Sequences Of Genome Segments Research Articles

First report of pepper chlorosis-associated virus infecting tobacco (Nicotiana tabacum) plants in Sichuan Province in China.

Tobacco (Nicotiana tabacum) is an economically important crop in China, and more than 30 viruses have been reported to infect tobacco (Yin et al. 2022). In July 2022, we observed interveinal necrosis on tobacco leaves in fields in Sichuan Province (N 27.9172, E 105.6662) (Fig. 1). Total RNA was isolated from multiple leaves of one plant using an RNAprep Pure Polysaccharide Polyphenol Plant Total RNA Extraction Kit (TIANGEN, Beijing, China). Total RNAs were pooled, and a TruSeq Stranded Total RNA with RiboZero Gold Kit (Illumina, San Diego, CA, USA) was used to eliminate ribosomal RNA. An RNA-Seq library was constructed using VAHTS Universal V6 RNA-seq Library Prep (Nanjing Vazyme, China). High-throughput sequencing was performed on the Illumina DNBseq platform (BGI-ShenZhen, China), which yielded 20,102,087 reads with an average length of 150 nt (total size >6 Gb). Unaligned reads were assembled de novo using SPAdes (Bankevich et al. 2012). Contigs with length ≥200 nt were subjected to local BLASTn and BLASTx analyses against the GenBank nt and nr databases, respectively (Wang et al. 2022). A total of 23 contigs were identified through BLASTx (e-value cut-off = 10 -3), ranging from 631 to 1555 bp long, with 82% to 96% coverage to partial genomic sequences of pepper chlorosis-associated virus (PepCaV-Higashitsuno_2021; Accessions: LC719619 to LC719621) and one contig (6459 bp) with 99% similarity to tobacco mosaic virus (Accession: OP525281) isolate DSMZ PV-0109 from Germany. The complete genome sequence of PepCaV was obtained using primers based on the assembled contigs. The 5'- and 3'-terminal regions of the RNA genome were obtained by 5'- and 3'-rapid amplification of cDNA ends. These amplicons were cloned using the pEASY-Blunt Zero Cloning Kit (TRANSGEN, Nangjing, China) and sequenced by Sanger sequencing. Complete genome sequences of tripartite PepCaV from tobacco samples were 7697, 1808, and 1557 nucleotides long (Accession: OR451987 to OR451989) and showed genome organization typical of the genus Ophiovirus in the family Aspiviridae. The complete sequences of RNA1, RNA2 and RNA3 genome segments shared 92.36%, 90.43%, and 95.24%, nucleotide sequence identities, respectively, with the isolate PepCaV-Higashitsuno_2021 pepper isolate (Accession: LC719619 to LC719621) (Shimomoto et al. 2023), but PepCaV has not been reported to infect N. tabacum. In June 2023, 10 plants collected from each place of Macheng (N 27.9094, E 105.6740), Xiangyang (N 28.0936, E 105.6249) and Moni (N 27.8899, E 105.5936) showing interveinal necrosis symptoms were tested using RT-PCR using PepCaV-MP610-F (5'-TGTTCTCTGCTATGCGGTTG -3') and PepCaV-MP610-R (5'-AGCAATCTCGCACCTGAAGT-3') to product 610bp amplicon. Twenty-five tobacco plants were positive for PepCaV. Single sequence from each location was submitted to GenBank (Accession: PP728631 to PP728633). Sap extracts from the original field leaf samples collected from Sichuan Province were used to mechanically inoculate tobacco plants (10 plants) at the four-leaf stage. After 7 days, leaf samples were tested using RT-PCR assay specific to PepCaV and TMV while samples were positive only for TMV but failed to transmit PepCaV by mechanical inoculation. According to previous literature, ophioviruses may be transmitted though soilborne fungus (Jeong et al. 2014). Further research is needed to understand the transmission, epidemiology, and pathological properties of the PepCaV. To our knowledge, this is the first report documenting natural PepCaV infection of tobacco plants in China, providing a scientific basis for PepCaV infection control in tobacco plantations.

