Sequence virus genome

Two different extraction methodologies were compared to investigate the suitability of the RNA obtained. Both brain and TCSN were sequenced. For analysis see Table 2. Details of brain and BHK cultured samples, relating to the extraction method used and RNA concentration during preparation stages resulting in number of reads obtained and outcome of obtaining full viral genome sequence.

Brain tissue samples ordered by concentration after the depletion process. Furthermore, de novo assembly on two of the three samples RV and RV failed to align viral reads into contigs for further analysis resulting in only host contigs being identified Table 2.

Interestingly, this sample was part of a cohort of samples that were highly degraded upon receipt, therefore the majority of RNA had already been degraded.

Otherwise a reduction of concentration between 3-fold and fold was observed Table 2. We investigated the requirement to deplete gDNA and rRNA in cultured viral samples after RNA extraction, since the amount of cellular material would be minimal in these supernatant preparations.

The virus titer of RV and RV20 has been calculated previously [ 23 , 24 ] with RV EBLV-2 approximately 1 log lower than RV20, therefore the difference in the percentage of viral reads is likely to be a reflection of this. Despite the marked difference between the percentage of viral reads of RV20 and RV, the difference within samples regarding whether the RNA was depleted or not, is not so obvious. Indeed, for both samples, the RNA sequenced directly without depletion provided more viral-specific reads.

The success of the methodology for both tissue material and cell cultured material, particularly the ability to detect viral sequence after de novo assembly, is illustrated in Table 2.

Apart from the column-extracted samples, all sequences obtained were sufficient to obtain viral specific reads either as a single contig, or a number of contigs, which subsequently can be identified by a BLAST search. The depth of viral read coverage of samples from which a single genome contig was obtained was investigated Figure 1.

Brain tissue sample read depths varied from RV50, total viral reads , maximum read depth 16, and average read depth of 8. RV61 did have a higher number of viral reads, but from two runs, therefore not directly comparable. Depth of sequence obtained from a infected brain samples and b from cultured samples. Due to the processes involved, each read is obtained from a single cDNA strand, and therefore where multiple reads cover a region viral heterogeneity can be detected within the reads indicating the presence of a heterologous viral population.

Although this methodology is not optimized for investigation of low level viral populations, which would require viral read depths of over 10,, it is still possible to observe dominant or high level single nucleotide polymorphisms SNPs.

Substitutions were identified throughout the genome, although only one was identified outside of a coding region RV20 at position in the M-G untranslated region. Analysis of currently available lyssavirus glycoprotein sequences, indicates this residue is Isoleucine in the majority of lyssavirus species.

RV61 glycoprotein sequence is available in Genbank, deposited separately by two independent laboratories. No original material is available for analysis and the published sequences are from passaged viruses. RV50, a US isolate isolated from Eptesicus fuscus , was genome sequenced using Sanger sequencing as part of a larger dataset [ 6 ], providing a unique opportunity to analyze the same virus isolate, propagated and sequenced independently.

RV was sequenced both from the host brain tissue directly and from cell culture supernatant after 6 passages in BHK cells. To investigate this variation further, specific primers were designed to amplify this region from the original brain sample and the passaged TCSN.

The original brain material PCR amplicon had a read depth of reads, This deep sequence analysis is a useful tool to investigate certain SNPs of interest indicated from the consensus data. RV, a raccoon-dog RABV from Estonia, had good coverage across the genome average read depth 44, maximum read depth and also had the most SNPs observed within a tissue sample Table 3.

The remaining two were both silent, one in the glycoprotein gene genome position and the other in the L-gene genome position Table 3. Full genome sequence had previously been generated using overlapping PCR products and Sanger sequencing [ 1 ]. Published sequences of all lyssavirus species including the recently identified IKOV; [ 22 ] have Gly The read coverage at this position was 6 reads, 5 of which contained the Glu 57 SNP and 1 contained the original Gly 57 , present in the Sanger sequencing and all other lyssaviruses.

The genomic termini sequences from data were obtained with varying success. Often de novo assembly failed to incorporate reads which contained the genomic termini. These reads were only incorporated after splicing the missing sequence, from a published similar genomic sequence, to the consensus sequence deduced from the de novo assembly and subsequently mapping reads against the spliced reference using GS mapper.

Subsequent to this dataset, in an attempt to increase the population of genomic end viral reads, the sample preparation methodology was modified by the addition of 1pmol of N and LRACEF2 at the hexamer cDNA synthesis stage, to enhance the cDNA population containing the genomic termini.

This modification was trialed on a genome sample highly related to RV, where the genomic ends were obtained data not shown. This simple modification will be used for future genome sequences. For samples where the genomic termini were still absent, RT-PCR was performed on the depleted RNA, using primers designed against the highly conserved genomic termini to obtain PCR products which were sequenced directly using Sanger sequencing.

