Multi-epitope based subunit vaccine construction against Banna virus targeting on two outer proteins (VP4 and VP9): A computational approach.

Recently, RNA viruses have gained a mammoth concern for causing various outbreaks, and due to pandemics, they are acquiring additional attention throughout the world. An emerging RNA as well as vector-borne Banna Virus (BAV) is a human pathogen resulting in encephalitis, fever, headache, muscle aches, and severe coma. Besides human, pathogenic BAV was also detected from pigs, cattle, ticks, midges, and mosquitoes in Indonesia, China, and Vietnam. Due to high mutation tendency and dearth of a species barrier, this virus will consider as a significant threat in the near future throughout the planet, particularly in Africa. Despite of severe human case fatalities in several countries, there are no specific therapeutics, available vaccines, and other preventive measures against BAV. Thus, to find out the effective therapeutics and preventive strategies are crying exigency. In the present study, a unique multi-epitope-based peptide vaccine candidate is constructed using bioinformatics' tools that efficiently instigate immune cells for generating BAV antibodies. The potential vaccine candidates were developed using both T and B -cell epitopes. UniprotKB database was used to retrieve of two outer proteins (VP9 and VP4), and homologous sequences of BAV taxid: 7763, 649,604, 77,763, and 8453 were searched by NCBI BLAST. These serotypes are the most closely associated with the disease. Then combining the best-selected epitopes in various combinations with different adjuvants, three distinct vaccine candidates were formed. The validity tests were performed for the screened vaccine candidate regarding stability, allergenicity, and antigenicity parameters. Moreover, molecular dynamic simulations of the selected vaccine with TLR-8 immune receptor confirmed the stability of the binding pose and showed a significant response to immune cells. Thus, the results established that the designed chimeric peptide vaccine could enhance the immune response against BAV.

[Isolation and identification of Banna virus from mosquito for the first time in Inner Mongolia].

OBJECTIVE: To identify the virus isolated from a mosquito Culex modestus collected from Tongliao city of Inner Mongolia Autonomous Region. METHODS: A strain of virus isolated from mosquito in Tongliao city was identified by serological and molecular biological methods. The nucleotides of the virus isolate were amplified by RT-PCR, and the products were purified and sequenced. Multiple alignment, phylogenetic and amino acid (AA) analysis were carried out by software Clustal X, MEGA4 and MegAlign (DNAStar). RESULTS: The new isolate was identified to be Banna virus by serological and molecular biological methods. Phylogenetic analysis showed that the Chinese isolates were distributed within one cluster. The homologue of nucleotide and amino acid of 12 segments between the new isolate and other strains isolated from China were 89.6%-98.4% and 90.4%-98.6%. CONCLUSION: The virus isolated from Culex modestus in Inner Mongolia belonged to Banna virus, and it is the first time that Banna virus was isolated in this region.

Seroprevalence of tick-borne and mosquito-borne arboviruses in European brown hares in Northern and Western Germany.

Tríbec virus (Kemerovo serogroup, genus Orbivirus), Eyach virus (genus Coltivirus), and Tahyna virus (California encephalitis serogroup, genus Bunyavirus) are arthropod-borne viruses known to occur in Germany. These viruses are also suspected to cause human disease. So far, no information is available on their geographical distribution in Germany and their natural transmission cycles. A total of 166 sera from European brown hares (Lepus europaeus) collected in seven districts of the Federal State of Schleswig-Holstein and in four districts of the Federal State of North Rhine-Westphalia was tested by plaque reduction neutralization test (PRNT) for antibodies against Tríbec virus, Eyach virus, Tahyna virus, and Central European encephalitis virus. One out of 22 sera (4.5%) collected in the district Nord-Friesland in Schleswig-Holstein was found positive (PRNT(90) 1:10) against Tríbec virus. Neither did sera from other regions of Schleswig-Holstein nor from hares from North Rhine-Westphalia react against any of the arboviruses tested. For the first time, antibodies against Tríbec virus could be found in a European brown hare in Germany. The negative serological results for Central European encephalitis virus are in line with the current knowledge of its natural distribution within Germany. The negative serological results for Tahyna virus or Eyach virus argue against an autochthonous circulation of these viruses in the regions tested.