Full-length coding sequence analysis of genome segments A and B of infectious bursal disease virus in Thailand: identification of Chinese-like and recombinant virus in the field

ABSTRACT Infectious bursal disease virus (IBDV) causes immunosuppression in chickens, with distinctive bursal lesions. Genomic sequence information is essential to understand the epidemiology of antigenically and/or pathogenically variable IBDV strains. The present study investigated the full-length coding sequences of genome segments A and B of IBDV circulating in Thailand during 2010–2015. Five clinical samples of pooled bursas obtained from five commercial chicken farms experiencing problems with IBD in five different provinces of Thailand were identified by using a one-step reverse transcriptase polymerase chain reaction. The full-length coding sequences of genome segments A and B of three and five isolates, respectively, were successfully characterized. Phylogenetic analysis showed that two groups of IBDV were circulating in Thailand. The first group was similar to strain HLJ-504, genotype A3B3, isolated in China, but it had some minor variations in both genome segments A and B. The second group, which had a recombination event in segment A, was clustered with genotype A1B1. Its segment A originated from the intermediate vaccine W2512-like strain and isolate 9109, whereas segment B shared characteristics of a classical strain without recombination events. This study characterized the full-length coding sequences of genome segments A and B of Thai IBDV and demonstrated the presence of recombinant IBDV in Thailand for the first time. Therefore, the molecular characterization of the full-length coding sequences of both genome segments of IBDV is essential to monitor the emergence of new variant viruses, which is important for effective control and appropriate vaccine development. RESEARCH HIGHLIGHTS For the first time, this work demonstrated a recombinant IBDV strain in Thailand. Two genogroups of IBDV were found in Thailand: including HLJ-504-like and recombinant virus. Analysis of the full coding sequence is essential for monitoring emerging variant IBDV.

Diversity and Genetic Reassortment of Keystone Virus in Mosquito Populations in Florida.

Keystone orthobunyavirus (KEYV), a member of the genus Orthobunyavirus, was first isolated in 1964 from mosquitoes in Keystone, Florida. Although data on human infections are limited, the virus has been linked to a fever/rash syndrome and, possibly, encephalitis, with early studies suggesting that 20% of persons in the Tampa, Florida, region had antibodies to KEYV. To assess the distribution and diversity of KEYV in other regions of Florida, we collected >6,000 mosquitoes from 43 sampling sites in St. Johns County between June 2019 and April 2020. Mosquitoes were separated into pools by species and collection date and site. All pools with Aedes spp. (293 pools, 2,171 mosquitoes) were screened with a real-time reverse transcriptase polymerase chain reaction (rRT-PCR) assay that identifies KEYV and other closely related virus species of what was previously designated as the California encephalitis serogroup. In 2020, screening for KEYV was expanded to include 211 pools of Culex mosquitoes from sites where KEYV-positive Aedes spp. had been identified. rRT-PCR-positive samples were inoculated into cell cultures, and five KEYV isolates from Aedes atlanticus pools were isolated and sequenced. Analyses of the KEYV large genome segment sequences revealed two distinct KEYV clades, whereas analyses of the medium and small genome segments uncovered past reassortment events. Our data documented the ongoing seasonal circulation of multiple KEYV clades within Ae. atlanticus mosquito populations along the east coast of Florida, highlighting the need for further studies of the impact of this virus on human health.

Mammalian orthoreoviruses exhibit rare genotype variability in genome constellations.

Mammalian orthoreoviruses (reoviruses) are currently classified based on properties of the attachment protein, σ1. Four reovirus serotypes have been identified, three of which are represented by well-studied prototype human reovirus strains. Reoviruses contain ten segments of double-stranded RNA that encode 12 proteins and can reassort during coinfection. To understand the breadth of reovirus genetic diversity and its potential influence on reassortment, the sequence of the entire genome should be considered. While much is known about the prototype strains, a thorough analysis of all ten reovirus genome segment sequences has not previously been conducted. We analyzed phylogenetic relationships and nucleotide sequence conservation for each of the ten segments of more than 60 complete or nearly complete reovirus genome sequences, including those of the prototype strains. Using these relationships, we defined genotypes for each segment, with minimum nucleotide identities of 77-88% for most genotypes that contain several representative sequences. We applied segment genotypes to determine reovirus genome constellations, and we propose implementation of an updated reovirus genome classification system that incorporates genotype information for each segment. For most sequenced reoviruses, segments other than S1, which encodes σ1, cluster into a small number of genotypes and a limited array of genome constellations that do not differ greatly over time or based on animal host. However, a small number of reoviruses, including prototype strain Jones, have constellations in which segment genotypes differ from those of most other sequenced reoviruses. For these reoviruses, there is little evidence of reassortment with the major genotype. Future basic research studies that focus on the most genetically divergent reoviruses may provide new insights into reovirus biology. Analysis of available partial sequences and additional complete reovirus genome sequencing may also reveal reassortment biases, host preferences, or infection outcomes that are based on reovirus genotype.

