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Category Archives: Genome

Secrets of Night Parrot unlocked after first genome sequenced – CSIRO

Posted: February 18, 2024 at 10:06 am

14 February 2024 News Release

Researchers at CSIRO, Australias national science agency, have sequenced the first genome of the Night Parrot, one of the worlds rarest and most elusive birds.

The development will answer questions about population genetics and biology that could boost conservation hopes for the recently rediscovered species.

The genome will enable us to explore the genetic basis of why the Night Parrot is nocturnal, a very unusual feature in parrots. Well investigate faculties like navigation, smell, beak shape and its less-than-optimal night vision, Dr Leo Joseph, Director of CSIROs Australian National Wildlife Collection said.

Researchers will also be able to run statistical analyses on the genome of this individual to estimate past population sizes of Night Parrot populations in Australia.

Now, we have the capability to compare this annotated genome with other, closely related parrots, shedding light on the reasons behind its scarcity and limited distribution compared to many of its relatives.

CSIRO researchers sequenced the Night Parrot genome its genetic blueprint using tissue obtained from Dr Kenny Travouillon, Acting Curator of Ornithology at the Western Australian Museum, after Traditional Owners in the Pilbara found the deceased specimen and delivered it to the Museum Boola Bardip.

The specimen, which is the best-preserved on display in the world, is now open to public viewing at the WA Museum Boola Bardip.

Dr Gunjan Pandey, who led the Night Parrot genomics project, said access to high-throughput DNA sequencing technology under CSIROs Applied Genomics Initiative is accelerating genomics research in Australia.

We can now generate very high-quality genomes from really tiny tissue samples even as small as an ants head or a single mosquito, Dr Pandey said.

This level of quality and detail just wasnt possible even five years ago.

The genetic data can be used to ensure conservation programs maximise diversity, so the species is resilient and has the best chance of long-term survival.

Once more widespread in arid Australia, the Night Parrot declined due to environmental changes such as predation by cats and foxes.

It is now known only from localised parts of southwest Queensland and Western Australia.

A couple of dozen scientific specimens were collected during the nineteenth century and one in 1912. Then a specimen was found in 1990 in southwest Queensland, Dr Joseph said.

Live birds were reported from the same area in 2013, and a live parrot was finally caught and tagged in 2015.

While the Night Parrot genome is an exciting scientific resource to understand more about this bird, protecting the species from cats, foxes, fire and habitat loss is also crucial for their conservation.

The Night Parrot genome will open up numerous opportunities for further research to help conserve this species, Dr Pandey said.

This will empower scientists to develop a plan for saving the Night Parrot, which is the ultimate goal of sequencing the genome and making it publicly available.

Note to editors

The National Center for Biotechnology Information (NCBI) at the National Library of Medicine (NLM) annotated the genome sequence of the Night Parrot (Pezoporus occidentalis). The locations of individual genes were found using NCBIs Eukaryotic Annotation Pipeline (EGAP). The annotated genome is now available online as part of the NCBI Reference Sequence (RefSeq) Database through NCBI Datasets.

CSIRO together with the Threatened Species Initiative, supported by Bioplatforms Australia, will continue genetic studies to understand more about the Night Parrot and other closely related birds such as the Eastern Ground Parrot.

CSIROs Applied Genomics Initiative (AGI) uses high throughput sequencing technology to deliver reference genomes and large-scale diversity datasets for new insights and applied outcomes.

The genome was sequenced using Oxford Nanopore technology at the Biomolecular Resource Facility (BRF) at the Australian National University (ANU). BRF is a service node of Bioplatforms Australia which is made possible through investment funding provided by the Commonwealth Government National Collaborative Research Infrastructure Strategy (NCRIS). The AGI has successfully assembled over 100 genomes across diverse life forms in recent years, and many of these annotated genomes are accessible to the publicvia GenBank .

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CRISPR gene editing tool gets a revolutionary high-tech upgrade – Earth.com

Posted: at 10:06 am

In the realm of scientific innovation, the past decade has seen the CRISPR/Cas systems emerge as a groundbreaking tool in genome editing, boasting applications that span from enhancing crop yields to pioneering gene therapy.

The recent advent of CRISPR-COPIES by the Center for Advanced Bioenergy and Bioproducts Innovation (CABBI) marks a significant leap forward, refining CRISPRs flexibility and user-friendliness.