Although this approach resulted in a genome sequence still lacking the sequencing relating to the primer sites, for known lyssavirus species isolates this is acceptable. Whenever possible, consensus sequences where aligned together with genome sequences from the same lyssavirus species. Any potential insertions or deletions indels were investigated by analysis of the flowgrams in the GS program suite. A limitation of pyrosequencing processing of raw data is runs of homopolymers such as the GAAAAAAA poly A termination signal found at the end of each lyssavirus gene sequence Figure 3 a and b.

Burkholderia phage BcepC6B. Burkholderia phage BcepF1. Burkholderia phage BcepGomr. Burkholderia phage BcepIL Burkholderia phage BcepMigl. Burkholderia phage BcepMu. Burkholderia phage BcepNY3. Burkholderia phage BcepNazgul. Burkholderia phage BcepSaruman.

Burkholderia phage BcepSauron. Burkholderia phage Bp-AMP1. Burkholderia phage DC1. Burkholderia phage FLC5. Burkholderia phage JG Burkholderia phage KL3. Burkholderia phage KS Burkholderia phage KS5. Burkholderia phage KS9.

Burkholderia phage Mana. Burkholderia phage Mica. Burkholderia phage ST Burkholderia phage phib. Burkholderia phage phi Burkholderia phage phiE Burkholderia virus phi Butyrivibrio virus Arawn. Callitrichine gammaherpesvirus 3. Campylobacter phage CP Campylobacter phage CP30A. Campylobacter phage CPX. Campylobacter phage NCTC Campylobacter phage PC Campylobacter virus CP Campylobacter virus CPt Canid alphaherpesvirus 1.

Caprine alphaherpesvirus 1. Caprine gammaherpesvirus 2. Caulobacter phage CcrBL Caulobacter phage CcrBL9. Caulobacter phage CcrColossus. Caulobacter phage CcrPW. Caulobacter phage CcrSC. Caulobacter phage Cr Caulobacter phage Lullwater. Caulobacter phage Percy. Caulobacter phage Sansa. Caulobacter phage Seuss. Caulobacter virus Karma. Caulobacter virus Magneto. Caulobacter virus Rogue. Caulobacter virus Swift.

Caulobacter virus phiCbK. Caviid betaherpesvirus 2. Cebine betaherpesvirus 1. Celeribacter phage PL. Cellulophaga phage phi Cellulophaga phage phiSM. Cellulophaga phage phiST. Cercopithecine alphaherpesvirus 2. Cercopithecine alphaherpesvirus 9. Cercopithecine betaherpesvirus 5.

Papio ursinus cytomegalovirus. Cervid alphaherpesvirus 1. Cervid alphaherpesvirus 2. Cervid alphaherpesvirus 3. Chimpanzee herpesvirus strain Citrobacter phage CF1 DK Citrobacter phage CR44b. Citrobacter phage CR8.

Citrobacter phage CVT Citrobacter phage Margaery. Citrobacter phage Merlin. Citrobacter phage Michonne. Citrobacter phage Miller. Citrobacter phage Moogle. Citrobacter phage Moon. Citrobacter phage Mordin.

Citrobacter phage PhiZZ Citrobacter phage PhiZZ6. Citrobacter phage SH1. Citrobacter phage SH2. Citrobacter phage SH3. Citrobacter phage SH4. Citrobacter phage Sazh. Citrobacter phage Stevie. Citrobacter phage phiCFP Citrobacter virus HCF1. Clavibacter phage CMP1. Clavibacter phage CN1A. Clostridium phage CDKM Clostridium phage CDKM9. Clostridium phage CDMH1. Clostridium phage CPD2. Clostridium phage CPS1. Clostridium phage CPS2. Clostridium phage CpV1.

Clostridium phage PhiS Clostridium phage c-st. Clostridium phage phi CD Clostridium phage phi24R. Clostridium phage phi Clostridium phage phiB1. Clostridium phage phiCD Clostridium phage phiCDHM Clostridium phage phiCP13O. Clostridium phage phiCP26F.

Clostridium phage phiCP34O. Clostridium phage phiCPO. Clostridium phage phiCP7R. Clostridium phage phiCPV4. Clostridium phage phiCTA. Clostridium phage phiCTB. Clostridium phage phiCTC. Clostridium phage phiCTC2A. Clostridium phage phiCTC2B. Clostridium phage phiCTP1. Clostridium phage phiMMP Clostridium phage phiSM Clostridium phage phiZP2. Clostridium phage susfortuna. Clostridium virus phiC2. Clostridium virus phiCD Columbid alphaherpesvirus 1. Falconid herpesvirus 1. Colwellia phage 9A.

Common bottlenose dolphin gammaherpesvirus 1 strain Sarasota. Corynebacterium phage Adelaide. Corynebacterium phage BFK Corynebacterium phage C3PO. Corynebacterium phage Darwin. Corynebacterium phage Dina. Corynebacterium phage Juicebox. Corynebacterium phage Kimchi Corynebacterium phage Lederberg. Corynebacterium phage P Corynebacterium phage Poushou.

