Genetic diversity of Chinese rabies viruses: evidence for the presence of two distinct clades in China.

There have been three major rabies epidemics in China since the 1950s. To gain more insights into the molecular epidemiology of rabies viruses (RVs) for the third (the current) epidemic, we isolated RV from dogs and humans in major endemic areas, and characterized these isolates genetically by sequencing the entire glycoprotein (G) gene and the G-L non-coding region. These sequences were also compared phylogenetically with RVs isolated in China during previous epidemics and those around the world. Comparison of the entire G genes among the Chinese isolates revealed up to 21.8% divergence at the nucleotide level and 17.8% at the amino acid level. The available Chinese isolates could be divided into two distinct clades, each of which could be further divided into six lineages. Viruses in clade I include most of the Chinese viruses as well as viruses from southeast Asian countries including Indonesia, Malaysia, the Philippines, Thailand, and Vietnam. The viruses in the other clade were found infrequently in China, but are closely related to viruses distributed worldwide among terrestrial animals. Interestingly, most of the viruses isolated during the past 10 years belong to lineage A viruses within clade I whereas most of the viruses isolated before 1996 belong to other lineages within clades I and II. Our results indicated that lineages A viruses have been predominant during the past 10 years and thus are largely responsible for the third and the current epidemic in China. Our results also suggested that the Chinese RV isolates in clade I share a common recent ancestor with those circulating in southeast Asia.

Genetic analysis of hantaviruses carried by reed voles Microtus fortis in China.

To gain more insights into the epidemiology of hantaviruses in China, the hantaviral S, M and L segment sequences were recovered from two Microtus fortis captured in Shenyang and four M. fortis trapped in Yuanjiang in China. Genetic analysis revealed that Shenyang sequences are closely related to the sequences of Fusong strains of Vladivostok virus (VLAV). Interestingly, the complete S segment and partial L segment sequences from Yuanjiang were quite distinct from those of Shenyang and Fusong strains, with up to 18% nucleotide (nt) and 5% amino acid (aa) sequence divergences. The partial M segment sequences (nt 2676-3650) from Yuanjiang were even more divergent from Shenyang and Fusong sequences (>20% and 8%, respectively). On the phylogenetic trees based on the S and partial M and L segment sequences, the Shenyang strains grouped together with Fusong strains. In contrast, four Yuanjiang sequences formed a distinct group that was a sister taxon to the Vladivostok-Fusong-Shenyang group. Our data indicated that the virus carried by M. fortis in Shenyang belong to VLAV. The newly characterized sequences from Yuanjiang might represent a novel distinct hantavirus species. Our results also demonstrated the great genetic diversity and complexity of the M. fortis-associated hantaviruses in China.

Factors influencing the routine immunization status of children aged 2-3 years in China.

OBJECTIVES: To examine the factors associated with the routine immunization status of children aged 2-3 years in China for gaining a better understanding of the Expanded Program on Immunization and to provide evidence for formulating specific strategies to guide the allocation of health resources. METHODS: We analyzed data from 45095 children aged 2-3 years in the 2013 National Immunization Coverage Survey to identify the sociodemographic and provider-associated factors affecting the full immunization status of children. Univariate and multiple logistic regression analyses were performed. RESULTS: The immunization rate for children aged 2-3 years ranged from 95.9% (diphtheria and tetanus toxoid with pertussis vaccine, 4th dose) to 99.5% (Japanese encephalitis vaccine, 1st dose) and was 93.1% for full immunization. In terms of sociodemographic factors, male children [adjusted OR (AOR): 1.115; 95% confidence interval(CI):1.016-1.222], minority children (AOR: 1.632; 95% CI: 1.457-1.828), children of fathers with less than high school education (AOR: 1.577; 95% CI: 1.195-2.081), those born at home (AOR: 4.655; 95% CI: 3.771-5.746), those who immigrated from an adjacent county (AOR: 2.006; 95% CI: 1.581-2.546), and those living in urban-rural fringe areas (AOR: 1.807; 95% CI: 1.475-2.214) or mountainous areas (AOR: 1.615; 95% CI: 1.437-1.814) had significantly increased odds of not being fully immunized. In terms of provider-associated factors, administration of vaccines at home (AOR: 2.311; 95% CI: 1.316-4.059), household reminders (AOR: 2.292; 95% CI: 1.884-2.789), and travel time to vaccination providers of >40 minutes (AOR: 1.622; 95% CI: 1.309-2.010) were negatively associated with immunization rates. In addition, compared to 3-year-old years, 2-year-old children (AOR: 1.201; 95% CI: 1.094-1.318) were less likely to be fully immunized. CONCLUSIONS: All included factors except maternal education level and distance from home to vaccination providers significantly affected immunization rates. Appropriate reminders and accessibility of immunization services played key roles in improving the immunization status. More attention to high-risk groups identified in this study may reduce the disparities in routine childhood immunization in China.

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.

Molecular characterization of rabies virus isolates in China during 2004.

Human rabies cases have been on the rise during the past few years in China and a total of 2651 cases were reported in 2004. To better understand the current rabies epidemics in China, we isolated rabies viruses from dogs and humans from five provinces and characterized these isolates genetically by sequencing the entire nucleoprotein (N) gene. Comparison of the N genes among these isolates revealed 86.6-99.9% homology and these viruses can be grouped into three lineages. Phylogenetic analysis indicates that all the Chinese isolates have a close relationship with viruses circulating in Asian canine population. When compared with rabies viruses isolated previously, the three lineages were similar to three of the four groups. Thus, our data suggest that rabies viruses currently circulating in China were similar, if not identical, to those reported in the previous epidemics.