Recovering influenza genomes from wild bird habitats for better avian flu surveillance

Background: Avian influenza viruses (AIVs) cause severe disease in poultry and dangerous zoonotic infections in humans. Outbreaks disrupt trade, threaten food security, and jeopardize public health. They also allow novel flu viruses to adapt to human hosts, threatening future pandemics and new types of seasonal flu. A cornerstone of outbreak prevention and pandemic preparedness is surveillance of AIVs in wild waterfowl, their natural animal reservoir. Current surveillance efforts focus on testing dead or captured birds, but rarely provide warning of dangerous strains. We have developed an alternative surveillance strategy using sediment specimens from wild bird habitats; since this is where virus-laden feces from the whole wild bird community accumulate, it likely contains an extensive collection of locally-circulating viruses. Here, we report a custom laboratory method to recover influenza genomes from sediment using targeted genomic sequencing. Methods and materials: A proof-of-concept experiment was conducted on 4 sediment specimens collected near Vancouver, Canada during the fall of 2016. Total RNA was extracted from these sediments and prepared into metagenomic sequencing libraries. Hemagglutinin, neuraminidase, and matrix segments of the influenza A virus genome were enriched using 600 custom hybridization probes, then sequenced on an Illumina MiSeq. qPCR targeting the matrix gene was used to estimate the fold-enrichment provided by the probes. Consensus sequences of hemagglutinin and neuraminidase genome segments were generated by mapping sequencing reads to subtype-specific references using the Burrows-Wheeler Aligner. Reference sequences were selected by querying the reads from each library against a BLAST database containing 8555 AIV sequences. Results: Influenza genome targets were enriched 19 million-fold following two sequential captures using our custom hybridization probe method. 17 haemagglutinin and neuraminidase genome segments were recovered from these sediment samples, 9 of which were at least 80% complete. Sequence analysis identified 6 subtypes of haemagglutinin (H3, H4, H6, H9, H10, H11) and 4 subtypes of neuraminidase (N1, N2, N6, N8, and N9). Phylogenetic analysis revealed they were closely-related to other AIV sequences previously isolated from wild birds in the same region of North America in recent years. Conclusion: Targeted genomic sequencing of wetlands sediment has demonstrated its potential as a viable complement to current AIV surveillance practices.

Prevalence of the Puumala orthohantavirus Strains in the Pre-Kama Area of the Republic of Tatarstan, Russia.

Puumala orthohantavirus (PUUV) causes nephropathia epidemica (NE), a mild form of hemorrhagic fever with renal syndrome (HFRS) commonly diagnosed in Europe. The majority of HFRS cases in the European part of Russia are diagnosed in the Volga Federal District, which includes the Republic of Tatarstan (RT). The current study aims to analyze the genetic variability of PUUV in Pre-Kama region of the RT bounded by the Volga, Kama, and Vyatka rivers. In 2017, bank voles were caught in seven isolated forest traps in the Pre-Kama region and for the 26 PUUV-positive samples, the partial small (S), medium (M), and large (L) genome segment sequences were obtained and analyzed. It was determined that all identified PUUV strains belong to the Russian (RUS) genetic lineage; however, the genetic distance between strains is not directly correlated with the geographical distance between bank vole populations. One of the identified strains has S and L segments produced from one parental strain, while the M segment was supplied by another, suggesting that this strain could be the reassortant. We suggest that the revealed pattern of the PUUV strains distribution could be the result of a series of successive multidirectional migratory flows of the bank voles to the Pre-Kama region in the postglacial period.

Characterization of the Puumala orthohantavirus Strains in the Northwestern Region of the Republic of Tatarstan in Relation to the Clinical Manifestations in Hemorrhagic Fever With Renal Syndrome Patients.

