Aedes albopictus mosquitos collected in August and September 2019 in Ticino, a region in southern Switzerland, to address the question regarding the risk of the local transmission of zoonotic viruses. A total of 13 viruses from seven different virus families and several unclassified viral taxa were identified. Reads of insect-specific flaviviruses were present in all pools, and a complete genome of aedes flavivirus was assembled and phylogenetically analysed. The most abundant virus was Wenzhou sobemo-like virus, assembled from 1.3 × 105 to 3.6 × 106 reads in each pool. In a pool of male mosquitos, a complete genome of aedes Iflavi-like virus was detected and phylogenetically analysed. Most importantly, genomes of human pathogenic viruses were not found. This is the first study to determine the virome of Ae. albopictus from Switzerland and forms a baseline for future longitudinal investigations concerning the potential role of Ae. albopictus as a vector of clinically relevant viruses.A metagenomic study was performed on 498 female and 40 male  Aedes albopictus plays an important role as a vector of arboviruses, many of which can severely affect human health, including dengue virus (DENV), Zika virus (ZIKV), yellow fever virus (YFV), and chikungunya virus (CHIKV) [Ae. albopictus, originally endemic in South-East Asia, has spread over the past approx. 40 years throughout large parts of the Americas, Africa, Australia, and Southern Europe [Ae. albopictus was spotted for the first time in 2003 in canton Ticino, where since 2007 it has been firmly established; occasionally it is also found in northern parts of Switzerland [Ae. albopictus outside of a tropical area was reported in 2007 in Italy [Ae. albopictus as a vector in France as well [Ae. albopictus appears to retain its competence to harbour and transmit zoonotic viruses outside of the tropical regions.The Asian tiger mosquito  (CHIKV) . Changinn Europe . In Swittzerland ,6,7. To in Italy . Several as well ,10. HencWolbachia sp. may limit the ability of mosquitos to transmit specific zoonotic viruses [In addition to zoonotic viruses, mosquitos are known to host many different insect-specific viruses (ISVs) which persistently infect mosquitos, but not vertebrates , althoug viruses .Ae. albopictus is endemic in Ticino and progressively invades other regions of Switzerland, and because the presence of the vector facilitates the possibility that zoonotic viruses may be locally transmitted to humans, we addressed the question as to whether these mosquitos indeed harbor such viruses. Specifically, using next-generation sequencing and metagenomic analyses, we determined the full, unbiased virus population diversity of Ae. albopictus collected in five municipalities of Lugano in Ticino. A similar study has not previously been performed in Switzerland.As n = 40; females n = 498) were collected, divided into 15 pools according to collection date, place, and gender , canton Ticino, Switzerland . Mosquitg for 5 min, and the supernatants were passed through a 0.45 µm syringe filter . To enrich nucleic acids that are protected by a virus capsid, 14 µL of micrococcal nuclease buffer, 1 µL of micrococcal nuclease , and 1 µL of ribonuclease A from bovine pancreas  were added to 134 µL of each filtrate from the previous step and incubated for 15 min at 45 °C and 1 h at 37 °C. Total RNA and DNA were extracted using the QIAmp Viral RNA mini kit  without RNA carrier. β-mercaptoethanol  was added at a final concentration of 1% in order to inactivate nucleases. RNA was transcribed using 2.5 µM of random primer with a known 20 nucleotide (nt) tag sequence at the 5′ end (SISPA-N: GTTGGAGCTCTGCAGTCATCNNNNNN) and the RevertAid First Strand H minus cDNA Synthesis Kit  following the manufacturer’s recommended protocol. Then, 1 µL of RNase H  was added to degrade remaining RNA. A premix of 0.8 µM SISPA-N primer, 10× Klenow buffer, and 0.2 mM dNTP was added to 45.5 µL of the first-strand DNA. Following denaturation at 95 °C for 1 min and cooling down on ice, the second strand was synthesized using Klenow polymerase  for 15 min at 25 °C followed by 1 h at 37 °C. An additional step of second-strand synthesis using Klenow polymerase was performed at the same conditions, followed by DNA purification using the PureLink® PCR Micro Kit . Then, dsDNA was amplified non-specifically by sequence-independent single primer amplification (SISPA). For this, the HotStarTaq DNA polymerase  and the SISPA primer (GTT GGA GCT CTG CAG TCA TC) were used under the following conditions: 15 min of activation at 95 °C, 18 cycles of 30 s at 94 °C, 30 s at 58 °C and 1 min at 72 °C, followed by 10 min at 72 °C and cooling down to 4 °C. Finally, the amplified products were purified using the QIAquick PCR purification kit . The total DNA was quantified on the Agilent 4200 TapeStation . Sequencing libraries were made using NEBNext Ultra II DNA library prep kit and NEBNext® Multiplex Oligos for Illumina®  . After quantifying at the TapeStation and pooling at equal molar concentrations, libraries were sequenced using the Illumina NovaSeq system in a single-end 1 × 100 nt run at the Functional Genomics Center Zurich .To each pool, 500 µL of phosphate-buffered saline (PBS)  and a stainless-steel bead  were added, and samples were mechanically homogenised in a TissueLyser II  at 20 Hz for 2 min. Then, homogenates were centrifuged at 16,060× https://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Raw reads were pre-processed using Trimmomatic (version 0.36) to trim off PCR primers, sequencing adaptors, and low-quality ends  window) [ftp://ftp.ncbi.nlm.nih.gov/blast/db/) using BLASTN (version 2.6.0+) [−5 and bit score ≥ 100 were kept for contig taxonomy annotation. The naïve best-hit method was used to obtain the specific taxa assignment per contig after manual inspection of the alignments. The LCA (lowest common ancestor) algorithm (contig mode) based on multiple BLAST hits was also applied to obtain a more accurate and general taxa assignment [http://rmarkdown.rstudio.com/). Additionally, de novo assembled contigs were further investigated in the metagenomic pipeline of SeqMan Ultra software , viral sequences were analyzed with NCBIs ORF finder (https://www.ncbi.nlm.nih.gov/orffinder/), and phylogenetic trees were built in MEGA X [p value ≤ 0.05).Individual metagenomes per sample and the metagenome of all samples combined were assembled and analyzed using the same pipeline. In detail, the technical quality of Illumina single-end (SE) DNA-seq reads was evaluated using FastQC version 0.11.7 ( window) . Quality window) . To anno 2.6.0+) . Hits wesignment . To quansignment . Mapped signment . Unmappen MEGA X . MUSCLE The raw sequence reads generated in this study are available at the NCBI sequence read archive (SRA) database under Bioproject accession PRJNA638077. Virus genomes generated have been deposited in GenBank under the accession numbers MT577804, MT577805, and MT591567.6 sequencing reads per pool) and applied to reference alignment and de novo analysis to construct contigs. In each mosquito pool, between 1.9% and 36.1% of the total generated sequences were classified as viral reads .Interestingly, in pool MO15, the only pool with male mosquitos, a contig from the order Picornavirales with 9666 nt in length has been constructed using 165,192 sequencing reads  . Further5 and 3.6 × 106 sequenced reads  (Between 1.3 × 10l reads)  have beel reads) . A hypotl reads)  were assAedes dorsalis in Washington. Moreover, in seven pools, sequencing reads of two unclassified viruses were assembled with high nt identity to viruses detected in Brazilian mosquitos. In seven pools, 25,294 reads with 99–100% nt identity to the putative glycoprotein gene of Guato virus (GenBank acc: KT966486.1) have been assembled [Two contigs belonging to Rebdoviridae, particularly Arboretum virus, were detected in pools MO8 and MO10. Contigs of 1064 nt and 1235 nt in length, generated from 144 and 125 sequenced reads, respectively, showed 79.6% nt identity to novel rhabdoviruses isolated from mosquitos in Peru (GenBank acc: KC994644.1) . One conAphis nerii and several other insects species [In pool MO4, three contigs with lengths ranging from 307 to 794 nt showed 93.3%–99% nt similarity to members of the family Dicistroviridae, in particular to Aphid lethal paralysis virus isolate ALPV-CE (GenBank acc: MK704471.1), and Aphid lethal paralysis virus isolate ALPV-An (GenBank acc: JX480861.1) identified in  species . That waTwo contigs of 526 and 839 nt length showing 81% and 90% nt identity, respectively, to Lake Sarah–associated circular virus-48 were generated in pool MO12. This virus belongs to the CRESS DNA viruses identified from Lake Sarah in New Zealand (GenBank acc: KP153505.1) . Viral rAe. albopictus is naturally infected with Wolbachia strains.Finally, since only a fraction of the reads was found to have a viral origin (1.9% to 36.1%), we also investigated the presence of other infectious agents, Wolbachia in particular. Indeed, between 290 and 14,763 reads were assembled to Wolbachia wAlbB in each pool, thereby confirming that Ae. albopictus and Ae. Aegypti, are vectors for several medically important viruses such as ZIKAV, DENV, CHIKV, and WNV [It is well known that arthropods constitute a major reservoir for many different viruses and therefore play an important role in virus spread and evolution ,39. High and WNV ,43,44,45 and WNV ,47. TherAe. albopictus collected in Southern Switzerland. Importantly, genomes of medically relevant viruses were not detected. The most abundant virus genomes detected were Wenzhou sobemo-like virus and AEFV. Wenzhou sobemo-like virus has previously been found in mosquitos [Ae. albopictus collected in Northern Italy, with a prevalence of 77.5% in 2008 and 16.8% in 2012 [In the current study, we used viral metagenomic sequencing to determine the virome of osquitos , but plaosquitos . AEFV isosquitos ,51,52. W in 2012 ,54.Culex flavivirus, two other members of the ISFVs, are known to inhibit WNV, ZIKV, and DENV [It would be interesting to investigate whether a high prevalence of ISFVs such as AEFV in Southern Switzerland (this study) and Northern Italy ,54 correand DENV ,55,56. Oand DENV ,58.Wolbachia sp. is an intracellular endosymbiont that reduces the ability of Ae. aegypti to transmit ZIKV and DENV by restricting their replication [Wolbachia-infected Ae. aegypti mosquitos have been released into the environment in Australia with the aim to supress the spread of DENV [Wolbachia reads have been detected in all our mosquito pools. This was expected, as previous studies revealed a prevalence of Wolbachia in Ae. albopictus of >95% [Not only viruses have been shown to inhibit the replication of other viruses. lication . Indeed, of DENV . Wolbach of >95% ,61,62.Culex spp. in California, there is no explanation why in the present study iflavi-like virus has been detected only in a pool of male Ae. albopictus. However, the number of mosquitos collected at different locations varies greatly, and the presence of the virus may be location-specific. While it is rather unlikely that only a single male mosquito in the pool hosted the virus when considering the large number of assembled reads, this possibility cannot be excluded. The transmission of ISVs is not fully understood yet, and the transmission occurs mainly vertically from the female to progeny or on the breeding sites [An interesting observation of this study was that Aedes iflavi-like virus genomes were found exclusively in a pool of male mosquitos. The most closely related viruses have been detected in mosquitos collected in Spain and California ,63. Consng sites ,64. FurtAe. albopictus and sets the basis for future metagenomic analyses to explore the spatial and temporal dynamics of the virus diversity in these mosquitos. This kind of study may also contribute to a better understanding of virus–virus interactions and thereby support novel strategies to prevent arbovirus diseases. The world of the ISFVs and ISVs remains largely unexplored but, if better known, may yield important insights into viral evolution and the role of these viruses in the emergence and transmission of pathogenic viruses.In conclusion, this study presents a snapshot of the virome of established Swiss populations of