CRISPR-COPIES represents a cutting-edge solution designed to swiftly pinpoint ideal chromosomal sites for genetic modification across any species.

It will accelerate our work in the metabolic engineering of non-model yeasts for cost-effective production of chemicals and biofuels, explains Huimin Zhao, a prominent figure at CABBI and the University of Illinois.

The essence of gene editing lies in its ability to precisely alter genetic codes, enabling the introduction of novel traits such as pest resistance or enhanced biochemical production.

While CRISPR/Cas systems have facilitated targeted genetic modifications, the challenge of identifying optimal genomic integration sites persisted as a significant bottleneck, often involving cumbersome manual screening and testing processes.

Enter CRISPR-COPIES, the Computational Pipeline for the Identification of CRISPR/Cas-facilitated Integration Sites.

This innovation transforms genome-wide neutral integration site identification into a rapid, efficient process, taking mere minutes to accomplish what once was a daunting task.

Finding the integration site in the genome manually is like searching for a needle in a haystack, said Aashutosh Boob, a ChBE Ph.D. student at the University of Illinois and primary author of the study.

However, with CRISPR-COPIES, we transform the haystack into a searchable space, empowering researchers to efficiently locate all the needles that align with their specific criteria.

The versatility and efficiency of CRISPR-COPIES were showcased in a study published in Nucleic Acids Research, demonstrating its application across various species to enhance the production of valuable biochemicals.

Moreover, the creation of a user-friendly web interface makes this tool accessible to researchers with limited bioinformatics background, democratizing the advanced capabilities of CRISPR/Cas systems.

A primary goal of CABBI is to harness non-model yeasts for the sustainable production of chemicals and fuels from plant biomass.

Traditional genome-editing techniques, hindered by their labor-intensive nature and the scarcity of genetic tools, posed significant challenges to this endeavor.

CRISPR-COPIES addresses these issues by offering a streamlined approach for the rapid identification of stable integration sites, thereby facilitating the engineering of strains for enhanced biochemical yields and crop traits.

This innovative software is poised to significantly accelerate the strain construction process, offering a boon to researchers worldwide in both academic and industrial settings.

By simplifying genetic engineering tasks, CRISPR-COPIES not only saves time and resources but also opens new avenues for the development of transgenic crops and the efficient conversion of biomass to valuable chemicals.

In summary, CRISPR-COPIES stands as a monumental advancement in the field of genetic engineering, offering researchers a powerful and accessible tool for precision genome editing.

By streamlining the identification of optimal genetic integration sites, it accelerates the pace of scientific discovery and innovation while advancing new possibilities to address some of the most pressing challenges in agriculture, biofuel production, and gene therapy.

As this technology continues to evolve and become more integrated into various fields of research, CRISPR-COPIES promises to drive forward the boundaries of whats possible.

This new technology gives the world with a significant leap towards a future where genetic engineering can be conducted more efficiently, accurately, and with greater impact than ever before.

The full study was published in the journal Nucleic Acids Research.

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Ancient retroviruses played a key role in the evolution of vertebrate brains – EurekAlert

Posted: at 10:06 am

image:

A myelinating oligodendrocyte(green)

Credit: Peggy Assinck, Altos Labs-Cambridge Institute of Science

Researchers report February 15 in the journal Cell that ancient viruses may be to thank for myelinand, by extension, our large, complex brains. The team found that a retrovirus-derived genetic element or retrotransposon is essential for myelin production in mammals, amphibians, and fish. The gene sequence, which they dubbed RetroMyelin, is likely a result of ancient viral infection, and comparisons of RetroMyelin in mammals, amphibians, and fish suggest that retroviral infection and genome-invasion events occurred separately in each of these groups.

Retroviruses were required for vertebrate evolution to take off, says senior author and neuroscientist Robin Franklin of Altos Labs-Cambridge Institute of Science. If we didnt have retroviruses sticking their sequences into the vertebrate genome, then myelination wouldnt have happened, and without myelination, the whole diversity of vertebrates as we know it would never have happened.

