Molecular evolution of emerging Banna virus.

Banna virus (BAV) is an emerging pathogen that causes human viral encephalitis and has been isolated from types of blood-sucking insects and mammals in Asia. However, there are no reported systematic studies that describe the origin and evolution of BAV. Here, a phylogenetic analysis of BAVs isolated from a variety of potential vectors and vertebrate hosts worldwide revealed that BAVs emerged in the beginning of the 20th century and do not exhibit a species barrier. The mean substitution rate of BAVs was 2.467×10-2substitution/site/year (95% HPD, 1.093×10-3 to 5.628×10-2). The lineage is mainly composed of BAVs from high-latitude regions, which are the most recently emerged viruses with significantly higher substitution rates compared with the lineage comprised of the isolates from middle or low-latitude regions. The genetic differences between BAV strains are positively correlated with the geographic distribution. Strains from the same latitude regions are almost 100% identical, whereas the differences between strains from long distance regions with different latitudes could be >60%. Our results demonstrate that BAV is an emerging virus at a stage that involves rapid evolution and has great potential for introduction into non-endemic areas. Thus, enhanced surveillance of BAV is highly recommended worldwide.

Emergence of genotype I of Japanese encephalitis virus as the dominant genotype in Asia.

Japanese encephalitis virus (JEV), a mosquito-borne zoonotic pathogen, is one of the major causes of viral encephalitis worldwide. Previous phylogenetic studies based on the envelope protein indicated that there are four genotypes, and surveillance data suggest that genotype I is gradually replacing genotype III as the dominant strain. Here we report an evolutionary analysis based on 98 full-length genome sequences of JEV, including 67 new samples isolated from humans, pigs, mosquitoes, midges. and bats in affected areas. To investigate the relationships between the genotypes and the significance of genotype I in recent epidemics, we estimated evolutionary rates, ages of common ancestors, and population demographics. Our results indicate that the genotypes diverged in the order IV, III, II, and I and that the genetic diversity of genotype III has decreased rapidly while that of genotype I has increased gradually, consistent with its emergence as the dominant genotype.

Banna virus, China, 1987-2007.

Banna viruses (BAVs) have been isolated from pigs, cattle, ticks, mosquitoes, and human encephalitis patients. We isolated and analyzed 20 BAVs newly isolated in China; this finding extends the distribution of BAVs from tropical zone to north temperate climates and demonstrate regional variations in BAV phylogeny and mosquito species possibly involved in BAV transmission.

[Isolation and identification of arboviruses from mosquito pools in some regions of Liaoning province, China].

OBJECTIVE: To isolate and identify arboviruses from mosquito pools in some regions of Liaoning province. METHODS: Mosquitoes were collected from Shenyang, Yingkou, Panjin, Jinzhou and Dandong cities of Liaoning province in 2006. Viruses were isolated by inoculating the specimens onto C6/ 36 and BHK-21cells. The new isolates were identified using serological and molecular biological methods. RESULTS: 5410 mosquitoes were collected from the five cities in total. Three isolates produced CPE in C6/ 36 cell and five isolates produced CPE in both C6/36 and BHK-21 cell. Three isolates (LN0684, LN0688 and LN0689) were identified as Banna virus and one isolate (LN0636) was identified as Getah virus. Phylogenetic analysis showed that the three Banna virus strains were clustered into the same evolution branch as the other Chinese isolates. The identity of nucleotide sequence was between 91.2% and 94.7%, compared with other Banna virus strains. The new isolated Getah virus was clustered into the same branch with the strain of South Korea (swine). The identity of nucleotide sequence was 99.2%, when comparing with the strain of South Korea and was 95% to 99% with the strains from Russia, mainland of China and Taiwan region. Conclusion Eight virus isolates, including three Banna virus, one Getah virus and four unknown virus strains were isolated from mosquitoes in Liaoning province. Banna virus and Getah virus were reported for the first time in Liaoning province, while Getah virus showed the highest nucleotide homology with the South Korea strains.

[The first report of Kadipiro virus isolation in China].

