OBJECTIVETo investigate the predominance ticks and the infectious status of severe fever with thrombocytopenia (SFTSV) in Penglai and Laizhou counties, Shandong province.

METHODSTwo towns with high incidence rate were selected in Penglai and Laizhou, respectively, then three villages were selected in each towns. Parasitic ticks were collected from the host skin by hand manually and free ticks manually with white cloth from the grassland, monthly, during April to December in 2011. Samples were classified by original, varieties, developmental stages, then extracted RNA, using Realtime RT-PCR to test severe fever thrombocytopenia syndrome virus, S fragments were amplified with nested PCR, then isolated virus. By neighbor joining method in the phylogenetic tree, the minimum infection rate (MIR) was used to represent the infection status of ticks in novel bunyavirus.

RESULTSA total of 3 145 ticks were collected totally from 5 categories, there were 3 048(96.92%) of Haemaphysalis longicornis, 73(2.32%) of Rhinpicephalus sanguineus, 10(0.32%) of microplus Boophilus, 9(0.29%) of Haemaphysalis campanulata, 5(0.16%) of Dermacentor sinicus, respectively. The positive rate of nucleic acid of 2 044 samples was 6.16% (126/2 044), minimum infection rate (MIR) was 4.01%, there were 122(96.83%) of Haemaphysalis longicornis, 3(2.38%) of Rhinpicephalus sanguineus, and 1(0.79%) of microplus Boophilus, MIR was 4.00%, 4.11%, and 10.00%, respectively. There were no nucleic acid positive samples in Haemaphysalis campanulata and Dermacentor sinicus. The 11 S segments were amplified in 126 positive samples, the homology of S fragment was 95.6%-99.9% with 11 strains isolated from the identified SFTS cases in local area, 3 strains isolated from animals, and 11 strains isolated from other areas. There was no significant difference among original, varieties and developmental stages.

CONCLUSIONHaemaphysalis longicornis was the predominant species in Penglai and Laizhou counties, it could be propagation medium with Rhipicephalus sanguineus and microplus Boophilus, S sequence in ticks was higher homology with virus isolated from local SFTS cases.

Animals ; China ; Phlebovirus ; isolation & purification ; Phylogeny ; Real-Time Polymerase Chain Reaction ; Ticks ; classification ; virology

Genus Phlebovirus is single negative-strand RNA virus, and belongs to family bunyaviridae. Its genomes have three segments including L, M and S encoding RNA-dependent RNA polymerase, envelope glycoprotein and nucleoprotein respectively. Phlebovirus is arbovirus and can be disseminated by arthropod. More than 70 types of Phlebovirus so far have been reported, and 68 known serotypes are divided into groups Sandfly fever and Uukuniemi, of which a few members are closely related to human diseases. In addition, new emerging viruses of genus Phlebovirus are discovered recently. In this review, the latest research progress in molecular characteristics, epidemiology, diagnosis, treatment and emerging viruses of Phlebovirus is summarized.
Animals ; Humans ; Phlebotomus Fever ; diagnosis ; epidemiology ; therapy ; virology ; Phlebovirus ; classification ; genetics ; isolation & purification ; physiology

Animals ; Biological Assay ; Nucleic Acid Amplification Techniques ; Phlebovirus/isolation & purification* ; Psychodidae/virology* ; Recombinases/metabolism* ; Reverse Transcription

No abstract available.
Animals ; Humans ; Insect Bites and Stings/virology ; Phlebotomus Fever/*etiology/prevention & control/transmission ; Phlebovirus/isolation & purification/physiology ; Ticks/*virology

To understand the immunogenicity of purified inactivated severe fever with thrombocytopenia syndrome bunyavirus (SFTSV), concentration by ultrafiltration as well as molecular-sieve chromatography (MSC) were used for purification of inactivated SFTSVs. Inactivated viruses in purified samples were analyzed and identified by western blotting and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the glycoprotein (GP) and nucleoprotein (NP) antigen titers of which were detected using a double-sandwich enzyme-linked immunosorbent assay (ELISA). Purified inactivated SFTSVs were enriched and observed by electron microscopy, and the total protein concentration detected using the bicinchoninic acid assay. Purified inactivated SFTSVs were applied to New Zealand rabbits via two immunization programs to evaluate immunogenicity and to compare the immune effect. After SFTSVs were inactivated and concentrated by ultrafiltration, MSC revealed two typical elution peaks. The sample of one peak was identified as inactivated virions, in which GP and NP were detected by SDS-PAGE, western blotting and ELISA. Main corponent of the other peak was NP. After concentration by ultrafiltration, purified inactivated SFTSVs with purity >90% and total protein concentration of 1. 1 mg/mL were obtained, and the typical electron microscopy of bunyavirus was observed. In the sera of animals immunized with purified inactivated SFTSVs, SFTSV-specific IgG antibody and neutralizing antibody were detected at high titers. However, antibody titers were affected by the immunization program. Effect of immunization on days 0, 14 and 28 was significantly better than that on days 0, 7 and 28. Our work revealed that cultivation of SFTSVs contained intact virus particles and large amounts of free NP. Using MSC, purified inactivated SFTSVs of high purity could be obtained. Purified inactivated SFTSVs induced high titers of neutralizing antibody and virus-specific IgG antibody showing satisfactory immunogenicity, which provides important clues for further study on a vaccine for the inactivated virus.
Animals ; Antibodies, Neutralizing ; immunology ; Antibodies, Viral ; immunology ; Bunyaviridae Infections ; immunology ; virology ; Humans ; Neutralization Tests ; Phlebovirus ; classification ; genetics ; immunology ; isolation & purification ; Rabbits