[Safety and immunogenicity of measles attenuated live vaccine prepared by master seed lot vaccine of Shanghai-191 strain].

OBJECTIVE: To evaluate the safety and immunogenicity of a new measles vaccine prepared by master seed lot original vaccine of Shanghai-191 strain. METHODS: According to selection and exclusion criteria, 8-10-month healthy children, who have no history of measles, measles vaccine inoculation and vaccination contraindication were selected. The children selected were vaccinated 0.5 ml measles attenuate live vaccine prepared by master seed lot vaccine of the Shanghai-191 strain. They were observed the local and systemic response in 30 min and 6h, 24h, 48h, 72h, 7d, 14d, 21d, 28d after inoculation, and immune serum were collected before immunization and after 28 days. Measles virus antibody positive rate and geometric mean titer (GMT) were detected by hemagglutination inhibition. RESULTS: There were not serious adverse reactions. Measles virus antibodies in serum positive rate was 100% after 28 days of immunization, and measles virus antibody GMT was 1:50.66. CONCLUSION: New measles vaccine prepared by master seed lot original vaccine of the Shanghai-191 strain at 8-10-month children was safe and immunogenicity.

[An epidemiologle investigation of hantaviruses carried by rodent hosts in Hunan province].

OBJECTIVE: To explore the hantavirus infection and their genotype in rodents in Hunan. METHODS: Hantavirus antigens in the rat lungs from Hunan province were detected by immunofluorescence assay. Partial S and M segment in antigen-positive samples were amplified by RT-PCR, and then sequenced. The phyologenetic trees were constructed for the analysis of genetic characters of hantavirus. RESULTS: A total of 344 rats were trapped in the main epidemic area of Hunan province, and hantavirus antigens were found in 6 of the 344 rats( 1.74% ).The phylogenetic trees constructed by partial S segment( nt 620-990) or partial G2 segment (nt 2001- 2301) showed that the hantaviruses carried by Rattus norvegicus, R . flabipectus and R. rattoides from Xiangxiang district were genetic subtype SEOV4. The virus carried by R. norvegicus in Ningyuan district was phylogenetically different from the known SEOV. The hantavirus carried by Mus musculus from Shimen district was genetic subtype HTNV4. CONCLUSION: The hantaviruses in the main epidemic areas in Hunan province mainly belonged to SEOV, and R. flabipectus and R. rattoides carried the same genotype of SEOV as R. norvegicus.

[Molecule character of rabies virus in 3 counties in Hunan China].

OBJECTIVE: To study the rabies molecular biology features in animals between high incidence area of rabies and no rabies cases area in Hunan. METHODS: detect saliva of dogs and brains of dogs and cats by direct immunofluorescence assay, review positive samples by RT-PCR, sequencing extract RNA virus for genetic analysis. RESULTS: 12 were detected rabies virus antigen and positive nucleoside acid in 82 dogs from Wugang city also 1 in 17 from Dongkou county; the positive rate: Wugang 14.63 percent, Dongkou 5.88 percent. No rabies virus was detected in 67 samples of dog brains from Fenghuang County. Also none in 28 samples of cat brains. Amplificating N gene of rabies virus from positive samples of dog brain's tissue (No Wg13, Dk13) by RT-PCR, it shows that homology of nucleoside acid between two strain of virus is 99.4 percent; also 99.1 percent of amino acid. The homology of nucleoside acid (amonio acid) among Wg13 stain and Chinese strain CTN and aG strain is 89.4 percent (98.2 percent) and 86.1 percent (95.1 percent); The homology of nucleoside acid (amonio acid) among Dk13 stain Chinese strain CTN and aG strain is 89.1 percent (98.0 percent), 86.1 percent (94.9 percent).Compare with isolated rabies virus from abroad, the homology between two strains and Indonesia is 92.8 percent and 93.2 percent, the most similar of them. The strains isolated from other countries including Japan, Sri Lanka and India are relatively lower; The sequence of gene Wg13 and Dk13 were taken replacement of amino acid. CONCLUSION: Two strains are belong to type I rabies virus, comparing its N gene with current using vaccine strains, both are in same group, and homology are relatively higher.

[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.

[Analysis on the epidemiology of rabies in Hunan province in the past 10 years].

OBJECTIVE: To explore the possible factors causing the increase of rabies cases and to provide references for the development of related prevention and control strategy in Hunan. METHODS: Data was collected and analyzed on epidemic situation of rabies in the past ten years, and studies were carried in some counties. RESULTS: From 1994 to 1999, the annual cases sustained between 17 and 78. However, the number of cases has continued to increase since 2000. The annual reported cases in 2001 and 2002 were 311 and 313, and accounted for 34.8% and 30.0% of the total cases in the whole country respectively. The epidemic areas were mainly located in the south and middle parts of Hunan. Furthermore, the epidemic areas had been expanding. In 1997, human rabies cases were only reported in 7 cities but increased to 12 cities in 2004. CONCLUSION: Factors as increasing number but low inoculating rate to dogs, and incorrect treatment on the wounds etc. might have served important roles for the recurrence of rabies. Hence, it is necessary to take comprehensive preventive measures to control and prevent the epidemics of rabies in Hunan.