Myelin is a complex, fatty tissue that ensheathes vertebrate nerve axons. It enables rapid impulse conduction without needing to increase axonal diameter, which means nerves can be packed closer together. It also provides metabolic support to nerves, which means nerves can be longer. Myelin first appeared in the tree of life around the same time as jaws, and its importance in vertebrate evolution has long been recognized, but until now, it was unclear what molecular mechanisms triggered its appearance.

The researchers noticed RetroMyelins role in myelin production when they were examining the gene networks utilized by oligodendrocytes, the cells that produce myelin in the central nervous system. Specifically, the team was investigating the role of noncoding regions including retrotransposons in these gene networkssomething that hasnt previously been explored in the context of myelin biology.

Retrotransposons compose about 40% of our genomes, but nothing is known about how they might have helped animals acquire specific characteristics during evolution, says first author Tanay Ghosh, a computational biologist at Altos Labs-Cambridge Institute of Science. Our motivation was to know how these molecules are helping evolutionary processes, specifically in the context of myelination.

In rodents, the researchers found that the RNA transcript of RetroMyelin regulates the expression of myelin basic protein, one of the key components of myelin. When they experimentally inhibited RetroMyelin in oligodendrocytes and oligodendrocyte progenitor cells (the stem cells from which oligodendrocytes are derived), the cells could no longer produce myelin basic protein.

To examine whether RetroMyelin is present in other vertebrate species, the team searched for similar sequences within the genomes of jawed vertebrates, jawless vertebrates, and several invertebrate species. They identified analogous sequences in all other classes of jawed vertebrates (birds, fish, reptiles, and amphibians) but did not find a similar sequence in jawless vertebrates or invertebrates.

Theres been an evolutionary drive to make impulse conduction of our axons quicker because having quicker impulse conduction means you can catch things or flee from things more rapidly, says Franklin.

Next, the researchers wanted to know whether RetroMyelin was incorporated once into the ancestor of all jawed vertebrates or whether there were separate retroviral invasions in the different branches. To answer these questions, they constructed a phylogenetic tree from 22 jawed vertebrate species and compared their RetroMyelin sequences. The analysis revealed that RetroMyelin sequences were more similar within than between species, which suggests that RetroMyelin was acquired multiple times through the process of convergent evolution.

The team also showed that RetroMyelin plays a functional role in myelination in fish and amphibians. When they experimentally disrupted the RetroMyelin gene sequence in the fertilized eggs of zebrafish and frogs, they found that the developing fish and tadpoles produced significantly less myelin than usual.

The study highlights the importance of non-coding regions of the genome for physiology and evolution, the researchers say. Our findings open up a new avenue of research to explore how retroviruses are more generally involved in directing evolution, says Ghosh.

###

This research was supported by the Adelson Medical Research Foundation, the UK Multiple Sclerosis Society, the Wellcome Trust, and the Altos Labs-Cambridge Institute of Science.

Cell, Ghosh et al., A retroviral link to vertebrate myelination through retrotransposon RNA mediated control of myelin gene expression https://cell.com/cell/fulltext/S0092-8674(24)00013-8

Cell (@CellCellPress), the flagship journal of Cell Press, is a bimonthly journal that publishes findings of unusual significance in any area of experimental biology, including but not limited to cell biology; molecular biology; neuroscience; immunology; virology and microbiology; cancer; human genetics; systems biology; signaling; and disease mechanisms and therapeutics. Visit http://www.cell.com/cell. To receive Cell Press media alerts, contact press@cell.com.

Experimental study

Animals

A retroviral link to vertebrate myelination through retrotransposon RNA-mediated control of myelin gene expression

15-Feb-2024

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Natural selection and genetic diversity maintenance in a parasitic wasp during continuous biological control application – Nature.com

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Hopes elusive parrots genome will provide answers – news.com.au

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MicroRNA is the master regulator of the genome researchers are learning how to treat disease by harnessing the … – The Conversation

Posted: November 30, 2023 at 8:35 pm

The Earth formed 4.5 billion years ago, and life less than a billion years after that. Although life as we know it is dependent on four major macromolecules DNA, RNA, proteins and lipids only one is thought to have been present at the beginning of life: RNA.

It is no surprise that RNA likely came first. It is the only one of those major macromolecules that can both replicate itself and catalyze chemical reactions, both of which are essential for life. Like DNA, RNA is made from individual nucleotides linked into chains. Scientists initially understood that genetic information flows in one direction: DNA is transcribed into RNA, and RNA is translated into proteins. That principle is called the central dogma of molecular biology. But there are many deviations.