Studies of coltivirus in China.

OBJECTIVE: The purpose of this article is to review the developments of studies of Coltivirus in China. DATA SOURCES: The data used in this review was obtained mainly from the studies of Coltivirus reported from 1990 to 2003 in China. STUDY SELECTION: Relevant articles on studies of Coltivirus in domestic and foreign literature were selected. DATA EXTRACTION: Data were maily extracted from the articles which are listed in the reference section of this review. RESULTS: Many Coltiviruses have been isolated not only from blood samples of patients with unknown fever or from cerebrospinal fluid of patients with encephalitis in Xishuangbanna area in Yunnan province, but also from mosquitoes collected in many areas in China. In some patients diagnosed as Japanese encephalitis or unknown fever, an increase of Coltivirus IgG antibody of fourfold, or more, has been detected using ELISA. Similarly, Coltivirus IgM antibody was positive in some patients with Japanese encephalitis or viral encephalitis. From most Chinese patients, except the northeastern, the isolates of Coltiviruses belong to subgroup B2, according to RT-PCR amplification of the ninth and twelfth segments of the isolates and sequence analysis of their amplicons. Some biological properties of Chinese Coltiviruses isolates are different from that of North American Coltiviruses. CONCLUSIONS: The isolates of Coltiviruses from Chinese patients are one of the common agents causing viral encephalitis and unknown fever in summer-autumn season. It might be an important public health problem due to its high isolation rate and wide distribution in China. Mosquito is the main transmission vector of the virus.

Recombinant VP6-based enzyme-linked immunosorbent assay for detection of immunoglobulin G antibodies to Eyach virus (genus Coltivirus).

BACKGROUND: Eyach virus (EYAV) is a tick-borne virus belonging to genus Coltivirus, family Reoviridae. It was isolated in Germany and France and has been suspected to be responsible for neurological diseases in humans. To date, there has been no relatively rapid and relatively specific serological assay for EYAV. OBJECTIVES: To develop an ELISA for EYAV, suitable for epidemiological and/or diagnostic purposes. This ELISA should allow to distinguish between infections with EYAV and the related Colorado tick fever virus (CTFV). STUDY DESIGN: VP6, VP7 and VP12 of Eyach virus (the three proteins most divergent between EYAV and CTFV) were expressed in bacteria using the pGEX-4T-2 vector. A partial sequence of VP6 (designated pVP6) was chosen to develop an ELISA for detecting anti-EYAV IgG antibodies in serum. This choice was based on two observations: (i) the homologous VP7 protein of CTFV was successfully used as a target for detecting antibodies to CTFV (the VP7 showed the highest reactivity to an anti-CTFV antibody among all CTFV expressed proteins); (ii) to distinguish infection with EYAV from a CTFV infection: the expressed sequence was chosen within a region which is highly divergent (49% of amino acid identity) from the homologous VP7 sequence of CTFV. RESULTS AND CONCLUSIONS: pVP6 was shown to be the most reactive among the three expressed proteins. The elaborated pVP6 ELISA was evaluated with 340 sera of French blood donors, and found to exhibit a specificity of 100% (no false positives). Furthermore, no cross reaction was detected with antibody to CTFV, thus permitting us to distinguish between infections by either virus. The use of this recombinant protein for serological assays is a good alternative to the use of native EYAV antigen due to the extremely low productivity of the virus in cell culture, and the requirement for suckling mice. This ELISA will be useful to clarify the epidemiological status and the suspected pathogenicity of the virus.

Recombinant VP9-based enzyme-linked immunosorbent assay for detection of immunoglobulin G antibodies to Banna virus (genus Seadornavirus).