Over 1,000 cases of hemorrhagic fever with renal syndrome (HFRS) were recorded in the Republic of Tatarstan (RT) in 2015. HFRS is a zoonotic disease caused by several different Old World hantaviruses. In RT, Puumala orthohantavirus (PUUV) is a prevalent etiological agent of HFRS. We looked for the genetic link between the PUUV strains isolated from the bank voles and from the infected humans. In addition, possible correlation between the genetic makeup of the PUUV strain involved and different clinical picture of HFRS was investigated. Partial PUUV small (S) genome segment sequences were retrieved from 37 small animals captured in the northwestern region of RT in 2015. Phylogenetic analysis revealed that 34 PUUV sequences clustered with strains of the previously identified “Russia” (RUS) genetic lineage, while 3 remaining PUUV sequences clustered with the known lineage from Finland (FIN). Sequence comparisons showed that the majority of the S-segment sequences isolated in the current study displayed 98.2–100.0% sequence identity when compared with the strains isolated earlier from the HFRS patients hospitalized in Kazan city. HFRS patients infected with PUUV strains of either RUS or FIN genetic lineages were observed to have consistent differences in clinical presentation of the disease and laboratory findings. These findings indicated a strong genetic link between the infected bank voles and human HFRS cases from the same localities. Thus, S-segment sequences of the PUUV strains isolated from HFRS patients could serve as a molecular marker for determining the likely geographic area where infection occurred.

Complete Genome Segment Sequences of Tomato Chlorotic Spot Virus from Peanut in Haiti.

Tomato chlorotic spot virus (TCSV) is emerging as a significant constraint to vegetable and legume crops in the Americas. The complete genome sequence of a TCSV isolate naturally infecting peanut (Arachis hypogea) in Haiti was determined in the effort to build epidemiological knowledge of the virus.

Gga-miR-454 suppresses infectious bursal disease virus (IBDV) replication via directly targeting IBDV genomic segment B and cellular Suppressors of Cytokine Signaling 6 (SOCS6)

MicroRNAs (miRNAs), as post-transcriptional regulators, play important roles in the process of viral infection through inhibiting virus replication or modulating host immune response. However, the role of miRNAs in host response against infectious bursal disease virus (IBDV) infection is still unclear. In this study, we found that gga-miR-454 of the host was decreased in response to IBDV infection and that transfection of DF-1 cells with miR-454 inhibited IBDV replication via directly targeting the specific sequence of IBDV genomic segment B, while blockage of endogenous miR-454 by inhibitors enhanced virus replication. Furthermore, gga-miR-454 increased the expression of IFN-β by targeting Suppressors of Cytokine Signaling 6 (SOCS6), enhancing the antiviral response of host cells. These findings highlight a crucial role of gga-miR-454 in host defense against IBDV infection.

Phylogenetic analysis of Puumala virus strains from Central Europe highlights the need for a full-genome perspective on hantavirus evolution.

Puumala virus (PUUV), carried by bank voles (Myodes glareolus), is the medically most important hantavirus in Central and Western Europe. In this study, a total of 523 bank voles (408 from Germany, 72 from Slovakia, and 43 from Czech Republic) collected between the years 2007-2012 were analyzed for the presence of hantavirus RNA. Partial PUUV genome segment sequences were obtained from 51 voles. Phylogenetic analyses of all three genome segments showed that the newfound strains cluster with other Central and Western European PUUV strains. The new sequences from Šumava (Bohemian Forest), Czech Republic, are most closely related to the strains from the neighboring Bavarian Forest, a known hantavirus disease outbreak region. Interestingly, the Slovak strains clustered with the sequences from Bohemian and Bavarian Forests only in the M but not S segment analyses. This well-supported topological incongruence suggests a segment reassortment event or, as we analyzed only partial sequences, homologous recombination. Our data highlight the necessity of sequencing all three hantavirus genome segments and of a broader bank vole screening not only in recognized endemic foci but also in regions with no reported human hantavirus disease cases.

Complete cryspovirus genome sequences from Cryptosporidium parvum isolate Iowa.