One major example of an exception to the central dogma is that some RNAs are never translated or coded into proteins. This fascinating diversion from the central dogma is what led me to dedicate my scientific career to understanding how it works. Indeed, research on RNA has lagged behind the other macromolecules. Although there are multiple classes of these so-called noncoding RNAs, researchers like myself have started to focus a great deal of attention on short stretches of genetic material called microRNAs and their potential to treat various diseases, including cancer.

Scientists regard microRNAs as master regulators of the genome due to their ability to bind to and alter the expression of many protein-coding RNAs. Indeed, a single microRNA can regulate anywhere from 10 to 100 protein-coding RNAs. Rather than translating DNA to proteins, they instead can bind to protein-coding RNAs to silence genes.

The reason microRNAs can regulate such a diverse pool of RNAs stems from their ability to bind to target RNAs they dont perfectly match up with. This means a single microRNA can often regulate a pool of targets that are all involved in similar processes in the cell, leading to an enhanced response.

Because a single microRNA can regulate multiple genes, many microRNAs can contribute to disease when they become dysfunctional.

In 2002, researchers first identified the role dysfunctional microRNAs play in disease through patients with a type of blood and bone marrow cancer called chronic lymphocytic leukemia. This cancer results from the loss of two microRNAs normally involved in blocking tumor cell growth. Since then, scientists have identified over 2,000 microRNAs in people, many of which are altered in various diseases.

The field has also developed a fairly solid understanding of how microRNA dysfunction contributes to disease. Changing one microRNA can change several other genes, resulting in a plethora of alterations that can collectively reshape the cells physiology. For example, over half of all cancers have significantly reduced activity in a microRNA called miR-34a. Because miR-34a regulates many genes involved in preventing the growth and migration of cancer cells, losing miR-34a can increase the risk of developing cancer.

Researchers are looking into using microRNAs as therapeutics for cancer, heart disease, neurodegenerative disease and others. While results in the laboratory have been promising, bringing microRNA treatments into the clinic has met multiple challenges. Many are related to inefficient delivery into target cells and poor stability, which limit their effectiveness.

One reason why delivering microRNA treatments into cells is difficult is because microRNA treatments need to be delivered specifically to diseased cells while avoiding healthy cells. Unlike mRNA COVID-19 vaccines that are taken up by scavenging immune cells whose job is to detect foreign materials, microRNA treatments need to fool the body into thinking they arent foreign in order to avoid immune attack and get to their intended cells.

Scientists are studying various ways to deliver microRNA treatments to their specific target cells. One method garnering a great deal of attention relies on directly linking the microRNA to a ligand, a kind of small molecule that binds to specific proteins on the surface of cells. Compared with healthy cells, diseased cells can have a disproportionate number of some surface proteins, or receptors. So, ligands can help microRNAs home specifically to diseased cells while avoiding healthy cells. The first ligand approved by the U.S. Food and Drug Administration to deliver small RNAs like microRNAs, N-acetylgalactosamine, or GalNAc, preferentially delivers RNAs to liver cells.

Identifying ligands that can deliver small RNAs to other cells requires finding receptors expressed at high enough levels on the surface of target cells. Typically, over one million copies per cell are needed in order to achieve sufficient delivery of the drug.

One ligand that stands out is folate, also referred to as vitamin B9, a small molecule critical during periods of rapid cell growth such as fetal development. Because some tumor cells have over one million folate receptors, this ligand provides sufficient opportunity to deliver enough of a therapeutic RNA to target different types of cancer. For example, my laboratory developed a new molecule called FolamiR-34a folate linked to miR-34a that reduced the size of breast and lung cancer tumors in mice.

One of the other challenges with using small RNAs is their poor stability, which leads to their rapid degradation. As such, RNA-based treatments are generally short-lived in the body and require frequent doses to maintain a therapeutic effect.

To overcome this challenge, researchers are modifying small RNAs in various ways. While each RNA requires a specific modification pattern, successful changes can significantly increase their stability. This reduces the need for frequent dosing, subsequently decreasing treatment burden and cost.