Currently, next generation sequencing (NGS) is the mainly used approach for identification and monitorization of viruses with a potential public health threat in clinical and environmental samples. To facilitate detection in NGS, the sequence-independent, single-primer-amplification (SISPA) is an effective tool for enriching virus sequences. We performed a preliminary assessment of SISPA-nanopore sequencing as a potential approach for screening tick-borne viruses in six specimens with detectable Crimean-Congo hemorrhagic fever virus (CCHFV) and Jingmen tick virus (JMTV) sequences. A comparison of unbiased NGS and SISPA followed by nanopore sequencing was carried out in 4 specimens with single and pooled ticks. The approach was further used for genome sequencing in culture-grown viruses. Overall, total/virus-specific read counts were significantly elevated in cell culture supernatants in comparison to single or pooled ticks. Virus genomes could be successfully characterized by SISPA with identities over 99%. Genome coverage varied according to the segment and total read count. Base calling errors were mainly observed in tick specimens and more frequent in lower viral loads. Culture-grown viruses were phylogenetically-related to previously-reported local viruses. In conclusion, the SISPA + nanopore sequencing was successful in generating data comparable to NGS and will provide an effective tool for broad-range virus detection in ticks. In the current global environment struck by the ongoing pandemic from a novel virus, identification of agents with a potential public health threat have never gained more attention. Widespread availability of massively parallel sequencing platforms and related protocols  have enabled metagenomic investigation on viruses and facilitated the discovery of several novel agents . MoreoveViruses transmitted by ticks comprise a large group in vector-borne viruses. Crimean-Congo hemorrhagic fever virus (CCHFV) is the causative agent and the most notable tick-borne infection due to its widespread distribution and health impact . LackingThe impact of SISPA-based enrichment and nanopore sequencing has not yet been investigated for vector-borne viruses transmitted by ticks. This study was carried out as a preliminary effort to assess the impact of this approach for screening particular tick-borne viral pathogens.The tick specimens were pooled according to species and collection site up to a maximum of 50 individuals, ground by vortexing with tungsten carbide or stainless-steel beads , in 500–700 μL of Eagle’s minimal essential medium, supplemented with 5% fetal bovine serum and 1% L-glutamine and. Each pool was subsequently centrifuged at 4000 rpm for 4 min, and the supernatant was aliquoted and stored at −80 °C. In addition to the tick pools, African green monkey kidney  cell culture supernatants with detectable target viruses on the first passage were evaluated. Fresh RNA from processed tick specimens and cell cultures supernatants were prepared using QIAamp viral RNA kit  without carrier RNA in an individual elution volume of 60 µL. For Illumina sequencing without previous SISPA, cDNA and double strand cDNA synthesis was performed using Superscript IV reverse transcriptase  and Nondirectional RNA Second Strand Synthesis Module , The sequencing runs on the tick pools were carried out as described previously, using Illumina instruments  ,14. The The Fast5 sequences generated during sequencing were transcribed to FastQ sequences by using Guppy v.3.4.5 (Oxford Nanopore Technologies) on the MinION IT device (MNT-001). Computational separation of the barcoded samples was performed with Guppy v.3.4.5 for Windows with standard parameters. Adapter and primer sequences were removed by using Guppy v.3.4.5 and Geneious Prime 2.1 . The trimmed FastQ sequences for each sample were aligned to the corresponding virus reference if available with Guppy v.4.0.11 for Linux, and the resulting alignments were used for calculation of accuracy and genome coverage of the consensus sequence.Consensus sequences were handled using Geneious (Biomatters Ltd.). BLASTn and MEGABLAST algorithms were used for nucleotide similarity searches in the National Center for Biotechnology Information (NCBI) database . Alignmen = 4) and JMTV (n = 2) genome sequences, as processed ticks (n = 4) or cell culture supernatants (n = 2) (n = 2) and pooled (n = 2) ticks. Here, higher total or virus-specific read counts were observed in NGS, within a fold range of 101–104 (We tested six specimens with detectable CCHFV ( (n = 2) . A direc 101–104 . In indiGenome segments of both target viruses could be successfully identified and characterized by SISPA . In mostWe further performed SISPA to characterize the target viruses in cell culture supernatants. Here, this approach produced better sequence data, with few Indels and no ambiguities. We did not use an alternate method for genome sequencing in these specimens; therefore, the number and rate of possible base calling errors appearing as substitutions could not be determined. However, pairwise comparisons have revealed these viruses to be at least 93% identical to phylogenetically or geographically related viruses. The obtained CCHFV genome showed highest sequence identities with isolate Saf . It alsoIn this study, we present the findings of an application of the SISPA protocol in combination with nanopore sequencing for the detection of two tick-borne pathogenic viruses with segmented genomes. Although SISPA can be coupled with various NGS platforms, we preferred the third generation nanopore sequencing due to its portability, reduced costs and time for obtaining results. Overall, this strategy generated sufficient sequence output to identify and characterize the target viruses in single or pooled ticks and cell culture supernatants. However, differences in sequence yield, as observed in total and specific read counts, were apparent . MoreoveThe current study has several limitations to be addressed. First, we were compelled to use different aliquots for unbiased NGS and SISPA + nanopore sequencing, due to specimen availability. Although the homogenized aliquots employed in the study were kept securely at −80 without freeze/thaws, potential variations affecting quantitative and qualitative sequence output in SISPA experiments cannot be ruled out. Another shortcoming is the lack of standardized specimens with known copy number of viral genomes, evaluated in parallel with the specimens in the experiments. A more informative comparison would be possible using several spiked specimens of identical matrices, as tick pools and culture supernatants, enabling calculations on the limits of detection and optimal performance in the sequencing step. Moreover, the impact of SISPA enrichment for metagenomic virus detection could not be evaluated in this setting. Nevertheless, the study demonstrates a successful proof-of-concept that can produce sufficient sequence data even in a suboptimal setting. We are currently working on developing optimized protocols and pipelines targeting ticks as well as mosquitoes, using well-characterized specimens and several vector-borne viruses. In conclusion, the SISPA + nanopore sequencing approach was successful in generating virus genome sequence data from tick specimens and culture-grown tick-borne viruses. It will provide an invaluable tool for broad-range detection of viruses in vectors with further optimization, for which studies are underway.


Outbreaks of a vesiculopustular disease in dairy cattle and milkers have been frequently reported in Brazil since 1999 when the vaccinia virus strain Cantagalo was first isolated in the State of Rio de Janeiro. However, the genomic diversity of the viral isolates associated with these outbreaks is not well known, particularly in the southeastern states that represent the focal point of virus spread to other regions. Here, we report the genomic sequences and an analysis of the polymorphic site profiles and genotypic diversity of four clinical isolates of vaccinia virus strain Cantagalo collected from 1999 to 2006 in southeastern Brazil. Poxviridae.,,Vaccinia virus (VACV) is a large and complex double-stranded DNA-containing virus that belongs to the family ,In Brazil, outbreaks of a vesiculopustular disease in dairy cattle and milkers have been frequently reported since 1999.,,,,,,,,,,,After the first outbreaks in 1999, the number of reported cases increased with the rapid spread of the infection to the other southeastern states, i.e., Minas Gerais (MG) and Espírito Santo (ES).de novo assembly using Velvet vs 1.2.10 with a kmer of 149.,Outbreaks of vesiculopustular disease affecting dairy cattle were previously reported by the state secretariats for animal health surveillance and their affiliated agencies in the municipalities of Miracema (RJ), Vieiras (MG), Campos de Goytacazes (RJ), and Alegre (ES) in 1999, 2001, 2003, and 2006, respectively . Scab sa,The Supplementary data (Table).The median-joining network explores the relatedness of the field isolates of VACV in greater detail and zooms in on the SNP-based distances among them . We can The most striking difference observed in the base-by-base profile is an insertion of 3,753 bp in the left end of the genomes of CTGV-ALEH2 and Serro2 viruses  from MI233 (RJ) and ALEH2 (ES). However, ALEH2 is more distant genetically from CG04 than MI233 is. Additionally, the deletion in the region of the CPXV 77 kDa and C9L genes present in the ALEH2 genome is not shared with the 3 RJ and VI04 isolates, but is shared with Serro2 virus, which was collected in the previous year but approximately 500 km apart. However, although sharing this structural feature, ALEH2 and Serro2 genomes have accumulated 81 SNPs. Taken together, our data suggest a different event of virus spread into ES, not originated from the Northwestern region of RJ and not directly originated from Serro2, reflecting the complex genomic diversity of CTGV isolates related to the first years of the outbreaks that emerged in the Southeastern region of Brazil in 1999. These studies will lead to further investigation of the focal point of VACV dissemination in Brazil, and the biological and virulence diversity of the isolates.