Banna virus (BAV, genus Seadornavirus, family Reoviridae) is an arbovirus suspected to be responsible for encephalitis in humans. Two genotypes of this virus are distinguishable: A (Chinese isolate, BAV-Ch) and B (Indonesian isolate, BAV-In6969) which exhibit only 41% amino-acid identity in the sequence of their VP9. The VP7 to VP12 of BAV-Ch and VP9 of BAV-In6969 were expressed in bacteria using pGEX-4T-2 vector. VP9 was chosen to establish an ELISA for BAV, based mainly on two observations: (i). VP9 is a major protein in virus-infected cells and is a capsid protein (ii). among all the proteins expressed, VP9 was obtained in high amount and showed the highest immuno-reactivity to anti-BAV ascitic fluid. The VP9s ELISA was evaluated in three populations: French blood donors and two populations (blood donors and patients with a neurological syndrome) from Malaysia, representing the region where the virus was isolated in the past. The specificity of this ELISA was >98%. In mice injected with live BAV, the assay detected IgG-antibody to BAV infection 21 days post-injection, which was confirmed by Western blot using BAV-infected cells. The VP9 ELISA permits to determine the sero-status of a population without special safety precautions and without any requirements to propagate the BAV. This test should be a useful tool for epidemiological survey of BAV.

[Preparation and storage of coltivirus antigen and its application in detection of coltivirus antibodies in serum from patients].

OBJECTIVE: To prepare purified and concentrated coltivirus high titer antigen in order to further detect antibodies against coltivirus in serum sample of patients. METHODS: The coltivirus in C6/36 cells was cultured and harvested at different time, and the titer was titrated. The virus was purified and concentrated by polyethylene glycol (PEG), and stored at -20 degrees and 4 degrees, with and without glycerol, respectively, then the titer of coltivirus antigen was tested by indirect ELISA. By using the antigen, coltivirus antibodies in serum samples from both suspected Japanese encephalitis (JE) and viral encephalitis (VE) patients were detected. RESULTS: The highest titer of coltivirus was found at 3-4 weeks of culturing. The antigen titer could be maintained at least for 6 months, especially antigen with glycerol either at 4 degrees or at -30 degrees even for two years. Totally 1141 serum samples from patients diagnosed clinically as JE and VE were tested. The results showed that 130 samples were coltivirus IgM antibody positive and the average positive rate was 11.4% (130/1141). Among 41 samples of paired-serum from patients in Guangzhou Children's Hospital, 9 samples were positive, the positive rate was 22.0% (9/41) in which 5 samples were diagnosed clinically as VE. CONCLUSIONS: Stable and purified coltivirus antigen was obtained in order to test coltivirus antibodies as well as development of kits. Coltivirus probably can cause summer-autumn encephalitis in China.

[Isolation of coltivirus from mosquitoes in Beijing and it's biological characteristics].

Two strains (Beijing 95-70 and 95-75) of coltivirus were isolated from mosquitoes collected in Beijing during summer-autumn in 1994. The viruses caused cytopathogenic effect on C6/36 cells but not on BHK-21 and Vero cells, and were not lethal for new born mice and three week old mice. The isolates were resistant to 5'-IdU and ether, but sensitive to acid pH 3.0. There were 12 segments RNA shown by polyacrylamide gel electrophoresis (PAGE) and PAGE profile of the two isolates were similar (6-6). The PAGE profile was also similar to that of the TRT2 strain isolated in 1991, but there were at least 8 segments slightly different between the new isolates and TRT2 in band migration distance. Tissue culture cross-neutralization test showed that the new isolates were antigenically related to TRT2 strain identified as coltivirus. Growth curve of the two isolates on C6/36 cells showed that virus titer began to rise at the first day after infection and continued to increase up to 7.0 Log TCID50 per 0.1 ml and maintained a high level until the 21st day after infection. It seems that the virus may cause persistent infection on C6/36 cell.