For example, modified GalNAc-siRNAs, another form of small RNAs, reduces dosing from every few days to once every six months in nondividing cells. My team developed folate ligands linked to modified microRNAs for cancer treatment that reduced dosing from once every other day to once a week. For diseases like cancer where cells are rapidly dividing and quickly diluting the delivered microRNA, this increase in activity is a significant advancement in the field. We anticipate this accomplishment will facilitate further development of this folate-linked microRNA as a cancer treatment in the years to come.

While there is still considerable work to be done to overcome the hurdles associated with microRNA treatments, its clear that RNA shows promise as a therapeutic for many diseases.

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"Ground-Breaking" Release of World’s Largest Whole Genome Resource – Inside Precision Medicine

Posted: at 8:35 pm

Entire genome sequences for nearly half a million people have been released by the UK Biobank, representing the largest dataset of its kind in the world.

The resource has the potential to offer new insights into the causes of major common diseases and guide the choice of potential therapeutic targets.

It has hailed as a step change in genomics and is available to approved researchers around the world through the UK Biobank Research Analysis Platform.

This is a veritable treasure trove for approved scientists undertaking health research, and I expect it to have transformative results for diagnoses, treatments and cures around the globe, said UK Biobank principal investigator Sir Rory Collins, PhD.

Executive vice president for innovative medicine research and development at industry partner Johnson & Johnson John Reed, PhD, maintained the findings could pave the way for more efficient clinical development and drive progress towards precision medicine.

This landmark dataset will enable us to leverage the power of artificial intelligence and machine learning for rapidly identifying novel disease targets and helping researchers predict how a candidate medicine might impact certain subpopulations of patients, based on their genetics, he said.

The UK Biobank whole genome sequencing (WGS) consortium was formed in 2018 with the goal of sequencing the genomes of all UK biobank participants.

The five-year project cost 200m, involved 11 partners and took 350,000 hours of sequencing time to create 27.5 petabytes of genetic data. At its peak, over 20,000 whole genomes, each with around three billion base pairs of DNA, were being sequenced each month. It resulted in the genomes of 491,554 UK Biobank volunteers being sequenced overall.

Half the funding came from the U.K. government and the Wellcome research organisation. The remaining 100 million was given by the biopharmaceutical and healthcare companies Amgen, AstraZeneca, GlaxoSmithKline, and Johnson & Johnson.

In return for their 25m investment, each of the four companies received a nine-month head start with the data before its public release.

The large-scale biomedical database and research UK Biobank resource follows the health of half a million volunteers recruited in 2006 and has already provided numerous clinical insights.

Data collected on over 10,000 variables, including blood pressure, cognitive function, diet and bone density, have been studied to examine why having the same genetic predisposition for a disease can result in different outcomes, reactions and side-effects to identical treatments.

It has led to thousands of scientific studies being published, and major insights such as the discovery that Type 1 diabetes is as common in adults as children.

Executive vice president of research and development at Amgen David Rees, PhD, said: This ground-breaking dataset allows scientists to explore how genetics affect levels of proteins, metabolites and other physiological factors, more closely than ever before, promising to accelerate our understanding of the genetic underpinnings of disease.

Chief executive of UK Research and Innovation (UKRI) professor Dame Ottoline Leyser, PhD, noted: Researchers can now apply to access de-identified full genome data from half a million participants, alongside a rich combination of medical, biochemical, lifestyle and environmental data from volunteers involved.

Today marks an important milestone in UKRIs commitment to realise the potential of genetics for biomedical research, innovation and translation to the clinic.

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Pangenome analysis reveals genomic variations associated with domestication traits in broomcorn millet – Nature.com

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Global genetic diversity, introgression, and evolutionary adaptation of indicine cattle revealed by whole genome … – Nature.com

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Genome characteristics of atypical porcine pestivirus from abortion cases in Shandong Province, China – Virology Journal – Virology Journal

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Viral metagenomic analysis

The number of clean reads was 21,157,543 for the RNA sample and 26,789,502 for the DNA sample. For RNA, the data were assembled to a total sequence length of 2,337,534, with 60.92% GC content. The length of the largest contig was 11,556 nt, which was identified as APPV (Table1), and named as APPV-SDHY-2022 for further analysis in this study. For DNA, the data were assembled with a total sequence length of 38,447,346 and 41.71% GC content. Other viruses, including Getah virus, porcine picobirnavirus, porcine kobuvirus, porcine sapovirus, Po-Circo-like virus, porcine serum-associated circular virus, porcine bocavirus 1, porcine parvovirus 1, porcine parvovirus 5 and porcine circovirus 3 were also identified by sequence alignment ((Table1), however, most contigs of these viruses were less than 500bp (see Additional file 2: Table s2 & Table s3). No other known pathogens (PRRSV, PPV2-4/68, CSFV, PCV2 and Japanese encephalitis virus) related to abortion were sequenced.