Zika virus has recently spread to South- and Central America, causing congenital birth defects and neurological complications. Many people at risk are flavivirus pre-immune due to prior infections with other flaviviruses (e.g. dengue virus) or flavivirus vaccinations. Since pre-existing cross-reactive immunity can potentially modulate antibody responses to Zika virus infection and may affect the outcome of disease, we analyzed fine-specificity as well as virus-neutralizing and infection-enhancing activities of antibodies induced by a primary Zika virus infection in flavivirus-naïve as well as yellow fever- and/or tick-borne encephalitis-vaccinated individuals.Antibodies in sera from convalescent Zika patients with and without vaccine-induced immunity were assessed by ELISA with respect to Zika virus-specificity and flavivirus cross-reactivity. Functional analyses included virus neutralization and infection-enhancement. The contribution of IgM and cross-reactive antibodies to these properties was determined by depletion experiments.Pre-existing flavivirus immunity had a strong influence on the antibody response in primary Zika virus infections, resulting in higher titers of broadly flavivirus cross-reactive antibodies and slightly lower levels of Zika virus-specific IgM. Antibody-dependent enhancement (ADE) of Zika virus was mediated by sub-neutralizing concentrations of specific IgG but not by cross-reactive antibodies. This effect was potently counteracted by the presence of neutralizing IgM. Broadly cross-reactive antibodies were able to both neutralize and enhance infection of dengue virus but not Zika virus, indicating a different exposure of conserved sequence elements in the two viruses.Our data point to an important role of flavivirus-specific IgM during the transient early stages of infection, by contributing substantially to neutralization and by counteracting ADE. In addition, our results highlight structural differences between strains of Zika and dengue viruses that are used for analyzing infection-enhancement by cross-reactive antibodies. These findings underscore the possible impact of specific antibody patterns on flavivirus disease and vaccination efficacy. The explosive spread of Zika virus, a flavivirus, to South- and Central America underscores the potential threat of newly emerging arthropod-borne viruses. Zika virus infection can cause congenital birth defects and neurological complications. Many people at risk are flavivirus pre-immune because of prior infections with other flaviviruses  or vaccinations  and have non-protective cross-reactive antibodies at the time of infection. Since pre-existing immunity can modulate the specificity and functional activity of antibody responses, and cross-reactive antibodies have been implicated in disease enhancement, we compared the specificities of serum samples from flavivirus-naïve and vaccinated individuals after primary Zika virus infections. Prior immunity led to a strong booster of cross-reactive antibodies that did not neutralize Zika virus. Importantly, we could also show that newly formed IgM antibodies contributed significantly to virus neutralization and prevented infection enhancement by other antibodies. Our data thus show how pre-existing cross-reactive immunities can alter the specificities and functional activities of antibody responses in flavivirus infections, which may affect flavivirus-induced disease and the efficacy of vaccinations. Epidemics of infections with Zika virus, a mosquito-borne flavivirus, first emerged in Pacific islands  followed by a big outbreak in South and Central America starting in 2015 . EspeciaIn sequential infections with different flaviviruses, anamnestic responses to shared epitopes can lead to the phenomenon of original antigenic sin, resulting in a strong boost of cross-reactive antibodies at the expense of type-specific antibodies to the newly infecting virus , 8. ThisThe main target of flavivirus neutralizing and protective antibodies is the envelope protein E , which cThe immune response to primary flavivirus infections is usually characterized by a rapid rise of specific IgM antibodies, which appear to play an important protective role in the early phase of infection , 25. In Since the modulation of antibody responses by pre-existing immunity can have profound biological consequences, we investigated the effect of pre-existing vaccine-induced immunity on IgM and IgG profiles after recent primary Zika virus infection. For this purpose, we analyzed samples from Zika patients, who were either flavivirus naïve or had previously been vaccinated against TBE and/or YF. We demonstrate that virus-specific IgM antibodies contribute substantially to Zika virus neutralization in the early phase of infection and play an important role in the prevention of IgG-mediated enhancement of Zika virus infection of Fcγ receptor-positive cells. Importantly, the formation of IgM antibodies is reduced in individuals with pre-existing cross-reactive flavivirus immunity. Pre-vaccinated individuals displayed a strong anamnestic response of cross-reactive IgG antibodies, which caused ADE of dengue but not of Zika virus infection in vitro. Our findings contribute to the understanding of the complex interplay of antibody populations in transient early stages of flavivirus infections, which can affect the clinical outcome of infections and the efficacy of vaccines.Human serum samples from 62 Zika patients were sent to the Center for Virology of the Medical University of Vienna for diagnostic purposes. As negative controls, 32 flavivirus-negative samples from a previous diagnostic study, in which we analyzed TBE antibody responses, were included . The samWe analyzed serum samples from 62 patients who were diagnosed with an acute symptomatic Zika virus infection after returning to Europe from Zika-endemic areas. They developed Zika virus-specific IgM and neutralizing antibodies, and 30 patients were Zika PCR positive . Samples from only two of these Zika patients exhibited a low degree of broad flavivirus cross-reactivity of IgM, as revealed by Rio Bravo (RB) and dengue IgM assays. Previous flavivirus immunity (YF and/or TBE vaccinations) was confirmed by neutralization assays (see below); the time points of vaccinations were not known to us. All serum samples were heat-inactivated for 30 min at 56°C prior to serological testing. Pools were prepared from equal aliquots of serum samples.TBE and YF NTs were performed as described previously , 36, 37.2. Stably transfected cells were selected with blasticidin (Fisher Bioreagents). After induction of protein expression by CuSO4, proteins were affinity purified with Strep-Tactin columns  according to manufacturer’s instructions.The flavivirus E proteins used in this study  were proPurified proteins were quantified with the Pierce BCA Protein Assay Kit , (Thermo Fisher Scientific) according to manufacturer’s protocol. Purity was verified with 15% SDS-PAGE according to Laemmli  and/or AZika and WN virus E proteins were analyzed by rate zonal ultracentrifugation in sucrose gradients as described previously , 47. TheSize-exclusion chromatography of the Zika and WN virus E proteins was carried out with a Superdex 200 Increase 3.2/300 column using the ÄKTA FPLC system  according to the manufacturer’s instructions in TAN buffer pH 8.0.25 ng/well strep-tagged E protein were added to Strep-Tactin-coated microplates  and incubated for 1.5 h at 37°C in phosphate-buffered saline (PBS) pH 7.4, 2% sheep serum, 2% Tween 20. After blocking with 1% bovine serum albumin (BSA) in PBS pH 7.4, serial dilutions of samples were added and incubated for 1 h at 37°C. Serial dilutions of bound human antibodies  were detected with goat anti-human IgG labeled with HRP (Thermo Fisher Scientific). The starting dilution for ELISAs with single serum samples was 1:100 and that with pools 1:300. Absorbance was measured at 450 nm. Titers were determined by curve fitting with a four-parameter logistic regression with GraphPad Prism 8 (GraphPad Software Inc.). A positive control serum was included on each ELISA plate. This control serum was obtained from a Zika patient with YF and TBE pre-immunity and yielded similar titration curves with Zika, RB, TBE and dengue serotype 2 virus E proteins .Validation of the ELISAs was performed as follows: Proper display of the antigens in this ELISA format was verified by the use of a number of E-specific monoclonal antibodies (mabs). These included FL-specific antibodies  were coated with 28 ng/well polyclonal rabbit anti-human IgM (Dako) in carbonate buffer pH 9.6 . Diluted50).NTs were performed with Zika virus strain H/PF/2013 (European virus archive), dengue virus serotype 1 strain Hawaii , dengue virus serotype 2 strain NGC (European Virus Archive) and TBE virus strain Neudoerfl in black 96-well microtiter plates (Greiner Bio-One). The assays were carried out in principle as described previously for YF virus , but fluThe assays were validated with neutralizing mabs specific for the different viruses, as follows: Zika and dengue viruses were neutralized by the EDE-specific mabs C8 and C10, corresponding to their activities reported in the literature , 50. Den8 RNA copies/reaction, the last point 100 RNA copies/reaction . Fold enhancement of infection was calculated as the ratio between viral RNA copies in the presence of a positive serum and viral RNA copies in the presence of a flavivirus-negative control serum.Serial dilutions of heat-inactivated serum samples were pre-incubated with either Zika virus or dengue virus serotype 2  for 1 h at 37°C. Fcγ receptor-bearing K562 cells (ATCC) were added and incubated for 3 (Zika virus) or 4 (dengue virus) days. Then the cell culture supernatant was harvested and used for virus detection by qPCR. Viral RNA was isolated with the RNeasy Mini Kit (Qiagen) and cDNA was generated with the iScript cDNA Synthesis Kit (BioRad), according to manufacturers’ instructions. cDNA was mixed with TaqMan Universal PCR Master Mix (Applied Biosystems/Thermo Fisher Scientific), 25 pmol of primers and 10 pmol of probe (Eurofin Genomics). Primers and probes used in this study targeted the NS5 gene: Zika virus: forward primer: ACAAGGGGAATTTGGAAAGGC; reverse primer: GAATCCAAGGGCTTCGAACTC; probe: FAM-AGCCGCGCCATCTGGTATATGTGG-TAMRA. Dengue virus 2: forward primer: CAGATGGAGGGAGAAGGAGTC; reverse primer: CGCCCTACTCTTGCTAACCA; probe: FAM-ACAGTCACAGAAGAAATCGCCGTGCA-TAMRA. The samples were amplified under the conditions described in : 3 min aADE was validated with mab 4G2, which caused slight ADE of dengue virus infection as described in the literature , and we 2PO4, 300 mM NaCl, 0.05% Tween 20, pH 8.0). Columns were spun down for 30 s at 700 g and the flow-through was reloaded six times. After a wash step with binding buffer, the column was equilibrated with depletion buffer . Pre-diluted serum samples (1:10–1:15) were loaded onto the columns and incubated for 10 min at RT. To deplete completely Zika or RB virus-reactive antibodies a minimum of eight rounds was necessary. Depletion was verified by IgG ELISA with the respective E protein as antigen as described above. Mock depletion was performed with Strep-Tactin XT spin columns in the absence of E protein.For the depletion of Zika virus-specific or flavivirus broadly cross-reactive antibodies, 200 μg Zika or RB virus E protein were bound to Strep-Tactin XT spin columns , equilibrated with binding buffer  and centrifuged for 30 s at 100 g and 30 s at 300 g. 100 μl of 1:2 pre-diluted serum samples were loaded onto the columns and incubated for 75 min at RT under gentle shaking. The columns were then centrifuged for 30 s at 100 g. The IgG depleted eluate was collected and the column was washed twice with 75 μl binding buffer. The eluates were pooled resulting in an IgG-depleted serum with a final dilution of 1:10. Depletion was verified by the flavivirus IgG ELISA (described above), using the Zika virus E protein, and the presence of IgM in the final eluate was confirmed by Zika IgM ELISA (described above).IgG depletions were performed with Ab SpinTraps  containing Protein G Sepharose following the manufacturer’s instructions. For mock depletions, empty spin columns (Thermo Fisher Scientific Pierce) were loaded with Sepharose 4B beads (Sigma). Columns were equilibrated with binding buffer (20 mM Na1 ml anti-IgM agarose beads (Sigma) were mixed with 2 ml binding buffer  and were centrifuged for 5 min at 4,000 g. The supernatant was removed and the beads were washed with 2 ml binding buffer. Mock depletions were performed with Sepharose 4B beads (Sigma). Serum was diluted 1:2 in binding buffer and added to the beads. The mixture was incubated for 90 min at RT under gentle shaking. After centrifugation for 5 min at 4,000 g, the supernatant was subjected to control ELISAs. Depletion was verified by Zika