Corynebacterium phage SamW. Corynebacterium phage StAB. Corynebacterium phage Stickynote. Corynebacterium phage Stiles. Corynebacterium phage Zion.

Corynebacterium phage phi Cricetid gammaherpesvirus 2. Croceibacter phage PS. Croceibacter phage PY. Cronobacter phage CR3. Cronobacter phage CR5. Cronobacter phage CR8. Cronobacter phage CR9. Cronobacter phage CS Cronobacter phage Dev-CD Cronobacter phage Dev2. Cronobacter phage ENT Cronobacter phage ESSI Cronobacter phage GW1. Cronobacter phage JC Cronobacter phage PBES Cronobacter phage Pet-CM Cronobacter phage S Cronobacter phage phiES Cronobacter virus Esp Curvibacter phage PB.

Cyanophage a. Cyanophage MED Cyanophage P-RSM1. Cyanophage P-RSM6. Cyanophage P-SSP2. Cyanophage P-TIM Cyanophage PP. Cyanophage PSS2. Cyanophage S-RIM Cyanophage S-TIM4. Cyanophage S-TIM5.

Cyanophage SS Cynomolgus cytomegalovirus. Cynomolgus macaque cytomegalovirus strain Ottawa. Cyprinid herpesvirus 1. Cyprinid herpesvirus 2. Cyprinid herpesvirus 3. Deep-sea thermophilic phage D6E. Delftia phage PhiW Delftia phage RG Dickeya phage Dagda. Dickeya phage Katbat. Dickeya phage Luksen. Dickeya phage Mysterion. Dickeya phage Ninurta. Dickeya phage RC Dickeya virus Limestone. Dinoroseobacter phage DFL12phi1. Dinoroseobacter phage DSWs Dunaliella viridis virus SI2. Edwardsiella phage Edno5.

Edwardsiella phage GF Edwardsiella phage KF Edwardsiella phage MSW Edwardsiella phage PEi Edwardsiella phage eiAU. Edwardsiella phage eiAU Edwardsiella virus pEtSU. Eggerthella phage PMBT5. Elephant endotheliotropic herpesvirus 4. Elephant endotheliotropic herpesvirus 5. Elephantid betaherpesvirus 1. Enterobacter phage Arya. Enterobacter phage CC Enterobacter phage E Enterobacter phage EcP1. Enterobacter phage EcpYZU Enterobacter phage Enc Enterobacter phage EspM4VN. Enterobacter phage F Enterobacter phage KNP3.

Enterobacter phage KNP7. Enterobacter phage PG7. Enterobacter phage Tyrion. Enterobacter phage myPSH Enterobacter phage phiEap Enterobacter phage phiKDA1. Enterobacter phage phiTH. Enterobacteria phage Enterobacteria phage 9g. Enterobacteria phage ATK Enterobacteria phage Aplg8. Enterobacteria phage BP Enterobacteria phage ES Enterobacteria phage GEC-3S.

Enterobacteria phage GiZh. Enterobacteria phage HK Enterobacteria phage IME Enterobacteria phage JenK1. Enterobacteria phage JenP1. Enterobacteria phage JenP2. Enterobacteria phage Kha5h. Enterobacteria phage O Enterobacteria phage P4.

Enterobacteria phage P7. Escherichia phage P1. Enterobacteria phage P Enterobacteria phage RB Enterobacteria phage ST Enterobacteria phage Sf Enterobacteria phage SfI. Enterobacteria phage SfV. Enterobacteria phage T3.

Enterobacteria phage T6. Enterobacteria phage T7M. Enterobacteria phage VT2-Sakai. Enterobacteria phage YYZ Enterobacteria phage cdtI. Sign Up. Edit Profile. Subscribe Now. Your Subscription Plan Cancel Subscription. Sign out. Home India News Entertainment. Certainly, positive tests are no cause for celebration, but for Thielen and Mehoke, they are a key to learning more about the rapidly spreading virus. There are a lot of things we can glean from that.

Topping that list is the ability to see how quickly the virus mutates—integral information for mapping its spread, as well as developing an effective vaccine. Influenza, for example, mutates constantly. That's why it's necessary to vaccinate against different strains of the flu each year. Based on the mutation rate, early data indicates that this would likely be a single vaccine rather than one that needs to be updated each year, like the flu shot.

With the United States continuing to ramp up testing and mitigation capabilities, the ability to understand how outbreaks are linked gives public health departments another tool for evaluation.

Mutations can explain how long the virus may have gone undetected and the supposition that there are likely far more cases than diagnosed, and can advise on what measures to put in place such as the social-distancing efforts and closings that are ongoing nationwide.