APPV presence was confirmed in the pooled sample by RTPCR amplification targeting the NS3 gene (see Additional file 3: Fig.s1A). The assembled sequence of the PCR products was identical to that of APPV-SDHY-2022 (see Additional file 3: Fig.s1B). This provided additional evidence of APPV presence in the abortion cases.

The genome of strain APPV-SDHY-2022 (GenBank accession no. OP381297) contains 11,556 nucleotides (nt) and consists of a 5UTR (370 nt, positions 1 to 370), CDS (10,909 nt, 371 to 11,279), and 3UTR (277 nt, 11,280 to 11,556). The nucleotide and amino acid sequences of the individual proteins of the strains were aligned separately, and the homology between APPV-SDHY-2022 and the reference strains was determined (Table2). Sequence alignment based on APPV polyprotein CDS showed that the nucleotide identities of APPV-SDHY-2022 with Clade I, Clade II, and Clade III strains were 82.6-84.2%, 93.2-93.6%, and 80.7-85%, respectively, while the amino acid identities were 91.4-92.4%, 96.4-97.7%, and 90.6-92.2%, respectively. APPV-SDHY-2022 shared the highest nucleotide identity (93.6%) with APPV-China/GD-SHM/2016, and the highest amino acid identity (97.7%) with GD-YJHSEY2N. Among the 12 mature proteins, NS5A showed the lowest homology (77.6-93.3% at the nt level) with the reference strains.

Phylogenetic analysis was performed based on complete polyprotein CDS and NS5A nucleotide sequences. The results showed that APPV-SDHY-2022 belongs to a separate branch of Clade II (Fig.2A). Moreover, the results revealed that the homology of NS5A nucleotide sequences was above 94.6% for the same isoform, 84.7-94.5% for different isoforms of the same clade and 76.8-81.1% for different clades (Table3). Therefore, we proposed that Clade II strains can be further divided into three subclades and that APPV-SDHY-2022 belongs to subclade 2.3. APPV-China/GD-SD/2016 and APPV-China/GZ01/2016 belong to subclade 2.2, and the other Chinese strains among the Clade II cluster belong to subclade 2.1 (Fig.2B). Since Clade II strains were found only in China, this typing method can help us better analyze the evolution of Clade II strains.

Phylogenetic analysis of Chinese APPV strains. Phylogenetic trees based on the nucleotide sequences of the complete polyprotein CDS (A) and the NS5A gene (B) were constructed by the neighbor-joining (NJ) method with 1,000 bootstrap replicates in MEGA11 software. The APPV-SDHY-2022 strain reported in this study is indicated with a red dot

To further explore the genetic evolution of APPV, potential recombination events were identified using Recombination Detection Program version 4 (RDP4) and then examined using SimPlot version 3.5.1. Among all available APPV strains, 8 strains (GD-DH01-2018, GD-BZ01-2018, JX-JM01-2018A01, GD2, GD-HJ-2017.04, GD-LN-2017.04, GD-CT4, and GD-MH01-2018) had potential genetic recombination events. Although NGS of APPV-SDHY-2022 confirmed recombination events of JX-JM01-2018A01 and GD-HJ-2017.04 by RDP4 (see Additional file 4: Table s4), no obvious genetic recombination in APPV-SDHY-2022 strains was observed by SimPlot software in this study (Fig.3).

