV in nature were highly neurovirulent with no apparent difference. However the neuroinvasiveness of the JEV in nature was greatly different, which didn't relate to the years of isolation and genotypes, but most of the viruses isolated from patients showed higher neuroinvasiveness.

[An epidemiological study of rabies virus in domestic dogs, cats and wildlife and the immunogenicity study for rabies vaccines derived from different cell cultured virus strains].

For epidemiological investigation of the rabies virus carrier rates of domestic dogs, cats and wild animals like rodent animals and bats,three kinds of regions where rabies had higher incidence (Hunan and Guizhou Provinces), lower incidence (Jiangsu Province, Wuhan City) and provisionally rabies-free (Shenyang City) were selected. Then the antigenic types, the genovariation of the isolaled viruses and the currently vaccine matching of the virus strains were analyzed. The results showed that in China the principal host of rabies is dog,the total virus carrier rate of the captured dogs was 2.56%, and the highest positive isolation rate was 20.0% in some monitoring site. However,there was no evidence about the rabies virus carrier rate in rodent animals,bats or other wild animals. The rabies vaccines which prepared from aG and CTN strains have already been produced successfully in China. The research showed that the nucleotide sequences of the newly isolated viruses were more similar with the glycoprotein gene of CTN strain. In order to evaluate the safety and the efficacy of the vaccines currently used, two groups (50 people each) were injected with vaccine of aG strain and CTN strain respectively in five surveillance points. The neutralizing antibody tested were 0.49 IU/mL-0.52 IU/mL and 6.7 IU/mL-7.53 IU/mL after the 7 and the 14 days of vaccine injection respectively. In addition, the rates of antibody positive seroconversion were 45.1%-47.9% and 100% respectively, and there was no moderate or severe adverse reactions observed. These data showed the vaccines have satisfactory effect on safety and protection.

[Development of purified HFRS bivalent vaccine].

AIM: To develop the purified HFRS bivalent vaccine. METHODS: LR1 (type I) and R22 (type II) of HFRS virus were cultured respectively in Vero cells. The viral suspensions were harvested, inactivated with beta-propiolactone, concentrated by ultra-filtration, purified by zone centrifugation, and desucrosed by column chromatography. The qualified vaccine bulks of type I and type II were mixed equally and absorbed with AI(OH)3 for preparation of 3 lots vaccines. RESULTS: The 3 lots vaccines all passed examination carried out by ourselves and reexamination performed by National Institute for the Control of Pharmaceutical and Biological Products . One of 3 lots has been used in clinical trial. CONCLUSION: It is practical to develop the purified HFRS bivalent vaccine by Vero cell culture.

[Construction of infectious Japanese encephalitis virus clone based on the cDNA template of the attenuated live vaccine production strain SA14-14-2].

OBJECTIVE: To construct infectious Japanese encephalitis virus (JEV) based on the in vitro-ligated cDNA template of the vaccine strain SA14-14-2, and identify the virus. METHODS: Full-length genomic cDNA of JEV SA14-14-2 strain was ligated and then RNA was transcribed in vitro, the infective virus was obtained by transfecting the RNA into Vero cells and identified. RESULTS: The infective clone of JEV was constructed, the virulence was weaker than the wild virus. CONCLUSION: It was possible to construct infectious clone from the production strain of live attenuated Japanese B encephalitis vaccine.

[Research on Culex tritaeniorhynchus and Culex pipiens quinquefasciatus intrathoracically infected with attenuated Japanese encephalitis virus SA14-14-2 vaccine strain].

BACKGROUND: To determine if the attenuated Japanese encephalitis (JE) virus SA14-14-2 vaccine strain interacts efficiently with Culex tritaeniorhynchus and Culex pipiens quinquefasciatus, and further to acquire a new knowledge of its characteristics and safety for human beings. METHODS: Laboratory colonies of the two species of mosquitoes were set up and were inoculated intrathoracically with the attenuated vaccine virus and wild JE virus (Nak), both of which were used with different dilution from 10(-1) to 10(-9). Subsequently, the virus titers in the mosquitoes were detected by the plaque assay. RESULTS: Inoculated with the vaccine strain, two species of mosquitoes were infected with the titers ranged from 10(0)-10(-3), and the maximum titers in Culex tritaeniorhynchus and Culex pipiens quinquefasciatus were 4.48 logPFU/ml and 5.63 logPFU/ml, respectively. Inoculated with wild JE virus, Culex pipiens quinquefasciatus was infected with titers ranged from 10(0)-10(-5), and the maximum titer in the mosquitoes was 6.59; Culex tritaeniorhynchus was infected with titers ranged from 10(0)-10(-4) and the maximum titer was 5.74 logPFU/ml. CONCLUSION: By intrathoracic infection, the attenuated JE virus SA14-14-2 vaccine strain can replicate in both species of mosquitoes.

[Molecular characterization of hantavirus Shandong isolate JNL virus strain].

OBJECTIVE: To understand the molecular epidemiologic characteristics of hantavirus Shandong isolate JNL virus strain. METHODS: The complete M and S gene of the JNL virus isolated from Shandong Province was amplified by RT- PCR, and the purified PCR product was cloned into T vector for sequencing. RESULTS: The results revealed that the JNL M segment was 3615 bp in length, encoding 1135 amino acids, and the S segment was 1698 bp encoding 429 amino acids, JNL belongs to HTN virus. The comparison of homology with HTN and SEO types showed that the difference of M and S complete sequences between JNL and all other HTN virus strains reached 20.0%-20.6%, and 15.5%-16.0%, respectively. Phylogenetic tree also showed that the position of JNL is located at a different clade. CONCLUSIONS: HTN virus Shandong local isolate JNL strain is a new specific HTN subtype virus.