IgM ELISA (described above), and the presence of IgG was confirmed by the flavivirus IgG ELISA (described above), using the Zika virus E protein.50 titers <100 were therefore considered 50.Antibody titers that were less than the lower detection limit of the assay were assigned values equal to 50% of this limit for data analysis. IgG ELISA titers and NTTiters were log-transformed for statistical analysis. Antibody titers and units of pools were analyzed by one-way ANOVA with Dunnett’s post-hoc test (GraphPad Prism 8). All other statistics were performed with SPSS 25.0.Statistical comparisons of pool and single serum data of the analyses were performed by applying a general linear model (GLM) with the log-link function .Comparisons of the four pools  were done by application of the GLM with data from naïve pools as reference. Maximum likelihood estimates of parameters reflecting the difference to naïve pools were tested for significance by Wald’s tests. Deviations of residuals from normality were assessed by Shapiro-Wilk’s tests.Comparisons of mock versus real depletion within pools  were performed by applying a two-factor Analysis of Variance (ANOVA) and linear contrasts.Relative IgG avidity of pre-immune sera against naïve ones were tested by comparing the reduction of log titers using the sera untreated with urea as offset applying a GLM.Fold enhancement of Zika and dengue virus infectivity between depleted and mock-depleted sera was compared by computing the area under the log-dilution-log-fold-enhancement curves using GLM.Fold enhancement of Zika virus between depleted and mock-depleted sera (using a single serum dilution) was compared by applying a GLM with linear contrasts and a Bonferroni-Holm adjustment.Trends of antibody responses  were tested by fitting a polynomial function to the data and comparing the slopes of the linear component as well as the maximum of the response reached during the observation period using GLM.For all analyses, a p-value below 0.05 was considered significant.To assess the effect of pre-existing flavivirus immunity on the patterns of antibody responses in primary Zika virus infections, we analyzed serum samples from laboratory-confirmed Zika patients (travelers returning from Zika-endemic regions) with and without prior TBE and/or YF vaccination . Since tOur analyses included the quantification of Zika virus-specific and flavivirus cross-reactive IgM and IgG antibodies in ELISA and NT, as well as the determination of in vitro ADE of Zika and dengue virus infections. Because these experiments required relatively large volumes for the depletion of antibody subsets , these analyses were first carried out with pools of equal aliquots of serum samples (one sample per patient) for which at least 500 μl were available. The number of patient samples included in these pools from the different groups are shown in For the analysis of Zika virus-specific antibodies, we used a recombinant Zika virus E protein with a C-terminal strep-tag and lacking the stem-anchor region see also . This prThe stable dimeric structure of the Zika virus E protein in solution was verified by sedimentation analyses and size exclusion chromatography in comparison to a recombinant WN virus E protein , which wIn addition, we analyzed whether our Zika E dimer is recognized by the human EDE-specific mabs C8 and C10  in the cThe four serum pools and individual sera see  were tesTo obtain information about the contribution of IgM antibodies to virus neutralization, we depleted IgG antibodies from the four serum pools using protein G columns (Methods). Control experiments demonstrated that IgG depletion was >99.5% complete  and no sSince increased infection of Fcγ receptor-positive cells is the result of an interplay between neutralizing and enhancing effects of antibody populations present in a given serum sample , we alsoTo confirm these results, we carried out additional ADE experiments with single dilutions of the following preparations of the four pools: i. unmodified, ii. IgG depleted, iii. IgM depleted, iv. mixture of equal aliquots of IgG- and IgM-depleted pools . The resSince the pre-immune groups had significantly higher titers of broadly flavivirus cross-reactive antibodies , we quanThere is evidence that the neutralizability by broadly cross-reactive antibodies can vary between different flaviviruses . We therThe serum pools described above did not provide information about possible differences in the time course of antibody production in our cohorts. We therefore also compared individual serum samples from naïve and pre-immune Zika patients , for whiThe distribution of IgM antibodies appeared similar at first glance, with a rapid rise followed by a decline in both groups . StatistThe pattern of Zika virus NT appeared very similar with samples of the naïve and pre-immune groups ; reminisPre-existing cross-reactive immunity can affect the antibody response upon infection with antigenically related viruses, both with respect to the kinetics and extent of the IgM response and to the specificities of populations of IgG antibodies induced. In our study, we analyzed the effect of prior TBE and/or YF vaccination on antibody responses to primary Zika virus infections and observed a strong anamnestic response in pre-vaccinated individuals, resulting in a high proportion of broadly cross-reactive IgG antibodies that accounted for 84 to 94% of the total E-response . In naïvThe FL appears to be cryptic in the cryo-EM (electron microscopy) structures of mature flaviviruses . ConsistThe different properties of infectious Zika and dengue viruses with respect to the exposure of broadly cross-reactive epitopes are not only reflected in NTs but also in analyses of ADE of infection of Fcγ receptor-expressing cells Figs  and 9. LIn our analyses we used a stable Zika virus E dimer  which haAlthough Zika virus was not enhanced by broadly flavivirus cross-reactive antibodies in our study, infection enhancement of this virus was readily demonstrable with Zika virus-specific antibodies at sub-neutralizing concentrations, consistent with the generally accepted ADE mechanism . An extrAnalysis of virus neutralization before and after IgG depletion revealed that IgM accounted for approximately 30 to 50% of the total neutralizing activity in our samples of primary Zika virus infections, with a tendency of a lower contribution in the double-vaccinated group . These pIt appears that the prominent role of IgM antibodies in virus neutralization in the early phase of human flavivirus infections and its function in protecting from severe forms of disease has been underappreciated so far. This is especially important in the context of sequential flavivirus infections in which IgM responses may be delayed and/or strongly diminished , 26, 27.Overall, our study highlights the potential effect of flavivirus vaccine-induced pre-immunity on the patterns of antibody responses in early stages of primary infection with a distantly related flavivirus, in this case Zika virus. The modulation observed, albeit with relatively small groups  of adults, includes both the primary IgM response as well as the anamnestic booster of broadly cross-reactive IgG antibodies that are capable to mediate ADE of dengue virus infections in vitro. Considering the importance of IgM for protection early after infection and its ability to block IgG-mediated ADE, further studies on these phenomena in the context of different combinations of flavivirus infections and vaccinations appear to be warranted.S1 Fig(A) Titration curves of the positive control serum (described in Methods) and (B) results of 32 negative control sera (dilution 1:100) in ELISA with Zika, dengue serotype 2, RB and TBE virus E proteins. The cut-off used for titer calculations is shown as dotted line and corresponds to the mean absorbance value of the 32 negative samples plus 3 standard deviations.(TIF)Click here for additional data file.S2 Fig(A) Serum collected 3 weeks after disease onset and (B) after 6 months were tested with and without a step of urea exposure, as described in Methods.(TIF)Click here for additional data file.S3 Fig(A) Standard curve of the positive control serum (described in Methods) and (B) results of 32 negative control sera in ELISA with Zika virus E protein. The cut-off is shown as dotted line and corresponds to the mean arbitrary units of the 32 negative samples plus 4 standard deviations.(TIF)Click here for additional data file.S4 Fig(A) Rate zonal ultracentrifugation of untreated Zika virus E protein (red solid line), and after SDS-treatment (red dotted line). The monomeric WN virus E protein (blue solid line) served as a control. M and D indicate positions of monomers and dimers, respectively. (B) Size-exclusion chromatograms of Zika virus E (upper panel) and the monomeric WN virus E (lower panel). (C) ELISA with Zika virus E protein and EDE-specific monoclonal antibodies (C8 and C10), reacting with E dimers but not monomers .(TIF)Click here for additional data file.S5 Fig(A) Serum pools. (B) Individual serum samples. The % residual neutralization after depletion is indicated in the empty columns. Mean titers were calculated from two independent experiments and error bars represent the range. Colored columns: mock depletion (mock dep), empty columns: Zika virus E depletion (ZIKV dep). n.t., not tested, because of volume limitation of the YF+ pool. S1 to S4: individual sera from each of the 4 groups.(TIF)Click here for additional data file.S6 FigSerum pools were depleted with protein G columns as described in Methods. (A) Zika virus IgG ELISA showing more than 99% depletion of IgG. (B) Zika virus IgM ELISA to control for loss of IgM antibodies during this procedure. Colored columns: mock depletion, empty columns: IgG depletion. The dotted line indicates the cut-off (c.o.) of the assay. Mean values were calculated from two independent experiments and error bars represent the range.(TIF)Click here for additional data file.S7 FigSerum pools were depleted with anti-IgM agarose beads as described in Methods. (A) Zika virus IgM ELISA showing more than 99% depletion of IgM. (B) Zika virus IgG ELISA to control for loss of IgG antibodies during this procedure. Colored columns: mock depletion, empty columns: IgM depletion. The dotted line indicates the cut-off (c.o.) of the assay. Mean values were calculated from two independent experiments and error bars represent the range.(TIF)Click here for additional data file.S8 FigSerum pools were depleted with RB virus E protein as described in Methods. Colored columns: mock depletion, empty columns: RB depletion. The dotted line indicates the cut-off (c.o.) of the assay. Mean values were calculated from two independent experiments and error bars represent the range.(TIF)Click here for additional data file.S9 FigThe dotted line indicates the cut-off (c.o.) of the assay. Mean values were calculated from two independent experiments and error bars represent the range. mock dep, mock-depleted serum pool; RB dep, Rio Bravo virus E-depleted serum pool.(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


