Full genome characterization and evolutionary analysis of Banna virus isolated from Culicoides, mosquitoes and ticks in Yunnan, China.

Introduction: Banna virus (BAV), a potential pathogen that may cause human encephalitis, is the prototype species of genus Seadornaviru within the family Reoviridae, and has been isolated from a variety of blood-sucking insects and mammals in Asia. Methods: Culicoides, Mosquitoes, and Ticks were collected overnight in Yunnan, China, during 2016-2023 using light traps. Virus was isolated from these collected blood-sucking insects and grown using Aedes albopictus (C6/36) cells. Preliminary identification of the virus was performed by agarose gel electrophoresis (AGE). The full genome sequences of the BAVs were determined by full-length amplification of cDNAs (FLAC) and sequenced using next-generation sequencing. Results: In this study, 13 strains BAV were isolated from Culicoides, Mosquitoes and Ticks. Their viral genome consisted of 12 segments of double-stranded RNA (dsRNA), and with three distinct distribution patterns. Sequence analysis showed that Seg-5 of four strains (SJ_M46, SJ_M49, JC_M19-13 and JC_C24-13) has 435 bases nucleotide sequence insertions in their ORF compared to other BAVs, resulting in the length of Seg-5 up to 2128 nt. There are 34 bases sequence deletion in Seg-9 of 3 strains (WS_T06, MS_M166 and MS_M140). Comparison of the coding sequences of VP1, VP2, VP5, VP9 and VP12 of the 13 BAV strains, the results show that VP1, VP2 and VP12 are characterised by high levels of sequence conservation, while VP9 is highly variable, under great pressure to adapt and may be correlated with serotype. While also variable, VP5 appears to be under less adaptive pressure than VP9. Additionally, phylogenetic analysis indicates that the 13 BAV strains locate in the same evolutionary cluster as BAVs isolated from various blood-sucking insects, and are clustered according to geographical distribution. Conclusion: The data obtained herein would be beneficial for the surveillance of evolutionary characteristics of BAV in China and neighboring countries as well as extend the knowledge about its genomic diversity and geographic distribution.

Full Genome Sequencing of Three Sedoreoviridae Viruses Isolated from Culicoides spp. (Diptera, Ceratopogonidae) in China.

Sedoreoviridae is a family of viruses belonging to the order Reovirales and comprises six genera, two of which, Orbivirus and Seadornavirus, contain arboviruses that cause disease in humans and livestock. Areas such as Yunnan Province in southwestern China, have high arboviral activity due in part to warm and wet summers, which support high populations of biting flies such as mosquitoes and Culicoides. Three viral isolates previously obtained from Culicoides collected at cattle farms in Shizong County of Yunnan Province, China, between 2019 and 2020 were completely sequenced and identified as Banna virus (BAV) genotype A of Seadornavirus and serotypes 1 and 7 of epizootic hemorrhagic disease virus (EHDV) of Orbivirus. These results suggest that Culicoidestainanus and C. orientalis are potential vectors of BAV and EHDV, respectively, and represent the first association of a BAV with C. tainanus and of an arbovirus with C. orientalis. Analysis using VP9 generally agreed with the current groupings within this genus based on VP12, although the classification for some strains should be corrected. Furthermore, the placement of Kadipiro virus (KDV) and Liao ning virus (LNV) in Seadornavirus may need confirmation as phylogenetic analysis placed these viruses as sister to other species in the genus.

Evolutionary analysis of a newly isolated Banna virus strain from Yunnan, China.

Banna virus (BAV) is a typical arbovirus whose infection is associated with fever and viral encephalitis. The whole genome of BAV is composed of 12 RNA segments. BAVs, which have been divided into three genotypes (A, B, and C) based on phylogenetic analysis of segment 12 or segment 9, are currently undergoing rapid evolution. Recent studies have shown that BAV variation can exceed intraspecific limits and generate novel viruses. In the current study, a new BAV strain, named 113c5, was isolated from Culex tritaeniorhynchus and found to be a member of genotype A2 based on phylogenetic analysis of segments 9 and 12. The complete genome sequence of the new BAV strain described in the current study might contribute to the surveillance of evolutionary characteristics of BAVs.