Bisegmented dsRNA viruses that infect most or all isolates of apicomplexan parasite Cryptosporidium parvum are currently assigned to a single species, Cryptosporidium parvum virus 1, in genus Cryspovirus, family Partitiviridae. An analysis of existing sequence data suggested that the complete sequences of both cryspovirus genome segments, dsRNA1 and dsRNA2, had yet to be determined. We therefore set out to accomplish this for the virus strain that infects C. parvum isolate Iowa. The results suggest that several previous cryspovirus sequences are indeed truncated at one or both segment termini and also identify sequences at or near the termini that are conserved in both segments. Complete sequences of other cryspovirus strains, including ones from other Cryptosporidium species, are needed for refining their classification into one or more virus species.

Complete genome sequence and phylogenetic analyses of an aquabirnavirus isolated from a diseased marbled eel culture in Taiwan.

An aquabirnavirus was isolated from diseased marbled eels (Anguilla marmorata; MEIPNV1310) with gill haemorrhages and associated mortality. Its genome segment sequences were obtained through next-generation sequencing and compared with published aquabirnavirus sequences. The results indicated that the genome sequence of MEIPNV1310 contains segment A (3099 nucleotides) and segment B (2789 nucleotides). Phylogenetic analysis showed that MEIPNV1310 is closely related to the infectious pancreatic necrosis Ab strain within genogroup II. This genome sequence is beneficial for studying the geographic distribution and evolution of aquabirnaviruses.

Molecular technologies for detection and typing of bluetongue virus

Rapid and accurate diagnosis plays an important role in the implementation of effective measures to control the spread of disease. Historically, the laboratory diagnosis and typing of BTV were carried out by various serological and virological methods, including virus neutralization (VN) assay, ELISA, as well as virus isolation (VI) in cell cultures or in embryonated chicken eggs. At present, various molecular techniques to detect BTV genome are increasingly used as primary diagnostic tools for the serotyping and epidemiological investigations of BTV. Initially, the viral RNA was detected by simple nucleic acid hybridization technologies. Then, conventional RT-PCR assays were developed and evaluated for the detection of BTV serotypes based on nucleotide sequences of different genome segments. Although RT-PCR, with its increased sensitivity, has advantages over hybridization, it is almost impossible to quantify accurately by regular and multiplex PCR procedures, and regular PCR may produce false positive results. Over the recent years, a number of real-time RT-PCR (rRT-PCR) methods have been described. The rRT-PCR offers certain advantages over conventional RT-PCR assay, as it is more rapid, sensitive, and can provide quantitative as well as qualitative genetic information. It does not use agarose gel electrophoresis, decreases the risk of contamination because it is run within an enclosed tube, and is suitable for large-scale testing and automation. The target amplicon is usually smaller, reducing the potential for problems caused by target degradation. Loop-mediated isothermal amplification (LAMP), a novel rapid, accurate and cost effective gene amplification method, is an autocycling and strand displacement DNA synthesis method. LAMP assays have been applied as a method of detecting a variety of animal pathogens, including BTV. RT- LAMP assay can be a valuable tool complementing the routine laboratory diagnosis of BTV.

Molecular characterization of field infectious bursal disease virus isolates from Nigeria.

Aim:To characterize field isolates of infectious bursal disease virus (IBDV) from outbreaks in nine states in Nigeria through reverse transcription polymerase chain reaction (RT-PCR) and sequence analysis of portions of the VP2 and VP1 genes and to determine the presence or absence of reassortant viruses.Materials and Methods:A total of 377 bursa samples were collected from 201 suspected IBD outbreaks during 2009 to 2014 from nine states in Nigeria. Samples were subjected to RT-PCR using VP2 and VP1 gene specific primers, and the resulting PCR products were sequenced.Results:A total of 143 samples were positive for IBDV by RT-PCR. These assays amplified a 743 bp fragment from nt 701 to 1444 in the IBDV VP2 hypervariable region (hvVP2) of segment A and a 722 bp fragment from nt 168 to 889 in the VP1 gene of segment B. RT-PCR products were sequenced, aligned and compared with reference IBDV sequences obtained from GenBank. All but one hvVP2 sequence showed similarity to very virulent IBDV (vvIBDV) reference strains, yet only 3 of the VP1 67 VP1 sequences showed similarity to the VP1 gene of vvIBDV. Phylogenetic analysis revealed a new lineage of Nigerian reassortant IBDV strains.Conclusion:Phylogenetic analysis of partial sequences of genome segment A and B of IBDV in Nigeria confirmed the existence of vvIBDV in Nigeria. In addition, we noted the existence of reassortant IBDV strains with novel triplet amino acid motifs at positions 145, 146 and 147 in the reassorted Nigerian IBDV.