The content of superoxide anion (O2−) and malondialdehyde (MDA) in virus-free seedlings was lower than infected seedlings at the main growth stage. With increased development, the activities of the antioxidant enzymes superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) were gradually higher in virus-free seedlings than infected seedlings. Our results indicate that virus-free seedlings may improve photosynthesis efficiency and promote photosynthesis by increasing chlorophyll content, photosynthetic key enzyme activity, and the gene expression levels in leaves. By increasing the activity of antioxidant enzymes, reducing the degree of membrane lipid peroxidation, and improving the stress resistance of chewing cane, the virus-free chewing cane seedlings increased yield and quality. Our findings provide a scientific and theoretical basis for the promotion and application of virus-free chewing cane seedlings.The effects of increasing yield and quality of virus-free chewing cane seedlings and their physiological and molecular basis were studied in this study. Results showed that compared with infected seedlings (the control), the yield of chewing cane stems grown from virus-free seedlings increased by 21.81–29.93%, stem length increased by 28.66–34.49 cm, internode length increased by 2.16–2.68 cm, the single stem weight increased by 20.10–27.68%, the reducing sugar increased by 0.91–1.15% , and sucrose increased by − 0.06–1.33% . The decrease in sucrose content did not reach significant level, but all other parameters were reached significant level. The chlorophyll content, photosynthetic parameters such as stomatal conductance (Gs), net photosynthetic rate (Pn) and transpiration rate (Tr), the activity of photosynthetic key enzymes ribulose-1,5-bisphosphate carboxylase (Rubisco) and phosphoenolpyruvate carboxylase (PEPC), and gene ( Saccharum cultivar is a high photosynthetic efficiency C4 crop composed of both sugar cane (Saccharum hybrids spp.) and chewing cane (Saccharum officinarum L.) that is widely grown in tropical and subtropical regions. Sugar cane is a hybrid species that contains a high fiber and sucrose content, and is primarily grown for raw material for sugar production. Chewing cane is grown of its low fiber content and high reducing sugar content. It is also rich in a number of essential amino acids and iron elements and is primarily consumed as a fresh fruit1. Chewing cane is widely planted in many countries around the world, but especially in Southeast Asia, the South Pacific, and China2. In China, the planting area of chewing cane reached 230,000 hm2 per year, and output exceeded 35 million tons. Chewing cane is an asexually propagated crop (using seed cane as propagator), and the most widely grown cultivar is ‘Badila’ and similar variant lines3. After several decades of continuous planting, it has been found that chewing cane can become infected with the viruses: Sugarcane mosaic virus (SCMV), Sorghum mosaic virus (SrMV), Sugarcane streak mosaic virus (SCSMV), Sugarcane bacilliform virus (SCBV), and Sugarcane yellow leaf virus (SCYLV), as well as other viruses. After which, serious problems can arise, including degeneration, reduction in yield, and deterioration of quality. Thus, the presence of viruses can negatively influencing the economic benefits of production4.6. Dewant et al.7 developed somatic embryo propagation technology to produce virus-free sugarcane seedlings, and were able to achieve a virus removal rate of 100% using growing medium containing Triazole Nucleoside (20 mg/L) and acyclovir (40 mg/L). A procedure for the in vitro elimination of SCMV, SrMV, SCSMV, and SCYLV from infected sugarcane has also been developed, and virus detection results found that the virus elimination rate following apical meristem culture was 61–92%, whereas the virus removal rate using axillary bud meristem culture was 90%8. In addition, SCSMV-free sugarcane was generated from infected plants using in vitro meristem tip culture technology9.Using virus-free seedlings is the primary way to control plant virus diseases2. Compared with virus-free sugarcane seedlings, sugarcane plants with virus symptoms had a decreased internode length and fresh weight, but not a lower sucrose concentration10. Compared with common seedlings, virus-free seedlings resulted in an average of 16.75% higher stem yield, and a sucrose content that was 0.6% higher 11. Wu et al.12 have also found that virus-free seedlings resulting from hot water treatment had a 9.5–14.7% greater stem yield and 0.68–1.69% greater sucrose content .Virus-free seedlings tend to have greater yield and quality. The biomass and sucrose content of virus-free sugarcane seedlings has been significantly greater than those of common seedlingsMost previous work has focused on virus-free technologies and the effect on yield and quality, but there are few reports on the physiological and biochemical basis for greater yield and quality among virus-free sugarcane seedlings. In particular, the effects of virus-free seedlings on the activity of photosynthetic enzymes and the expression of related genes have not been reported. In addition, results showing increased yield and quality among virus-free seedlings are for sugar cane, and there are only a limited number of studies with chewing cane. Our objective was to systematically evaluate the effect of starting with virus-free chewing cane seedlings on yield and quality, and to explore the physiological and molecular mechanisms behind any increases in yield and quality.The yield of the virus-free seedling No. 7 increased by 29.93% compared with that of infected seedling, and the stem length, stem diameter, internode length, and single stem weight increased by 28.66 cm, 0.52 cm, 2.16 cm, and 27.68%, respectively, all significantly greater than the No. 7 infected seedling  compared with the infected seedlings, but the difference was not significant. The reducing sugar content increased by 0.91%  in the virus-free seedling, significantly higher than the infected seedling. Furthermore, the fiber content increased by 0.07% in the virus-free seedling compared with the infected, but this difference was not significant.For the No. 8 seedlings, the sucrose content, brix content, and reducing sugar content were all significantly greater in the virus-free seedlings than the infected, by 1.33%, 1.29%, and 1.15% respectively. The fiber content was not significantly different between the virus-free and infected material.With the continuation of the growth period, chlorophyll content initially increased but then decreased Fig. a. The maWith the development of growth period, the net photosynthetic rate of leaves initially increased and then decreased, reaching the maximum value at the jointing stage Fig. b. The chThe stomatal conductance of leaves initially increased first over time, and then decreased, reaching the maximum at jointing stage Fig. c. The st2 concentration of leaves decreased first and then increased, reaching the maximum at seedling stage .The expression of the ges Fig. a. The hirbcS gene was detected in both virus-free seedlings and infected seedlings at different growth stages  was significantly greater than the infected seedling counterparts.The expression of the ges Fig. b. The grrbcL gene was detected in both virus-free and infected seedlings at four growth stages .The Oage Fig. a. The O2MDA in the leaves of virus-free seedlings increased first and then decreased, reaching maximum content at the tillering stage Fig. b. MDA inSuperoxide dismutase (SOD) activity in leaves of virus-free seedling No. 7, virus-free seedling No. 8, and infected seedling No. 8 reached the maximum activity at the seedling stage and then decreased gradually Fig. c. SOD acPOD activity of leaves increased first and then decreased, reaching the maximum activity at the tillering stage Fig. d. The chThe CAT activity in the leaves of virus-free and infected seedling No. 7 decreased gradually Fig. e. Meanwh13. Virus-free sugarcane seedling is an effective way to control sugarcane virus disease. It has been reported that virus-free sugarcane seedlings greater yield, but as much as 20%, and that the sucrose content can also be increased11. In this study, we compared virus-free and infected seedlings of chewing cane. Stem yield increased by 21.81–29.93%, stem length by 28.66–34.49 cm, internode length by 2.16–2.68 cm, single stem weight by 20.10–27.68%, reducing sugar by 0.91–1.15 , and sucrose content by − 0.06–1.33 . In this study, we recorded a greater increase in yield in chewing cane due to virus-free seedlings than has been shown in sugar cane. This may be due to the fact that chewing cane has poor stress resistance and is more vulnerable to viral infection than sugarcane45. It has been shown that this is particularly the case when it comes to specially the co-infection, where two or more kinds of viruses inhabit chewing cane and result in a significant decline in production1. Sugar cane is a hybrid progeny of S. officinarum and some wild species, and thus, its resistance to viruses is better than chewing cane14. Therefore, we believe that sugar cane may be less likely to be infected by certain viruses than chewing cane, the potential for increasing yield of virus-free seedlings of chewing cane is better than sugar cane.Sugarcane is an asexually propagated crop, which is vulnerable to various sugarcane viruses and result in the decline of sugarcane yield and poor quality15. Studies have shown that when viruses infect plants, they can cause organelle lesions, reduce the number of chloroplasts in cells, destroy the structure of chloroplasts, increase the activity of chlorophyll decomposition enzymes, and decrease the content of chlorophyll and photosynthetic rate17. After virus-free treatment, plants can increase leaf chlorophyll content and the net photosynthetic rate, enhancing photosynthesis37. When infected sugarcane is detoxified, the chlorophyll content and photosynthetic rate of virus-free sugarcane seedlings has increased, and the vitality of virus-free sugarcane plants also increased20. In this study, the chlorophyll content, and the Pn, Gs and Tr content of virus-free seedlings of chewing cane were higher than those of infected seedlings in each growth stage, and Ci was significantly lower than in infected seedlings. This indicates that after chewing cane seedlings are treated with virus-free treatment, the chlorophyll content, photosynthetic rate, transpiration rate, and the photosynthetic capacity increased. This finding is consistent with previous studies22. The increase of chlorophyll content and photosynthesis in virus-free seedlings of chewing cane is the basis of increasing chewing cane yield.Photosynthesis is the basis of plant growth and development, and the accumulation of photosynthate is the result of the interaction of Pn, Gs, and Ci. Tr can reflect the intensity of crop transpiration and it is the main driving force for crops to absorb and transport water and mineral elements. The photosynthetic and transpiration rates comprehensively reflect the intensity of plant vitality4 plants to improve the efficiency of photosynthesis and water use24. When plants are infected by pathogens, the activity of PEPC decreases, limiting photosynthesis and affecting plant growth25. Zhang et al.37 showed that compared with healthy plants, PEPC activity in sugarcane inoculated with Leifsonia xyli subsp. Xyli (Lxx), causing sugarcane ratoon stunting disease, decreased significantly, and that photosynthesis was reduced and growth was affected. In this study, PEPC activity of virus-free seedlings of chewing cane was significantly higher than infected seedlings during the tillering stage, jointing stage, and large growth stage. Our findings are similar to Zhang et al.37. We did not find a significant difference in the expression level of pepc gene between virus-free and infected seedlings at the seedling stage, but the expression level of pepc at the tillering stage, jointing stage, and the large growth stage was significantly higher in the virus-free seedlings than the infected seedings. Furthermore, the trend of expression of the pepc gene was consistent with the change trend of PEPC activity. We conclude that in chewing cane, the enhanced activity of PEPC in the virus-free seedlings is related to the up-regulation of the pepc gene transcription level.PEPC is a key enzyme involved in photosynthesis and has many physiological functions. It is responsible for fixing inorganic carbon in photosynthesis in C2 fixation and photorespiration during photosynthesis. It has been found that Rubisco is composed of large subunits (rbcL) and small subunits (rbcS)26 and that under stress such as pathogen infection, the subunits are down-regulated and Rubisco activity is significantly decreased, affecting plant photosynthesis27. Sampol et al.28 showed that compared with virus-free plants, viral infection caused a significant decrease in Rubisco activity and photosynthesis. Ji et al.29 reported that under phytoplasma infection, the expression of the rbcL gene in mulberry leaves was down-regulated, and that Rubisco activity and the photosynthetic rate were significantly reduced. In this study, the activity of Rubisco in the leaves of virus-free seedlings was significantly higher than infected seedlings, and the expression levels of rbcL and rbcS genes in virus-free seedlings were greater than infected seedlings. We also found that the expression levels of rbcL at the tillering, jointing, and large growth stages, as well as the expression levels of rbcS at the seedlings and jointing stages, were significantly different from infected seedlings. In this study, the activity of Rubisco in virus-free seedlings was significantly greater than infected seedlings, which may be related to the up-regulation of rbcL and rbcS gene expression in the virus-free seedlings. The up-regulation of the transcription level of photosynthetic genes and the enhancement of the activity of key photosynthetic enzymes are the photosynthetic physiology and molecular basis for increasing yield and quality of chewing cane virus-free seedlings.Rubisco is a key enzyme for carbon assimilation in plant photosynthesis, which regulates CO2− and MDA) in plant cells is in a dynamic balance with the removal of antioxidant enzymes  and does not cause damage to plants30. However, under stress, this balance is destroyed, resulting in the accumulation of reactive oxygen species, which directly or indirectly induce membrane lipid peroxidation31. Related studies have shown that plants can improve