Characterization of Novel Reoviruses Wad Medani Virus (Orbivirus) and Kundal Virus (Coltivirus) Collected from Hyalomma anatolicum Ticks in India during Surveillance for Crimean Congo Hemorrhagic Fever.

In 2011, ticks were collected from livestock following an outbreak of Crimean Congo hemorrhagic fever (CCHF) in Gujarat state, India. CCHF-negative Hyalomma anatolicum tick pools were passaged for virus isolation, and two virus isolates were obtained, designated Karyana virus (KARYV) and Kundal virus (KUNDV), respectively. Traditional reverse transcription-PCR (RT-PCR) identification of known viruses was unsuccessful, but a next-generation sequencing (NGS) approach identified KARYV and KUNDV as viruses in the Reoviridae family, Orbivirus and Coltivirus genera, respectively. Viral genomes were de novo assembled, yielding 10 complete segments of KARYV and 12 nearly complete segments of KUNDV. The VP1 gene of KARYV shared a most recent common ancestor with Wad Medani virus (WMV), strain Ar495, and based on nucleotide identity we demonstrate that it is a novel WMV strain. The VP1 segment of KUNDV shares a common ancestor with Colorado tick fever virus, Eyach virus, Tai Forest reovirus, and Tarumizu tick virus from the Coltivirus genus. Based on VP1, VP6, VP7, and VP12 nucleotide and amino acid identities, KUNDV is proposed to be a new species of Coltivirus Electron microscopy supported the classification of KARYV and KUNDV as reoviruses and identified replication morphology consistent with other orbi- and coltiviruses. The identification of novel tick-borne viruses carried by the CCHF vector is an important step in the characterization of their potential role in human and animal pathogenesis.IMPORTANCE Ticks and mosquitoes, as well Culicoides, can transmit viruses in the Reoviridae family. With the help of next-generation sequencing (NGS), previously unreported reoviruses such as equine encephalosis virus, Wad Medani virus (WMV), Kammavanpettai virus (KVPTV), and, with this report, KARYV and KUNDV have been discovered and characterized in India. The isolation of KUNDV and KARYV from Hyalomma anatolicum, which is a known vector for zoonotic pathogens, such as Crimean Congo hemorrhagic fever virus, Babesia, Theileria, and Anaplasma species, identifies arboviruses with the potential to transmit to humans. Characterization of KUNDV and KARYV isolated from Hyalomma ticks is critical for the development of specific serological and molecular assays that can be used to determine the association of these viruses with disease in humans and livestock.

First Isolation and Characterization of a Group C Banna Virus (BAV) from Anopheles sinensis Mosquitoes in Hubei, China.

Banna virus (BAV) is considered to be an emerging human pathogen that is transmitted by blood-sucking insects. BAV was isolated from various species of mosquitoes, midges, and livestock. It is widely distributed geographically, since it was identified in China, Vietnam, and Indonesia. Previously reported evolution studies of BAV indicated that BAV can be divided into two groups, including isolates from China and Vietnam clustered in group A, and Indonesian isolates in group B. In this study, we report the isolation of a new strain of BAV named HB14-71-01 from Anopheles sinensis mosquitoes from Hubei, China. An in vitro comparison study of the HB14-71-01 isolate and the group A BAV revealed differences based on observed cytopathic effect, plaque size, and viral growth rates. Additionally, the phylogenetic analysis indicated that the Hubei isolate belongs to a novel genotype of BAV and emerged nearly 105 years ago (95% highest posterior density (HPD): 35⁻434), unlike the two previously reported genotypes A and B. Our findings extend the knowledge about the genomic diversity and potential vectors/hosts of BAVs and will improve understanding of the relationships between genetic variation and pathogenicity.

Analysis of compositional bias and codon usage pattern of the coding sequence in Banna virus genome.