Genomic characterization of a broiler reovirus field strain detected in Pennsylvania

Avian reovirus (ARV) infections have been increasingly diagnosed in Pennsylvania (PA) from 2011 to the present time. A field ARV strain (Reo/PA/Broiler/05682/12) isolated from sick broilers suffered from severe arthritis/tenosynovitis of a PA broiler flock was conducted full genome sequence studies using RT-PCR and RACE techniques. The complete genome of the PA broiler ARV field strain was 23,494bp in length with an approximately 50% G+C content and 10 dsRNA segments encoding 12 viral proteins. The 10 genomic segments were ranged from 1192bp (S4) to 3958bp (L1) in length, which were almost identical to those of published reference strains. The amino acid (aa) alignments of the putative proteins encoded by the ORF in each segment revealed a high similarity to the counterpart proteins encoded by other avian orthoreoviruses in the Orthoreovirus genus, particularly with the reference strain AVS-B (89.4–98.9%). The phylogenetic analysis of the nucleotide sequences of all 10 genome segments of the PA broiler ARV revealed that a moderate to significant nucleotide sequence divergence from sequences of cognate genome segments of reference ARV S1133 and 138 strains. These genomic data demonstrate that this PA broiler ARV field strain is a unique tenosynovitis ARV and is different from the traditional ARV vaccine strains.

Molecular epidemiology studies on partial sequences of both genome segments reveal that reassortant infectious bursal disease viruses were dominantly prevalent in southern China during 2000-2012.

A molecular epidemiology study of infectious bursal disease viruses (IBDVs) isolated from seven provinces in southern China during the years 2000-2012 was performed based on partial sequences of genome segments A and B, namely the hypervariable region of the A-VP2 gene (A-vVP2) and the b fragment of VP1 gene (B-VP1b) from a total of 91 field isolates. Sequence analysis based on vVP2 revealed that 72 out of 91 isolates had the same characteristic amino acid (aa) sequences as vvIBDV. The mutation of D212N in A-vVP2 has become prevalent in the recent isolates. The origin of the field isolates with vvIBDV characteristic amino acid residues was complex, evidenced by the findings that more than one subgroup of strains prevailed in each province. When B-VP1b was analyzed, there were three lineages among the field isolates, and none of the isolates had a relationship to vvIBDV-related segment B. Phylogenetic analysis of both segments revealed that only a few isolates (13/91) had the same genetic relatives in consensus trees based on segments A and B, whereas the majority of the isolates (85.71%, 78/91) were identified to be naturally reassorted strains. Based on the origin of each segment, at least six types of reassortant IBDVs prevailed in southern China, three of which were shown to be dominant: segment A from vvIBDV and B from attenuated IBDV, segment A of vvIBDV and B from 002-73-like IBDV, and segment A of vvIBDV and B from HLJ0504 or a similar strain. Our findings suggest that both genomic segments of field IBDVs has been evolving, and continuous monitoring of the evolution of field IBDV genome is therefore urgently needed in the control of IBDV.

Molecular characterization of L class genome segments of a newly isolated turkey arthritis reovirus

Seven strains of turkey arthritis reovirus (TARV) isolated from cases of turkey arthritis were characterized on the basis of their L class genome segment sequences, which were then compared with those of turkey enteric reovirus (TERV) and chicken reovirus (CRV). All three L class gene segments of TARVs and TERVs and their encoded proteins λA, λB, and λC were similar in size to those of CRV reference strain S1133. The conserved motifs such as C2H2 zinc-binding motif and conserved polymerase region were present in λA and λB, respectively. A conserved motif for ATP/GTP-binding site and an S-adenosyl-l-methionine (SAM)-binding pocket for methyltransferase were observed in λC protein of TARVs and TERVs with only one substitution as compared to that in CRV. We propose a new genotype classification system for avian reoviruses (ARVs) based on the nt identity cut-off value for each of the L class. Based on this new genotype classification, all ARVs were divided into six, seven and eight genotypes in L1, L2 and L3 genes, respectively. Interestingly TARVs and TERVs grouped with three CRVs (two arthritic strains from Taiwan and one enteritic strain from Japan) in genotype L1-I and formed a different genotypes (L2-I, L3-I) from CRVs in L2 and L3 genes. The maximum nucleotide divergence was observed in genotypes of L1 and L2 genes but less at amino acid level indicates mostly changes were synonymous type. Compared to L1 and L2 genes, the nonsynonymous changes were more in L3 gene. Point mutations and possible reassortments among TARVs, TERVs and CRVs were also observed.