antioxidant enzyme activity, reduce free radical (O2−) content, and increase resistance to stress by virus-free treatment15. Kang32 showed that MDA content in virus-free potato seedlings was lower than common potato seedlings, and that the activities of protective enzymes such as CAT, SOD, and POD were significantly increased. Thus, this effectively prevented the damage of cell membrane structure by membrane lipid peroxidation. In this study, the antioxidant enzymes activities of SOD, POD, and CAT in leaves of virus-free chewing cane seedlings were higher than infected seedlings, and the content of O2− and MDA in leaves of virus-free chewing cane seedlings was lower than infected seedlings at all main growth stages. This indicates that the virus-free seedlings of chewing cane have a greater antioxidant capacity, have less membrane lipid peroxidation, and a greater stress resistance. In addition, previous studies34 show that under the stress of pathogens, sugarcane plants accumulate reactive oxygen species, destroy the redox system, and decrease antioxidant enzymes activities of POD, SOD, and CAT ,which also verified the results of our study from another perspective.Under normal conditions, the production of reactive oxygen species , and sucrose content increased by − 0.06–1.33% .The chlorophyll content, the activity of photosynthetic key enzymes, the expression levels of photosynthetic key genes, and photosynthesis of chewing cane were all enhanced in virus-free seedlings. In the virus-free seedlings, the content of O35. Plants with obvious symptoms of sugarcane mosaic disease were co-infected by SrMV and SCMV . Thirty kg of sugarcane field soil were used in each barrel, and the rates of available nitrogen, available phosphorous and available potassium were analyzed before planting, which were 72.62, 50.17 and 52.42 mg/kg, respectively. At the three-leaf stage, 4–5 seedlings of a similar size were retained in each barrel. Nine barrels of virus-free seedlings and nine barrels of infected seedlings were planted for both No. 7 and No. 8 cultivars. The experimental design was a single factor contrast test with three repetition. Thus, three barrels were one replicate. Planting management refers to the normal production and management of chewing cane37. At the maturity stage, 6 normal chewing cane stems (4 main stems and 2 tillers stems) were selected for each treatment , and brix, sucrose content, reducing sugar, and fiber content were measured using the methods outlined by Li and Zheng38.At the time of harvest, stem length, stem diameter, internode length, single stem weight, and stem yield per barrel were measured using similar methods as outlined by Zhang et al.2 (Ci), and transpiration rate (Tr) of the + 1 leaves of plants were determined using a chlorophyll meter  and portable photosynthetic apparatus LI-6400XT  from 9:00 to 11:00 am.Chlorophyll content and the photosynthetic parameters in leaves at the seedling stage, tillering stage, jointing stage, and large growth stage were determined. Chlorophyll content, net photosynthetic rate (Pn), stomatal conductance (Gs), intercellular COThe activity of phosphoenolpyruvate carboxylase (PEPC) and ribulose-1,5-bisphosphate carboxylase (Rubisco) within leaves were determined using the ELISA kit  according to the manufacturer's instructions.Total RNA was extracted from leaves using the TRlzon total RNA extraction kit  according to the manufacturer's instructions. The total RNA concentration and purity were determined using the ultra-micro spectrophotometer . The 1 μg of RNA was reverse-transcribed into cDNA using the HISCRIPT RT SuperMix for qPCR (+ g DNA wiper) kit  and stored at  − 20 °C for further use.pepc gene (GenBank Acc. No. AY135709), the small subunit gene rbcs of Rubisco (GenBank Acc. No. JN591757) and the large subunit gene rbcL of Rubisco (GenBank Acc. No. AE009947), the fluorescent quantitative specific primers of pepc, rbcs, and rbcL were designed using Primer 5.0 software. Primers with good linearity, high amplification efficiency, and specificity were screened for subsequent experiments . The experiment was carried by using cDNA as a template, preparing a fluorescence quantitative reaction system, and setting a reaction procedure according to the instructions. The amplification reaction was performed on a real-time fluorescent quantitative PCR instrument . The expression level of the gene relative to the internal control gene 41, and superoxide anion (O2−) content was determined using methods described by Wang and Luo42.MDA content in leaves was determined based on the methods outlined by Zeng et al.43 with slight modification. 1 g leaf sample was weighed and ground with liquid nitrogen, and then added to pre-cooled 2 mL 0.1 mol/L pH 8.8 boric acid buffer (containing 1 mmol/L EDTA-Na2 and 1% PVPP). The leaf was quickly ground. The homogenate was then transferred into a 10 mL centrifugal tube, and then the extract of 2 mL was taken to collect the homogenate adhering to the mortar and the mortar rod. After collection, the homogenate was transferred into the centrifugal tube and the volume was fixed to 10 mL with a buffer solution. The sample was centrifuged for 15 min  and the supernatant was collected and stored in a refrigerator at  − 20 °C for further enzymatic activity determination. The SOD activity was determined using the method outlined by Beauchamp and Fridovich44. The peroxidase (POD) activity was determined according to Xu et al.45, and the catalase (CAT) activity determination method is described by Aebi46.The crude enzyme solution was extracted according to Moerschbacher et al.p < 0.05. The figure draw using Origin 9.0 software .The same index for each treatment were measured three times, and the data statistics were performed in Excel 2010 . One-way ANOVA with Duncan's new multiple range test was used to analyze the significance of the differences between each group using IBM SPSS 19.0 statistical software . Means were thought to be significantly different and marked with lowercase letters , when Supplementary information


There are two errors in the article  that theActinia equina. The authors fail to note that this has previously been shown for Nematostella miRNAs in Supplementary table 1 in Grimson et al [Firstly, this article reports that miRNAs are likely to be 3' 2'-0-methylated in the cnidarian, In the Results section under the subheading “New virus-like sequences identified by metagenomic sequencing”, there is an error in the penultimate sentence.Specifically, the authors state with reference to Barns Ness dog whelk orthomyxo-like virus 1, that “the PB2 polymerase subunit falls between Infectious Salmon Anaemia virus and the Influenza/Thogoto virus clade”. This is incorrect, as PB2 should read PB1.The correct sentence is: These include the Caledonia dog whelk rhabdo-like virus 2 sequence, which is represented by a nucleoprotein that falls between the Rabies/Lyssaviruses and other rhabdoviruses, and Barns Ness dog whelk orthomyxo-like virus 1—for which the PB1 polymerase subunit falls between Infectious Salmon Anaemia virus and the Influenza/Thogoto virus clade .The authors do not report, or analyse, the PB2 subunit of Barns Ness dog whelk orthomyxo-like virus 1 in this paper. The sequences for the Barns Ness dog whelk orthomyxo-like virus 1 PB1 and PA subunits are available through GenBank .


However, deletion of the two virus host range genes, E3L and K3L, would render replication of the virus abortive in all the cell types tested, except the cells with defective PKR and RNase L activity. By expressing different poxvirus K3 ortholog proteins in the E3L and K3L double knockout vaccinia virus, we can construct a mutant vaccinia virus with modified host range. Here, using poxvirus K3 ortholog as a positive selection marker, we developed a proof-of-concept protocol to construct recombinant vaccinia viruses expressing foreign proteins. Such a recombinant virus has a modified host range in comparison to wild-type vaccinia virus. This protocol offers the following advantages:  Specifications Table➢Vaccinia Western Reserve strain (WR) E3L and K3L double deletion mutant virus (VACVΔE3LΔK3L) [3]➢Recombinant plasmid vector Fig. 1Sch➢E. coli, Taq DNA polymeraseRestriction enzymes, T4 DNA ligase, competent ➢Human lung carcinoma cells, A549 PKR and RNase L knockout (ko) cells [5]➢HeLa cells, BHK21 cells, HeLa/PKR ko cells [2]➢DMEM medium, 10X MEM, fetal calf serum (FCS)➢Attractene transfection reagent (Qiagen)➢Low melting point (LMP) agarose (Invitrogen)➢FLAG antibody (Sigma), vaccinia D12 antibody [6]➢Zeiss fluorescence microscope Axiovert 200 M➢12-well tissue culture plates➢NUCLISENS easyMAG (BioMerieux)➢Primers: K3FLF: AGAGCTCTGATTAGTTGCTGGCAACGA; K3FRR: ACTCGAGTACGTATATTTAGATGTTTTCA; and HHV8K8.1C: TCACACGATATAGGGCTTCTTTCT1.NC_006998) used for constructing the recombinant virus. For the proof-of-concept, the K3 ortholog of taterapox virus  driven by a sheeppox virus K3 ortholog promoter  was placed between the two flanking sequences and used as the host range selection marker. A gene of interest was cloned down stream of the early and late promoter BamHI and SmaI). As an alternative, the synthetic early/late promoter can be removed by HindIII digest and replaced with the gene of interest driven by a poxvirus promoter of choice.The structure of the recombinant shuttle vector and the parental virus, and the time course of the procedure are outlined in 2.The general protocol to infect the cells with the parental virus and transfect with the recombinant shuttle vector DNA is similar to the previously described 3.2 for 1 h. The virus was then removed and replaced with 1 ml of fresh DMEM with 2% FCS, glutamine, penicillin and streptomycin.24 h after infection and transfection A, the vi4.48 h post infection, the infected cells were examined under a fluorescence microscope. In comparison with the parental virus control, the recombinant virus  should develop noticeable virus plaques as shown in Poxvirus K3 ortholog based selection of recombinant vaccinia virus with modified host range5.2, the virus inoculum was removed, the cell monolayer was washed once with PBS and overlaid with MEM containing 2% FCS, 1x of penicillin-streptomycin-glutamine , and 1% LMP. The parental virus control was plated out in the same manner. 48 h post infection, the cells were examined under a fluorescence microscope and the plaques which express only green fluorescent protein, but not the mCherry, was marked with a marker, and the virus plaques are picked with a glass Pasteur pipette. In our experience, the purified recombinant virus expressing only the green fluorescent protein can be routinely obtained after 1 round of plaque purification.200 µl of a 5-fold serial dilution of the virus harvested above was added to BHK21 cell monolayers in a 12-well plate. Following 1 h incubation at 37 °C, 5% CO6.The plaque-purified recombinant virus is amplified once in BHK21 cells and the virus can be used to infect fresh cells for confirmation of expression of the protein of interest, either by Westernblot or PCR.As shown in Previously, using this selection system we have constructed several recombinant vaccinia viruses expressing genes of various interests: hepatitis E virus capsid protein  A FLAG tag was added to the C-terminus of the HHV8 K8.1 gene to facilitate the detection of the protein expression. The tagged HHV8 K8.1 gene was cloned under the control of the synthetic early/late promoter as shown in the 5 green and 1 red/green plaques (as an example to show it contains a mixture of recombinant viruses) were selected and grown up for examination of the expression of HHV8 K8.1 protein A. A549/PAlthough recombination between the recombinant shuttle vector and the poxvirus DNA genome can readily occur, selection and purification of a recombinant poxvirus can be laborious, mainly due to the recombinant virus accounting for only a tiny proportion in the total virus population. Using the vaccinia E3L and K3L double deletion mutant virus (VACVΔE3LΔK3L) as the parental virus and a shuttle recombinant vector targeting the K3L locus with a poxvirus K3L ortholog as a host range selection marker, this protocol allows the selection and purification of the recombinant virus after one round of amplification and one round of plaque purification. Using this protocol, we can routinely obtain a purified recombinant vaccinia virus within a week starting from the transfection with the recombinant shuttle vectors. Since the parental vaccinia virus used in this protocol does not have the E3L gene, which is critical for the virus pathogenicity, the recombinant virus generated should be highly attenuated. Different poxvirus K3 orthologs have been shown to have different host range activities (see Table 1 in reference The authors confirm that there are no conflicts of interest.