5 strains of virus isolated from Culex tritaeniorhynchus, Anopheles sinensis and Armigeres subalbatus, which caused cytopathic effect in C6/36 cells, had been obtained in the survey of arboviruses in Northwestern Yunnan Province. China. The virus particles displayed 70 nanometers diameter (n=7) with no envelope but spikes on the surfaces. RNA-PAGE of the genomes of the isolates showed 6-5-1 profile. A fragment of the 12th segment sequence was amplified by a pair of specific primers for Kadipiro virus strain JKT-7075 in RT-PCR. The full length of the 12th segment was 758 nucleotides, BLAST analysis revealed the highest identity was 90% to JKT-7075. Phylogenetic analysis demonstrated that the isolates appeared to be Kadipiro viruses (Family Reoviridae). It was the first report of kadipiro virus isolation in China.

[A new member of Brevidensovirus, 0507JS11 virus isolated from Culex mosquitoes collected in Xinjiang].

OBJECTIVE: To probe the primary characteristic of 0507JS11 virus isolated from Culex sp. and determine the classification of 0507JS11 virus in taxonomy. METHODS: 0507JS11 virus was cultured in Aedes albopictus C6/36 cells and cytopathic effects (CPEs) were recorded. Electro-microscopic morphology of 0507JS11 virus was observed. Total DNA extract of 0507JS11 virus was detected by 1% Agarose Gel Electrophoresis. Complete genomic sequence of 0507JS11 virus was sequenced and then made phylogenetic analysis. RESULTS: 0507JS11 virus could cause CPEs in Aedes albopictus C6/36 cells. Viral particles have no envelope and appear icosahedron symmetry with diameter of 20 nm. The genome of 0507JS11 virus was positive single strand DNA (ssDNA) with full length of 3977 nt. However, a DNA band about 4 kbp was observed in the electrophoresis of total DNA extract of 0507JS11 virus. The coding region of the genome included three ORFs, ORF1 and ORF2 code NSP1 and NSP2, ORF3 codes VP. Phylogenetic analysis of the complete genomic sequence of 0507JS11 virus indicated an independent linear in Brevidensovirus. CONCLUSION: 0507JS11 virus is a new member in Brevidensovirus.

Tahyna virus and human infection, China.

In 2006, Tahyna virus was isolated from Culex spp. mosquitoes collected in Xinjiang, People's Republic of China. In 2007, to determine whether this virus was infecting humans, we tested serum from febrile patients. We found immunoglobulin (Ig) M and IgG against the virus, which suggests human infection in this region.

[Molecular characterization of Japanese encephalitis virus isolated from Gansu province in 2008].

OBJECTIVE: To sequence PrM and E gene of the Japanese encephalitis virus isolated from Gansu province in 2008 and analysis the genotype of new JEV isolates and the molecular characterization of E gene. METHODS: Computer software was used to analyze nucleic acid sequence and deduced amino acid sequence, and draw phylogenetic trees, including ClustalX2.09, MegAlign and Mega4. RESULTS: The six JEV strains were clustered in genotype I. 87.5%-87.9% identity in nucleotide sequence and 96.8%-97.2% identity in amino acid sequence were found in E gene when compared with the vaccine strain SA14-14-2. Eleven common amino acid differences were observed in E protein between new isolates and the vaccine strain. CONCLUSION: Genotype I JEVs were isolated from mosquitoes collected in Gansu province. The amino acid difference occurred in sites that were not the key ones affecting the antigenic of JEV.

[Genotype 1 JEV was isolated again from Liaoning Province, China, 2007].

OBJECTIVE: To isolate Japanese encephalitis virus (JEV) from mosquitoes collected in Liaoning province and analysis the genotype of new isolated JEV strains and the characters of nucleotide and amino acid in the E gene. METHODS: Collected mosquitoes in Dandong Liaoning Province in August, 2006. Virus isolation was using issue culture cells. Isolated viruses were identified by using serological and molecular methods. RESULTS: Two new JEV strains, LNDG07-02 and LNDG07-16, were isolated from 1500 mosquitoes were belonging to genotype 1. The identity of nucleotide and amino acid of E gene between new JEV strains and live attenuated vaccine strain SA14-14-2 were 87.8-88% and 97.2%, respectively. Total 11 amino acid sites were differences in E gene between new isolates and SA14-14-2. However, there were no differentiation between the new JEV strains and the isolates in Donggang 2002. CONCLUSION: Genotype 1 JEV was isolated again from Donggang, since the first isolation of this genotype in 2002. Genotype 1 JEV continues in existence in Donggang Liaoning Province.