Recombination analysis of the complete genomes of the APPV-SDHY-2022 strain from Shandong Province. Potential recombination events were identified using Recombination Detection Program 4 (RDP4) and then examined using similarity plots and bootstrap analysis in Simplot 3.5.1. The major and minor parents were JX-JM01-2018A01 and GD-HJ-2017.04, respectively

Amino acid sequences of individual viral proteins of all the Chinese APPV strains were analyzed. No amino acid insertions or deletions were found in the APPV-SDHY-2022 strain. The amino acid sequences of the individual proteins were compared to identify those that differentiate Clade II from Clade I and Clade III, and 20 unique amino acids were found in Clade II strains (Fig.4), among which, most sites were distributed on NS5A(7H,16A,69Q,131Q,152M,189I,280A,397F,437A) and NS5B(77V,139P,193P,231K,274A), and the remaining sites were on Npro (85D,120E), C(90K), Erns(91K,139Y) and NS3(30T). Interestingly, the amino acids at these unique sites were identical between Clade I and Clade III strains, demonstrating that it is possible to determine the type of strain by measuring these specific amino acids alone.

The unique amino acids found in Clade II APPV strains. Amino acid sequences of viral proteins were aligned with reference strains using MEGA11 and BioEdit software

In this study, putative N-glycosylation sites in the three important glycoproteins, Erns, E1, and E2, in Chinese APPV strains were also predicted. APPV-SDHY-2022, along with most of the strains in Clade II, is heavily glycosylated, with a total of ten N-glycosylation sites (N104 in the E1 protein; N12, N26, N43, N64, and N99 in the Erns protein; N51,N64,N103, and N127 in the E2 protein) (Fig.5). All the Chinese APPV strains had a conserved putative N-glycosylation site at N104 with a consensus N-I-T motif in the E1 protein. The putative N-glycosylation sites in the Erns and E2 proteins differed greatly among strains in different subclades, and 9 patterns of putative N-glycosylation sites were observed in E2 proteins, including N51+N64+N103, N64+N103, N51+N64+N103+N141,N51+N64+N127+N103+N141,N51+N64+N103+N127,N64+N103+N127,N51+N127,N51+N64,N64(Fig.5). Among the N-glycosylation sites of E2 proteins, a putative site at N64 was highly conserved.

Putative N-glycosylation sites of Erns, E1 and E2 proteins. The putative N-glycosylation sites within the Erns, E1 and E2 sequences of Chinese APPV strains were predicted according to a glycosylation analysis algorithm, and are shown as a blue shaded box

To analyze the effect of glycosylation sites on the antigenicity of the E2 protein, the antigenic index was determined by the Jameson-Wolf method in this study, and the results showed that aa positions at 1~9, 15~28, 34~44, 49~55, 62~82, 118~130, 136~158, 174~184, 188~196 and 200~205 of the E2 protein were the potential immunodominant regions. A comparison of the antigenic index within Chinese strains with and without a specific putative site showed that the putative N-glycosylation site at N51 had a negative effect on the antigenicity of the corresponding region (Fig.6).

Antigenicity prediction for the E2 protein. The Jameson-Wolf algorithm, which combines secondary structure information with backbone flexibility to predict surface accessibility, was used to determine the predicted antigenic index, with a threshold value of 1.7. The putative N-glycosylation sites within the E2 sequences of Chinese APPV strains are shown as a blue arrow. Representative strains from different Clades/subclades or patterns of putative N-glycosylation sites were included, and the strains in each subclade with different patterns of putative N-glycosylation sites are underlined

To further analyze the effect of glycosylation sites on conformational epitopes of the E2 protein, BepiPred-3.0 was used to predict B-cell conformational epitopes. The results showed that the 15 most likely B-cell conformational epitope residues varied among different Clades/subclades or patterns of N-glycosylation sites, and 39E, 70R, 173R, 190K, and 191N were conserved residues among all Chinese strains (Table4) (see also the graphical representations of the predicted epitopes in Fig.7).

Conformational B-cell epitope prediction for the E2 protein. The potential B-cell conformational epitopes of the E2 protein in APPV Chinese strains were predicted by BepiPred-3.0, and the residues with scores above the threshold (default value is 0.1512) are predicted to be part of an epitope and colored in yellow on the graph (where Y-axes depict BepiPred-3.0 epitope scores and X-axes protein sequence positions). Shown is the graphical output of B-cell discontinuous epitope predictions for the E2 protein with APPV-SDHY-2022 as an example

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Genome characteristics of atypical porcine pestivirus from abortion cases in Shandong Province, China - Virology Journal - Virology Journal

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