Actinidia chinensis var. deliciosa ‘Miliang-1’ that had no disease symptoms using high-throughput sequencing. Using overlapping reverse transcription-polymerase chain reaction and rapid amplification of cDNA ends, the genomic sequences of AdV-1 v1 and v2 were confirmed as 17,646 and 18,578 nucleotides in length, respectively. The two complete genomes contained 9 and 15 open reading frames, respectively, coding for proteins having domains typical of Closteroviridae, such as RNA-dependent RNA polymerase (RdRp), heat shock protein 70 homolog (HSP70h) and coat protein (CP). Sequence analysis showed that the amino acid sequences of RdRp, HSP70h, and CP of the two variants exhibited high similarity (> 80%), while their genomic organization was somewhat different. This suggested that the two viral genomes identified here are variants of the family Closteroviridae in a single kiwifruit host. Furthermore, phylogenetic relationship analysis revealed that the two variants had a closer relationship with the unclassified virus Persimmon virus B (PeVB) and Actinidia virus 1 (AcV-1) than with other members of the family Closteroviridae, as did their genomic organization. It is speculated that the two variants, together with PeVB and AcV-1 belong to a new subfamily of Closteroviridae.Two distinct closterovirus-like genome sequences (termed AdV-1 v1 and v2) were identified in  Kiwifruit is a perennial vine plant that began domestication and cultivation at the beginning of the 20th century . It is cApple stem grooving virus in a number of kiwifruit seedlings imported from China [Apple stem grooving virus, Actinidia virus X, Alfalfa mosaic virus, Cucumber necrosis virus, Cucumber mosaic virus, Ribgrass mosaic virus, Turnip vein clearing virus and Tomato necrotic spot associated virus [Actinidia virus A, Actinidia virus B, Actinidia citrivirus, Actinidia virus 1, Actinidia chlorotic ringspot-associated virus and Actinidia seed-borne latent virus [Cherry leaf roll virus and Pelargonium zonate spot virus) [Betaflexiviridae, Bromoviridae, Virgaviridae, Tombusviridae, Bunyaviridae, Secoviridae, and Closteroviridae.Viral diseases in plants have a long incubation period, and therefore represent a significant hidden danger during production. In 2003, New Zealand researchers discovered for the first time the symptoms of viral infection caused by ed virus , six spent virus –10, and t virus) . These kClosteroviridae, were detected. The family Closteroviridae consists of single-stranded and long positive-sense RNA genomes, and is categorized into four genera: Closterovirus, Crinivirus, Ampelovirus and Velarivirus, according to their large differences in molecular and biological characteristics [Bulueberry virus A and Mint vein banding virus [In 2015, a deep-sequencing analysis was conducted to identify long-noncoding RNAs in kiwifruit grown in China by our laboratory. Interestingly, two new putative viral genome sequences that shared significant sequence similarity with viruses classified as belonging to the family eristics . In adding virus , 12.Closteroviridae. Finally, three phylogenetic trees were constructed based on the amino acid sequences of three typical viral proteins  to elucidate their phylogenetic relationship. To the best of our knowledge, this is the first report of variant genome sequences of Closteroviridae from Chinese kiwifruit, which should represent the basis for future studies of their pathogenesis.In the present study, the two new putative viral genome sequences were confirmed using overlapping reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE-PCR). Their genomic structure and organization were then analyzed and compared with other members of the family Actinidia chinensis var. deliciosa ‘Miliang-1’) that displayed no disease symptoms from an orchard in Gaozhen Village, Xikou Town, Jianning County, Sanming City, Fujian Province, China. Total RNA was extracted using an RNAprep Pure Plant kit . RNA contamination and integrity were monitored using 1.0% agarose gel electrophoresis and an RNA Nano 6000 assay kit for the Bioanalyzer 2100 system . A NanoPhotometer® spectrophotometer  was used to estimate RNA purity, and a Qubit® RNA assay kit used to measure the RNA concentration in a Qubit® 2.0 Flurometer .In September 2015, fruits with total soluble solids of 6.0–7.0% were collected from 4-year old kiwi plants , the rRNA-free residues cleaned using ethanol precipitation. Sequencing libraries were then generated using the rRNA-depleted RNA using a NEBNext® Ultra™ Directional RNA Library prep kit for the Illumina® platform , in accordance with the manufacturer’s instructions. Following the assessment of library quality, a cBot Cluster Generation System was used to cluster the index-coded samples using a TruSeq PE cluster kit v3-cBot-HS (Illumia), in accordance with the manufacturer’s recommendations. The libraries were then sequenced on an Illumina HiseqX10 platform from which 150 bp paired-end reads were generated.High-quality RNA was used for sequencing. Firstly, ribosomal RNA (rRNA) was removed using an Epicentre Ribo-zerode novo assembled into large contigs using Trinity software [https://blast.ncbi.nlm.nih.gov/) using BLASTN (nucleotide BLAST) and BLASTX (translated nucleotide BLAST) applications.After removing reads containing adapters, ploy-N, and low-quality reads, the high-quality reads were software . Assemblsoftware . The un-Actinidia chinensis var. deliciosa ‘Miliang-1’ were reverse transcribed (RT) using a SMART™ RACE cDNA Amplification kit  for reverse transcription-polymerase chain reaction (RT-PCR) and 5’/3’-rapid amplification of cDNA ends (RACE). From the two contig sequences obtained from deep-sequencing, 12 to 16 pairs of specific primers were designed for RT-PCR using DNAMAN software. The detailed RT-PCR primer sequences and fragment positions are displayed in To confirm the results of next-generation sequencing, high-quality RNAs extracted from mixed samples of leaves, stems and fruits of EasyPure® Quick Gel extraction kit  in accordance with the manufacturer’s protocol, then cloned using a pEASY®-T1 cloning kit . At least three clones were sequenced by Beijing Liuhe Huada Gene Technology Co., Ltd (China).Both RT-PCR and RACE-PCR were performed in 200 μl-tubes in a 50 μl-reaction system containing 20 ng RT products, 0.4 μM forward/reverse primers and 25 μl DreamTaq Green Master Mix . The amplification program consisted of: initial PCR activation at 94°C for 3 mins, followed by 34 cycles of denaturation of 94°C for 30 s, primer annealing at 51.6 to 60°C . Pairwise comparisons of the full-length nucleotide sequences/amino acid sequences of viruses were performed using fast alignment in DNAMAN software with default parameters. Multiple sequence alignments were performed with complete alignment using ClustalX software using default parameters [https://web.expasy.org/protparam/). Conserved domains and motifs were analyzed using the NCBI Blast application (https://blast.ncbi.nlm.nih.gov/Blast.cgi) and pfam (http://pfam.xfam.org/), respectively. Transmembrane helices of proteins were analyzed in ExPASy using the TMpred application . Amino acid sequences were used as input sequences to perform alignments using ClustalW in MEGA 10.1.8, from which phylogenetic trees were constructed using a neighbor-joining algorithm with Poisson correction, pairwise gap deletion and bootstrap with 1000 replicates [Nucleotide sequences of the viruses were analyzed using DNAMAN software, and their predicted open reading frames (ORFs) determined using Open Reading Frame Finder , and 19,7431 nt were retained. A BLASTN search of the NCBI database revealed that they had high sequence similarity to viral genome sequences. An additional BLASTX search demonstrated that both the two contigs contained ORFs that coded for three proteins with typical domains of Closteroviridae, such as RNA-dependent RNA polymerase, heat shock protein 70 homolog and coat proteins.A total of 96,299,434 clean reads were obtained by next-generation sequencing. These clean reads were then Closteroviridae viruses, overlapping RT-PCR was performed using 12 and 16 specific primer pairs  isolated from Actinidia chinensis Planch var. chinensis ‘Hort16A’ grown in Italy, a newly discovered member of the family Closteroviridae [Closteroviridae, having homology with Actinidia virus 1. Since the two new variants were isolated from A. chinensis var. deliciosa ‘Miliang-1’ grown in China, they were provisionally termed Actinidia deliciosa virus 1 variant 1 (abbreviated to AdV-1 v1), and variant 2 (AdV-1 v2), respectively.DNAMAN sequence alignments showed that the two new viruses shared 81.38%  and 65.0oviridae . FurtherClosteroviridae, their genomic structure and organization were also somewhat different  [Citrus tristeza virus  [losterovirus, even though BVA remains unclassified, the number of papain-like leader protease domains present in ORF1a was therefore not a distinguishing characteristic. Two papain-like leader proteases may have evolved by gene duplication which function synergistically to enhance virus RNA amplification [The first ORF  of AdV-1 v1 consisted of 9,558 nt encoding a protein of 3,185 amino acids (aa). Similar to ORF1a of AcV-1, the conserved domains of two papain-like leader proteases, a methyltransferase and a helicase domain were also present in ORF1a of AdV-1 v1. However, it was divided into two smaller ORFs (ORF1a1 and ORF1a2) in AdV-1 v2. ORF1a1 (nt positions 122–5329) of AdV-1 v2 encoded a 1,735-aa protein containing a papain-like leader protease and a methyltransferase domain, while ORF1a2 (nt positions 5362–9693) encoded a 1,443-aa protein with a helicase domain. In the family fication .Closteroviridae and responsible for viral RNA replication [UAGAA-3'5'-GUAAUAAGGUCACAAGCCGUUCAGGA , while RdRp (nt positions 9663–11188) of AdV-1 v2 may be expressed in a +1 ribosomal frameshift by the sequence UAGAA-3'5'-GCGUCAUAAGGCCGCAGGCCGUUCAGGA . RdRp from AdV-1 v1 shared high amino acid identity (> 80%) with that of AdV-1 v2 and AcV-1, but had low amino acid similarity (< 38%) with the RdRps of other members of the family Closteroviridae (The second ORF (ORF1b) of both AdV-1 v1 and v2 was 1,524 nt in length and encoded a 507-aa RNA dependent RNA polymerase RdRp, . Both Rdlication , 21. RdRoviridae . As withoviridae , resultiThere were three ORFs following ORF1b that coded for hypothetical proteins of unknown function. Their molecular masses were predicted to be 13.68, 25.56 and 5.58 kDa in AdV-1 v1, and 14.34, 25.83 and 5.67 kDa in AdV-1 v2, so these were