By using DNA Star, CUSP of EMBOSS, Codon W and IBM SPSS Statistics, nucleotide composition and codon usage pattern of 115 genes are researched in 37 BAVs. It shows that the composition of all genes prefers to AU, compared to CG, and for most of genes, the order is A, U, G and C in the virus. The ENC-values of the genes are slightly high which shows the weak codon bias, in which the codon bias of VP9 gene is the highest. The codon usage pattern of 12 different genes is different and related to their composition, their function or their host. For example, VP9 gene encoding viral spike protein in contact with different hosts spread dispersedly and VP1, VP2, VP3 and VP4 genes encoding the capsid protein are concentrated on first quadrant in correspondence analysis. The ENC of VP5 is correlated to GC3s in correlation analysis. The points of VP12 gene are tightly close to the expected curve in the ENC-plot analysis but GC12 is not related to GC3 in neutrality analysis. All analysis indicates the codon usage pattern of 12 genes is influenced by both natural selection and neutral mutation in a different extent in BAV. As a pathogen of viral encephalitis, compositional analysis and codon bias analysis of BAV can provide a theoretical basis for the disease control.

Identification and genetic analysis of Kadipiro virus isolated in Shandong province, China.

BACKGROUND: Kadipiro virus (KDV) belongs to the Reoviridae family, which consists of segmented, non-enveloped, double-stranded RNA viruses. It has previously been isolated from Culex, Anopheles, Armigeres and Aedes mosquitoes in Indonesia and China. Here, we describe the isolation and characterization of SDKL1625 from Anopheles sinensis mosquitoes in Shandong province, China. METHODS: In this study, we isolated Kadipiro virus in Aedes albopictus C6/36 cell culture and the complete genome sequencing was made by next generation sequencing. RESULTS: We isolated and characterized a Kadipiro virus from Anopheles sinensis mosquitoes in 2016 in Shandong province, China. Nucleotide and amino acid homology analysis of SDKL1625 showed higher levels of sequence identity with QTM27331 (Odonata, China, 2016) than with JKT-7075 (Culex fuscocephalus, Indonesia, 1981). The SDKL1625 has 86-97% amino acid identity with the JKT-7075, 88-99% amino acid identity with the QTM27331. Among the 12 fragments, VP1, VP2, VP4, VP6, VP7, VP9 and VP12 showed high amino acid identity (> 90%) and VP5 showed the lowest identity (86% and 88%). CONCLUSIONS: This is the first identification of KDV from mosquito in China. Virus morphology and genome organization were also determined, which will further enrich our understanding of the molecular biological characteristics of KDV and seadornaviruses.

Genome sequencing and phylogenetic analysis of Banna virus (genus Seadornavirus, family Reoviridae) isolated from Culicoides.

In an investigation of blood-sucking insects and arboviruses, a virus (YN12243) was isolated from Culicoides samples collected in the Sino-Burmese border region of Yunnan Province, China. The virus caused cytopathic effect (CPE) in C6/36 cells and passaged stably. Polyacrylamide gel analysis showed that the genome of YN12243 was composed of 12 segments of double-stranded RNA (dsRNA), with a distribution pattern of 6-6. The nucleotide and amino acid sequences of the coding region (1‒12 segments) were 17,803 bp and 5,925 amino acids in length, respectively. The phylogenetic analysis of VP1 protein (RdRp) revealed that YN12243 belonged to genus Seadornavirus of family Reoviridae, and further analysis indicated that YN12243 belongs to the Banna virus (BAV) genotype A2. Additionally, YN12243 was located in the same evolutionary cluster as BAV strains isolated from different mosquito species, suggesting that the BAV isolated from Culicoides does not have species barriers. These results indicate that Culicoides can also be a vector for BAV. In view of the hematophagous habits of Culicoides on cattle, horses, deer, and other large animals, as well as the possibility of spreading and causing a variety of animal arboviral diseases, it is important to improve infection detection and monitor the BAV in large livestock.

Isolation and characterization of Tarumizu tick virus: A new coltivirus from Haemaphysalis flava ticks in Japan.