Phylogenetic relationships of Iranian Infectious Pancreatic Necrosis Virus (IPNV) based on deduced amino acid sequences of genome segment A and B cDNA

Infectious Pancreatic Necrosis Virus (IPNV) is the causal agent of a highly contagious disease that affects many species of fish and shellfish. This virus causes economically important diseases of farmed rainbow trout, Oncorhynchus mykiss, in Iran which is often associated with the transmission of pathogens from European resources. In this study, moribund rainbow trout fry were collected during an outbreak of IPNV in three different fish farms in one northern province (Mazandaran), and two west provinces (Chaharmahal and Bakhtiari, and Kohgiluyeh and Boyer Ahmad) of Iran. We investigated full genome sequence of Iranian IPNV and compared it with previously identified IPNV sequences. The sequences of different structural and non-structural protein genes were compared with other aquatic birnaviruses sequenced to date. Our results showed that the Iranian isolate fall within genogroup 5, serotype A2 strain SP, having 99 % identity with the strain 1146 from Spain. These results suggest that the Iranian isolate may have originated from Europe.

Full-Genome Sequencing of Four Bluetongue Virus Serotype 11 Viruses.

Recently, a contamination incident was described in which the challenge inoculum used in a bluetongue virus serotype 8 (BTV-8) vaccination trial was contaminated with a BTV-11 virus that was closely related to the Belgian BTV-11 virus from 2008. This study reports the first complete genome sequences of four BTV-11 viruses: the BTV-11 contaminant, BTV-11 reference strain, BTV-11 vaccine strain and a recently isolated BTV-11 field strain from Martinique. Full-genome analysis showed that these viruses belong to serotype 11/nucleotype A and cluster together with other western topotype bluetongue viruses. Detailed comparisons of the genomes further indicated that the contaminant was derived from the BTV-11 reference strain, as they were distinguished by a single synonymous nucleotide substitution. The previously reported partial sequence of genome segment 2 of the Belgian BTV-11 was found to be identical to that of the BTV-11 vaccine strain, indicating that it most likely was the BTV-11 vaccine strain. These findings also suggest that the BTV-11 contaminant and the Belgian BTV-11 are not the same viruses. Finally, comparison of the reference and vaccine strain did not allow determining the amino acid substitutions that contribute to the attenuated phenotype.

Whole-genome consensus sequence analysis of a South African rotavirus SA11 sample reveals a mixed infection with two close derivatives of the SA11-H96 strain

Whole-genome, sequence-independent amplification and 454(®) pyrosequencing of a rotavirus SA11 cell culture sample with an unknown passage history yielded consensus sequences of twelve complete genome segments. Two distinct sequences for genome segment 8 (encoding NSP2) were present, indicating a mixed infection with two rotavirus SA11 strains. The genotypes of the viruses were G3-P[2]-I2-R2-C5-M5-A5-Nx-T5-E2-H5, where x was either 5 or 2. The strains were named RVA/Simian-tc/ZAF/SA11-N5/1958/G3P[2] and RVA/Simian-tc/ZAF/SA11-N2/1958/G3P[2]. The genotype (N2) and sequence of genome segment 8 of RVA/Simian-tc/ZAF/SA11-N2/1958/G3P[2] were identical to that of the bovine rotavirus O agent. Five novel amino acids were detected in minor population variants of three genome segments. Genome segment 1 (VP1) has a high nucleotide substitution rate, but the substitutions are synonymous. Distance matrices and Bayesian molecular clock phylogenetics showed that SA11-N2 is a reassortant containing genome segment 8 from the O agent, whereas SA11-N5 is a very close derivative of the prototype SA11-H96.