This article aimed to study the antigenicity of nucleocapsid proteins (NPs) in six pathogenic phleboviruses and to provide theoretical evidence for the development of serological diagnostic reagents. NPs of six pathogenic phleboviruses were expressed and purified using a prokaryotic expression system and rabbits were immunized with individual recombinant NPs. Cross-reactions among NPs and rabbit sera were determined by both indirect ELISA and Western blotting analyses, and the sera titer was determined by indirect ELISA. Furthermore, sera from SFTS patients were also detected by each recombinant NP as a coating antigen using indirect ELISA. The cross-reactions and the sera titer were subsequently determined. Both the concentration and purity of recombinant NPs of six pathogenic phleboviruses met the standards for immunization and detection. The results of indirect ELISA and Western blotting showed that each anti-phlebovirus NP rabbit immune serum had potential serological cross-reactivity with the other five virus NP antigens. Furthermore, the sera from SFTS patients also had cross-reactivity with the other five NP antigens to a certain extent. Our preliminary study evaluated the antigenicity and immune reactivity of six pathogenic phleboviruses NPs and laid the foundation for the development of diagnostic reagents.
Animals ; Antibodies, Viral ; immunology ; Antigens, Viral ; genetics ; immunology ; Cross Reactions ; Humans ; Nucleocapsid Proteins ; genetics ; immunology ; Phlebotomus Fever ; diagnosis ; immunology ; virology ; Phlebovirus ; classification ; genetics ; immunology ; isolation & purification ; Rabbits

Larvae, nymphs, and adult stages of 3 species of ixodid ticks were collected by tick drag methods in Seoul during June-October 2013, and their infection status with severe fever with thrombocytopenia syndrome (SFTS) virus was examined using RT-PCR. During the period, 732 Haemaphysalis longicornis, 62 Haemaphysalis flava, and 2 Ixodes nipponensis specimens were collected. Among the specimens of H. longicornis, the number of female adults, male adults, nymphs, and larvae were 53, 11, 240, and 446, respectively. Ticks were grouped into 63 pools according to the collection site, species, and developmental stage, and assayed for SFTS virus. None of the pools of ticks were found to be positive for SFTS virus gene.
Animals ; Female ; Fever/virology ; Humans ; Ixodidae/*classification/*virology ; Larva/pathogenicity ; Male ; Phlebotomus Fever/virology ; Phlebovirus/*isolation & purification ; Republic of Korea ; Thrombocytopenia/virology ; Tick Infestations/*virology

To understand the maintenance and transmission of SFTS virus, the potential vector ticks were collected from sheep, cattle and dogs in the endemic areas of SFTSV in Shandong Province. Among the collected ticks, the dominant species was H. longicornis ticks. Real-time PCR for RNA detection, virus isolation and characterization, genomic sequencing, phylogenetic and antigenic analysis were performed in this investigation. The results showed that the SFTS viral RNA was detected in 2.14% H. longicornis, and a SFTS virus was isolated from one of viral RNA positive ticks collected from sheep. Whole genome analysis of the SFTSV isolates with 11 human-origin SFTS virus revealed a highly pairwise similarity, and the growth curve analysis showed nearly identical in virus yield and the dynamic of virus reproduction compared to human derived viral isolates. Immunofluorescence and neutralization test showed identical serological reaction character of the two different origin viral strains. In this study, the characters of a SFTSV isolate was firstly described, which suggested that the tick species H. longicornis acting important vector role in the transmission of SFTS virus.
Animals ; Animals, Domestic ; parasitology ; Arachnid Vectors ; virology ; Bunyaviridae Infections ; transmission ; virology ; Cattle ; Cell Line ; Dogs ; Humans ; Livestock ; parasitology ; Molecular Sequence Data ; Phlebovirus ; classification ; genetics ; isolation & purification ; Phylogeny ; Sheep ; Ticks ; virology

In order to facilitate the detection of newly emerging or rare viral infectious diseases, a negative-strand RNA virus-severe fever with thrombocytopenia syndrome bunyavirus, and a positive-strand RNA virus-dengue virus, were used to investigate RNA viral genome unspecific amplification by multiple displacement amplification technique from clinical samples. Series of 10-fold diluted purified viral RNA were utilized as analog samples with different pathogen loads, after a series of reactions were sequentially processed, single-strand cDNA, double-strand cDNA, double-strand cDNA treated with ligation without or with supplemental RNA were generated, then a Phi29 DNA polymerase depended isothermal amplification was employed, and finally the target gene copies were detected by real time PCR assays to evaluate the amplification efficiencies of various methods. The results showed that multiple displacement amplification effects of single-strand or double-strand cDNA templates were limited, while the fold increases of double-strand cDNA templates treated with ligation could be up to 6 X 10(3), even 2 X 10(5) when supplemental RNA existed, and better results were obtained when viral RNA loads were lower. A RNA viral genome amplification system using multiple displacement amplification technique was established in this study and effective amplification of RNA viral genome with low load was achieved, which could provide a tool to synthesize adequate viral genome for multiplex pathogens detection.
Bunyaviridae Infections ; diagnosis ; virology ; Cell Line ; DNA Ligases ; metabolism ; DNA, Complementary ; analysis ; genetics ; DNA-Directed DNA Polymerase ; metabolism ; Dengue ; diagnosis ; virology ; Dengue Virus ; genetics ; isolation & purification ; Genome, Viral ; genetics ; Humans ; Phlebovirus ; genetics ; isolation & purification ; RNA, Viral ; analysis ; genetics ; Reference Standards ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Viral Load