During the course of tick-borne virus surveillance in Japan, three independent isolates of probably the same virus were obtained from three geographically distant populations of the hard tick Haemaphysalis flava. Genome analyses of the three isolates demonstrated that they were closely related but distinct strains of a novel virus, designated Tarumizu tick virus (TarTV), which has a genome of 12 double-stranded RNA segments. The development of the virus-induced cytopathic effects on BHK cells significantly varied according to virus strains. Ten out of 12 segments of TarTV appeared to encode putative orthologs or functional equivalents of viral proteins of Colorado tick fever virus (CTFV) and Eyach virus, suggesting that TarTV is the third member of the genus Coltivirus in the family Reoviridae. This was supported by the facts that the 5'- and 3'-terminal consensus sequences of coltivirus genomes were found also in TarTV genome, and segment 9 of TarTV had sequence and structural features that may mediate a stop codon read-through as observed in that of CTFV. However, segment 7 and 10 of TarTV had no significant sequence similarities to any other proteins of known coltiviruses. Electron microscopic analysis demonstrated that TarTV particle had a non-enveloped bilayer icosahedral structure, and viral inclusion bodies were formed in infected cells. TarTV could infect and replicate in several mammalian cell lines tested, but show no clinical symptoms in intracerebrally inoculated mice. Taken together, our findings provide new insights into genetic diversity and evolution of the genus Coltivirus.

The plasma virome of febrile adult Kenyans shows frequent parvovirus B19 infections and a novel arbovirus (Kadipiro virus).

Viral nucleic acids present in the plasma of 498 Kenyan adults with unexplained fever were characterized by metagenomics analysis of 51 sample pools. The highest to lowest fraction of plasma pools was positive for parvovirus B19 (75 %), pegivirus C (GBV-C) (67 %), alpha anellovirus (59 %), gamma anellovirus (55 %), beta anellovirus (41 %), dengue virus genotype 2 (DENV-2) (16 %), human immunodeficiency virus type 1 (6 %), human herpesvirus 6 (6 %), HBV (4 %), rotavirus (4 %), hepatitis B virus (4 %), rhinovirus C (2 %), Merkel cell polyomavirus (MCPyV; 2 %) and Kadipiro virus (2 %). Ranking by overall percentage of viral reads yielded similar results. Characterization of viral nucleic acids in the plasma of a febrile East African population showed a high frequency of parvovirus B19 and DENV infections and detected a reovirus (Kadipiro virus) previously reported only in Asian Culex mosquitoes, providing a baseline to compare with future virome studies to detect emerging viruses in this region.

[Analysis on molecular genetic evolution of Banna virus based on the 12th segment].

Objective: To understand the evolution characteristics of Banna viruses (BAVs) isolated worldwide from 1980 to 2012. Methods: In this study, a phylogenetic analysis using Bayesian Markov Chain Monte Carlo simulations was conducted on all available 12th segment of genes of BAVs isolated worldwide from 1980 to 2012 to investigate the evolutionary and epidemiologic dynamics of BAVs. Results: The Bayesian phylogenetic analysis of BAVs revealed that the common ancestor of BAVs appeared 315 (95%HPD: 63-619) years ago. The evolutionary rate of BAV based on the 12th segment gene was estimated to be 2.33×10-3 (95%HPD: 2.84× 10-4-8.52×10-3) substitution per site per year, indicating BAV belong to an emerging arbovirus with rapid evolution. Conclusion: The evolution of emerging BAVs is rapid and the distribution of BAVs has expanded with new variant being detected, so it is necessary to enhance the surveillance to fully understand the natural distribution and pathogenicity of BAVs.

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.

[Isolation and identification of a Banna virus strain from mosquitoes in Yunnan province].