Complete sequence characterization of isolates of Getah virus (genus Alphavirus, family Togaviridae) from China.

Ten virus isolates belonging to species Getah virus (GETV) have been obtained during surveys for arboviruses in China since 1964. Seven of these isolates (YN0540, YN0542, SH05-6, SH05-15, SH05-16, SH05-17 and GS10-2) were obtained during the current study. The full-length sequences of three Chinese isolates (M1, isolated in 1964; HB0234, isolated in 2002; YN0540, isolated in 2005) were determined. The full-length sequences of these isolates were respectively 11 696, 11 686 and 11 690 nt, and showed more than 97 % intraspecies identity. Deletions were found in the capsid protein of strain M1 and non-structural protein nsP3 of strain HB0234. The E2 gene and 3' UTR of all ten isolates were also characterized. The E2 gene of the Chinese GETV isolates showed nucleotide sequence identities of 98-100 % when compared with other GETV isolates. In the 3' UTR of the Chinese isolates, an insertion of 10 consecutive adenine residues (nt 189-198) appeared in strain M1, and 9 or 3 consecutive adenines were found towards the 3' end of the third RES in strains SH05-6 and SH05-15, respectively. The 3' UTRs of the Chinese isolates showed a deletion between positions 45 and 54 and nucleotide transitions at positions 43, 64 and 148. Sequence and phylogenetic analyses showed that there was a relatively high degree of conservation among GETV isolates. The isolation of GETV from various provinces in China and also in Russia and Mongolia (including regions of the northern tundra) are an indication of changes in the world distribution of this re-emerging virus.

Isolation and characterization of the full coding sequence of a novel densovirus from the mosquito Culex pipiens pallens.

During an investigation of arboviruses in China, a novel densovirus (DNV) was isolated from the adult female Culex pipiens pallens. The virus, designated Culex pipiens pallens densovirus (CppDNV), caused cytopathic effect in C6/36 cells. The virus particles were icosahedral, non-enveloped and had a mean diameter of 24 nm. The complete coding region of CppDNV was found to be 3335 nt and it contained three open reading frames (ORFs). CppDNV shares 82-93 % identical nucleotides with isolates of the Aedes albopictus densovirus [isolates AalDNV-1, AalDNV-2 (C6/36 DNV) and AalDNV-3], Aedes aegypti densovirus (AaeDNV) and Haemagogus equines densovirus (HeDNV). The nucleotide sequence identity among CppDNV isolates exceeds 98 %. Phylogenetic trees based on non-structural (NS1 and NS2) and capsid (VP) genes show that CppDNV clustered with the species AaeDNV and represents a novel variant of this species within the genus Brevidensovirus.

[Molecular analysis on the capsid gene and 3' untranslation region of three Getah viruses isolated in China].

To compare the molecular characteristics of the Capsid gene and 3' untranslation region (3'UTR) of Getah viruses (GETV) isolated in Hainan Province and Hebei Province of China,the viral RNAs were extracted from M1(Hainan), HB0215-3 and HB0234(Hebei) virus stocks. Capsid gene segments and 3' UTR segments from three strains of Chinese GETV were obtained by RT-PCR and then sequenced. The obtained nucleotide sequences were analyzed using the Clustal X(1.8), DNASTAR, MAGA3.1 programs. The full-length Capsid gene of the 3 strains of Chinese GETV were comprised of 801, 804 and 804 nucleotides each, encoding the protein of 267,268 and 268 amino acids each. Sequencing of Capsid gene fragments showed that two strains of Hebei isolates were identical and had homology of 97.6% at nucleotide level and 97.8% at amino acids level with M1. Their homologies when compared with strains isolated from other countries were also high at nucleotide levels (95.4%-99.6%). The 3'UTR from the three strains were comprised of 411, 401 and 401 nucleotides each, and had found specific deletion of 10 nt at position 44-54 and two specific nucleotide sites that was T at position 64 and C at position 148. GETV isolated in China presented relation of the year of virus isolation with the phylogenesis distance when compared with the other GETV strains and comprised a genetically highly conserved group.