termed p14, p26 and p6 in both AdV-1 v1 and v2  Tables. Mint virus 1 (MV-1), Bean yellow disorder virus (BYDV), Grapevine leafroll-associated virus 1 (GLRaV-1), and Cordyline virus 1 (CoV-1), respectively . Hsp70h (nt positions 12757–14511) of AdV-1 v1 had 88.87% amino acid identity with that of AdV-1 v2 (nt positions 12580–14334), and 93.15% with that of AcV-1, but only 33.28%, 35.18%, 24.32%, 23.72%, 24.83%, and 23.54% with those of PeVB v1, PeVB v2, ectively .Closteroviridae, such as PeVB v1 and PeVB v2. In AdV-1 v2, ORF7 was divided into two smaller ORFs (ORF7a and ORF7b). ORF7a (nt positions 14243–14950) and ORF7b (nt positions 15011–15766) of AdV-1 v2 had 40.63% and 43.79% amino acid identity with p59 of AdV-1 v1, respectively.The seventh ORF (nt positions 14420–15943) of AdV-1 v1 encoded a hypothetical protein of 58.80 kDa (p59) that is 1,524 nt in length, which had high amino acid similarity (93.69%) with heat shock protein 90 homolog  of AcV-1. However, no specific Hsp90h domain was detected in p59 of AdV-1 v1 when searched using both the pfam and NCBI databases. Additionally, p59 of AdV-1 v1 shared < 30% amino acid identity with Hsp90h of other members of the family Closteroviridae. However, ORF8 was also divided into two smaller ORFs (ORF8a and ORF8b) in AdV-1 v2. Both ORF8a (nt positions 15603–16091) and ORF8b (nt positions 16076–16345) of AdV-1 v2 shared high sequence similarity with ORF8 of AdV-1 v1 and thaumatin-like proteins, although no thaumatin-like motif was found in either ORF8a or ORF8b.ORF8 (nt positions 15780–16517) of AdV-1 v1 encoded a 245-aa, 28-kDa protein (p28), in which a motif of thaumatin-like family (PF00314) was present when searching in pfam database. A similar situation was also present in ORF8 of AcV-1 and ORF6 of the PeVB variants . This p2Closterovirus coat protein. Sequence identity analysis showed that CP in AdV-1 v1 (nt positions 16519–17250) had 88.07% amino acid sequence similarity with that of AdV-1 v2 (nt positions 16347–17078), 93.42% with CP of AcV-1, 21.25 to 23.08% with CP in PeVB variants, and < 21.00% with CP in other members of the family Closteroviridae , etc. [There were three short ORFs downstream of ORF9 in AdV-1 v2, encoding a 157-aa, a 70-aa, and a 191-aa hypothetical protein with unknown functions, respectively  Tables. proteins . Sequenc1), etc. , 22.Closteroviridae . The RdRp, HSP70h, and CP proteins encoded by AdV-1 variants were grouped with the unclassified virus variants PeVB v1 and PeVB v2, and AcV-1 in the same clade, consistent with their genomic organizations  of the family oviridae . The braizations . A wholeizations , which cHigh-throughput sequencing is not only used to analyze the expression levels of known genes from multiple organisms, it can also be used to detect new genes and new variants –26. CompClosteroviridae. Therefore, we hypothesized that AdV-1 v1 and v2 represent two variants of the family Closteroviridae, homologous to AcV-1. In addition, similar to AcV-1 and PeVB variants, results of BLAST sequence and alignment indicated that RdRp, HSP70h and CP in the AdV-1 variants displayed low sequence identity (< 35%) with those of other members of the four identified genera  collected in the Sichuan Province in China, were used to perform RT-PCR using primers designed from the CP gene of AcV-1, the results indicating that these isolates shared 89.8–90.2% nucleotide sequence identity and 94.7–95.9% amino acid sequence identity with the CP of AcV-1 [e.g. leaf mottle, chlorosis, or malformation) and asymptomatic leaves in Sichuan Province [A. chinensis var. deliciosa ‘Miliang-1’ in Fujian Province. Hitherto, this virus has only been found in Fujian in the southeast of China and Sichuan Province in the southwest, and it is unknown whether it exists elsewhere because of the lack of prior detection. Since Actinidia plants in the field are often co-infected with several viruses, the precise role of this virus as a causal agent remains unknown. Therefore, future studies should focus on its detailed distribution and pathogenesis to avoid a large-scale disease outbreak and economic loss.This is the first report of the genome sequence of n Citrus . Recentlof AcV-1 . This imProvince . SimilarS1 Appendix(DOC)Click here for additional data file.S1 Fig(EMF)Click here for additional data file.S2 Fig(EMF)Click here for additional data file.S3 Fig(JPG)Click here for additional data file.S4 Fig(JPG)Click here for additional data file.S1 Table(DOC)Click here for additional data file.S2 Table(DOC)Click here for additional data file.S3 Table(DOC)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(DOC)Click here for additional data file.


Thrombotic thrombocytopenic purpura (TTP) can often be life threatening and requires timely diagnosis and prompt initiation of plasmapharesis. Cobalamin deficiency can closely mimic TTP and distinguishing between the two diseases can prove to be a diagnostic challenge. Previously, cobalamin-related pseudo-TTP has been associated with pernicious anemia, dietary insufficiency and hereditary disorders of cobalamin activation. Here in, we discuss the first case of suspected metformin-induced cobalamin deficiency causing pseudo-TTP. Our patient was a 36-year-old female with type 2 diabetes mellitus on metformin for eight years who presented with hemolytic anemia, thrombocytopenia, schistocytes and mild acute renal failure. The initial impression was TTP; however, further workup revealed very low serum cobalamin levels and elevated methylmalonic acid levels. Apart from metformin use, no other cause of cobalamin deficiency was identified. We recommended upper gastrointestinal endoscopy to definitively rule out pernicious anemia.  Thrombotic microangiopathies are a heterogeneous group of diseases, which can be congenital or acquired, have an acute or gradual onset, and can manifest early or late in life . ThromboA 36-year-old non-vegan African American female, with a past medical history of childhood obesity, diabetes mellitus type 2 (DM2) for eight years and iron-deficiency anemia (IDA) for one year, presented to us with complaints of progressively worsening fatigue for six weeks, followed by upper and lower extremity cramping, joint stiffness and hand paresthesia over a five-week period. She reported a history of regular heavy menstrual bleeding requiring up to 10 pads per day. She was diagnosed with IDA a year ago after which she was started on oral iron supplementation. She denied any nausea, vomiting, melena, appetite or weight changes. She had no prior history of fibroids or prior blood transfusions.In the past couple of days, she had worsening fatigue and had a presyncopal episode, which prompted her to visit her primary care physician (PCP). The PCP ordered blood work, which showed severe anemia with a hematocrit of 10.8% and hemoglobin 3.7 g/dL with no other abnormalities. Therefore, she was asked to visit the hospital for further management.Her family history was significant for breast cancer in the maternal aunt. She had had five children and no history of miscarriages. She was a former smoker, drank alcohol occasionally and denied any drug use. Of note, she was taking metformin 1,000 mg twice daily for DM2 for eight years.2 saturation 99% on room air. Electrocardiogram showed sinus tachycardia. Repeat lab work revealed pancytopenia with white cell count 2.4 x 103/μL, hemoglobin 3.7 g/dL, hematocrit 11% and platelet count 94 x 103/μL. Peripheral blood smear showed schistocytes  4,320 U/L, haptoglobin <1 mg/dL, reticulocyte count percentage 1.7% and reticulocyte count number 19.3 x 10The patient received four packed red blood cells during the hospital stay and never received any plasmapheresis. As the patient was on high-dose metformin for many years and had no obvious other etiology to explain the cobalamin deficiency, we suspected that the patient’s symptoms and lab findings could be due to cobalamin deficiency from metformin use. We discontinued metformin and prescribed intramuscular (IM) cobalamin 1,000 μg daily for one week followed by the same dose IM cobalamin weekly for one month as the initial therapy. We also recommended upper gastrointestinal endoscopy to conclusively rule out pernicious anemia but the patient refused. Therefore, later we prescribed IM cobalamin 1,000 μg monthly life long. The patient improved clinically thereafter, and lab work at six-month follow-up is given in Table Even though cobalamin deficiency is common in the general population, its hematologic manifestations, which resemble TTP, are seen in only 2.5% of the cobalamin-deficient patients . The firThe mechanism for MICD has been controversial. The proposed theories have included inhibition of cobalamin absorption, changes in intrinsic factor levels, normal flora or gastrointestinal (GI) motility . The cubThe pathogenesis of cobalamin deficiency causing pseudo-TTP is also not completely understood. Cobalamin deficiency cannot only lead to ineffective erythropoiesis but can also increase red blood cell rigidity, which can in turn promote intramedullary destruction of red cell precursors . As the 3/μL, whereas the average platelet count associated with TTP is approximately 12.5 x 103/μL [Cobalamin is necessary for DNA synthesis and hematopoiesis Figure . Cobalamx 103/μL .Although ADAMTS13 is usually normal in cobalamin deficiency, some case reports have reported ADAMTS13 to be surprisingly low . This caThere is no indication for plasmapheresis in the management of pseudo-TTP. Treatment varies depending on the underlying cause. Treatment initially involves IM cobalamin with 1,000 μg daily for one week, followed by 1,000 μg weekly for four weeks. We recommended monthly life-long supplementation in this particular case as patient refused upper GI endoscopy; hence, pernicious anemia could not be completely ruled out. According to the prior literature, reticulocyte count starts trending upwards by day 3 of treatment and peaks at one week. The hemoglobin concentration starts going up within 10 days and reaches normal limits by eight weeks . These fPseudo-TTP can prove to be a diagnostic challenge and may incorrectly be treated with plasmapheresis which itself carries a high mortality rate. A carefully taken history and review of the medication list can help to avoid unnecessary interventions. It is important for clinicians to be aware of the link between metformin use and pseudo-TTP. Ordering simple bloods test like serum cobalamin level in a patient presenting with hemolysis and taking metformin as seen in our case can help to reach a timely diagnosis.