OBJECTIVE: To isolate and identify Banna virus (BAV) from mosquitoes collected in Mengla county of Yunnan province. METHODS: Mosquito samples were collected in houses and stock yards in Mengla county, 2008. Mosquitoes were homogenized and incubated onto both C6/36 and BHK21 cells. The new isolate was identified by using ELISA and RT-PCR. The sequences of segment 5, 8 and 11 of BAV were amplified by RT-PCR and determined. Phylogenetic analysis on the new BAV were performed using MEGA4 program. RESULTS: 1731 mosquitoes representing 7 species were collected with one strain of BAV isolated and identified. Phylogenetic analysis based on sequences of segment 8 showed the new isolate was closed to BAV strain isolated in Yunnan, but segment 11 sequence was closed to Vietnam strain. CONCLUSION: Results of phylogenetic analysis implied that the BAV re-assortment might have been occurred both in Chinese and Vietnam strains.

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.

[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.

Isolation and molecular characterization of Banna virus from mosquitoes, Vietnam.

We isolated and characterized a Banna virus from mosquitoes in Vietnam; 5 strains were isolated from field-caught mosquitoes at various locations; Banna virus was previously isolated from encephalitis patients in Yunnan, China, in 1987. Together, these findings suggest widespread distribution of this virus throughout Southeast Asia.

Complete characterisation of the American grass carp reovirus genome (genus Aquareovirus: family Reoviridae) reveals an evolutionary link between aquareoviruses and coltiviruses.

An aquareovirus was isolated from several fish species in the USA (including healthy golden shiners) that is not closely related to members of species Aquareovirus A, B and C. The virus, which is atypical (does not cause syncytia in cell cultures at neutral pH), was implicated in a winter die-off of grass carp fingerlings and has therefore been called 'American grass carp reovirus' (AGCRV). Complete nucleotide sequence analysis of the AGCRV genome and comparisons to the other aquareoviruses showed that it is closely related to golden ide reovirus (GIRV) (>92% amino acid [aa] identity in VP5(NTPase) and VP2(Pol)). However, comparisons with grass carp reovirus (Aquareovirus C) and chum salmon reovirus (Aquareovirus A) showed only 22% to 76% aa identity in different viral proteins. These findings have formed the basis for the recognition of AGCRV and GIRV as members of a new Aquareovirus species 'Aquareovirus G' by ICTV. Further sequence comparisons to other members of the family Reoviridae suggest that there has been an 'evolutionary jump,' involving a change in the number of genome segments, between the aquareoviruses (11 segments) and coltiviruses (12 segments). Segment 7 of AGRCV encodes two proteins, from two distinct ORFs, which are homologues of two Coltivirus proteins encoded by genome segments 9 and 12. A similar model has previously been reported for the rotaviruses and seadornaviruses.

[Detection of Banna virus-specific nucleic acid with TaqMan RT-PCR assay].

BACKGROUND: To develop a rapid and more sensitive TaqMan RT-PCR assay specific for Banna virus. METHODS: Total RNA of strains of Banna virus and other arboviruses were extracted and reverse transcribed to cDNAs. With the cDNAs as templates, the TaqMan RT-PCR assay was developed and optimized on ABI 7300 apparatus for specific-detection of Banna virus. The sensitivity, specificity and the possibility of this assay to detect Banna virus in pools of mosquitoes. Meanwhile, human sera samples added with different dilutions of Banna virus culture medium supernatant were evaluated. RESULTS: All the collected 12 Banna virus strains in our country were detected as positive, while the results of other arboviruses such as Japanese encephalitis virus (JEV), Batai virus, Sindbis virus and Orbiviruses were negative. This assay was more than 100 times sensitive than that of traditional PCR. About 0.5 CCID-50 of Banna virus in 50 microl samples could be detected with this assay. The sensitivity decreased by about 10 times when the dsRNA was denatured in 65 degrees C instead of 99 degrees C, and also when the virus seeds were kept at room temperature for 1 day or 1 week at 4 degrees C. There was no significant difference in the results of Banna virus detection between artificial human serum samples and positive control virus. Six were detected as positive for Banna virus-specific nucleic acids in 112 pools of mosquitoes, while 3 was positive with virus isolation. CONCLUSION: The Banna virus-specific TaqMan RT-PCR assay was proved to be highly sensitive, specific and showed a good reproducibility; the assay may be applied for surveillance of this virus in clinical samples and pools of mosquitoes screening.