Deletion Of Open Reading Frame Research Articles

The engineered ORF56–57 deletion mutant leads to attenuation of an Asian koi herpesvirus strain

Koi herpesvirus (KHV) poses a significant threat to the koi and common carp aquaculture industry due to its high mortality rate among infected fish. In this study, we successfully isolated an Asian KHV strain designated as SS09 and investigated the potential attenuation of the SS09 through the deletion of open reading frames 56 and 57 (ORF56–57). By utilizing homologous recombination technology, we generated a mutant strain with a deletion of ORF56–57, known as SS09 Δ56–57. The mutant strain exhibited slightly reduced viral replication efficiency in common carp brain (CCB) cells. Furthermore, challenge trials conducted on healthy koi populations revealed that fish infected with the SS09 Δ56–57 did not show mortality, whereas the mortality rate of fish infected with the wild strain was nearly 100%. This suggests that the deletion of ORF56–57 can lead to the attenuation of the Asian KHV strain. To further validate the feasibility of SS09 Δ56–57 as novel vaccine candidates or therapeutic interventions against KHV, we conducted immersion immunization of koi fish with three different doses of SS09 Δ56–57. Serum neutralizing antibody titers were determined, and challenge tests were performed three weeks after immunization. The results showed that SS09 Δ56–57 at different doses exhibited a relative percentage survival (RPS) ranging from 70.0% to 96.6%. The neutralization titer of anti-serum in fish following 3 weeks of inoculation also displayed a dose-dependent response. In summary, owing to its outstanding immunogenicity, safety, and efficacy, SS09 Δ56–57 can be regarded as a promising candidate vaccine strain for KHV disease.

A single-dose of intranasal vaccination with a live-attenuated SARS-CoV-2 vaccine candidate promotes protective mucosal and systemic immunity

An attenuated SARS-CoV-2 virus with modified viral transcriptional regulatory sequences and deletion of open-reading frames 3, 6, 7 and 8 (∆3678) was previously reported to protect hamsters from SARS-CoV-2 infection and transmission. Here we report that a single-dose intranasal vaccination of ∆3678 protects K18-hACE2 mice from wild-type or variant SARS-CoV-2 challenge. Compared with wild-type virus infection, the ∆3678 vaccination induces equivalent or higher levels of lung and systemic T cell, B cell, IgA, and IgG responses. The results suggest ∆3678 as an attractive mucosal vaccine candidate to boost pulmonary immunity against SARS-CoV-2.

SARS-CoV-2 ORF8 accessory protein is a virulence factor.

The relevance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ORF8 in the pathogenesis of COVID-19 is unclear. Virus natural isolates with deletions in ORF8 were associated with wild milder disease, suggesting that ORF8 might contribute to SARS-CoV-2 virulence. This manuscript shows that ORF8 is involved in inflammation and in the activation of macrophages in two experimental systems: humanized K18-hACE2 transgenic mice and organoid-derived human airway cells. These results identify ORF8 protein as a potential target for COVID-19 therapies.

Horizontal transfer and the widespread presence of Galileo transposons in Drosophilidae (Insecta: Diptera).

Galileo is a transposon notoriously involved with inversions in Drosophila buzzatii by ectopic recombination. Although widespread in Drosophila, little is known about this transposon in other lineages of Drosophilidae. Here, the abundance of the canonical Galileo and its evolutionary history in Drosophilidae genomes was estimated and reconstructed across genera within its two subfamilies. Sequences of this transposon were masked in these genomes and their transposase sequences were recovered using BLASTn. Phylogenetic analyses were employed to reconstruct their evolutionary history and compare it to that of host genomes. Galileo was found in nearly all 163 species, however, only 37 harbored nearly complete transposase sequences. In the remaining, Galileo was found highly fragmented. Copies from related species were clustered, however horizontal transfer events were detected between the melanogaster and montium groups of Drosophila, and between the latter and the Lordiphosa genus. The similarity of sequences found in the virilis and willistoni groups of Drosophila was found to be a consequence of lineage sorting. Therefore, the evolution of Galileo is primarily marked by vertical transmission and long-term inactivation, mainly through the deletion of open reading frames. The latter has the potential to lead copies of this transposon to become miniature inverted-repeat transposable elements.

Development of an attenuated vaccine against Koi Herpesvirus Disease (KHVD) suitable for oral administration and immersion

Since the end of the1990ies, Cyprinid herpesvirus 3 (also known as koi herpesvirus, KHV) has caused mass mortality events of koi and common carp all over the globe. This induced a high economic impact, since the KHV disease cannot be cured up to now, but only prevented by vaccination. Unfortunately, there is only one commercial vaccine available which is not approved in most countries. Therefore, there is an urgent need for new, safe and available vaccines. In this study, a live attenuated vaccine virus was generated by cell culture passages of virulent KHV, and shown to protect carp or koi after immersion or oral application against wild type challenge. An advantage of boost immunization was demonstrated, especially after oral application. Vaccination induced no or mild clinical signs and protecting antibodies have been measured. Additionally, the vaccine virus allowed differentiation of infected from vaccinated animals (DIVA) by PCR. The attenuation of the newly generated vaccine was tracked down to a partial deletion of open reading frame 150. This was confirmed by the generation of engineered ORF150 deletion mutants of wild-type KHV which exhibited a similar attenuation in vivo.

The Requirement for US28 During Cytomegalovirus Latency Is Independent of US27 and US29 Gene Expression.

The ability to establish a latent infection with periodic reactivation events ensures herpesviruses, like human cytomegalovirus (HCMV), lifelong infection, and serial passage. The host-pathogen relationship throughout HCMV latency is complex, though both cellular and viral factors influence the equilibrium between latent and lytic infection. We and others have shown one of the viral-encoded G protein-coupled receptors, US28, is required for HCMV latency. US28 potentiates signals both constitutively and in response to ligand binding, and we previously showed deletion of the ligand binding domain or mutation of the G protein-coupling domain results in the failure to maintain latency similar to deletion of the entire US28 open reading frame (ORF). Interestingly, a recent publication detailed an altered phenotype from that previously reported, showing US28 is required for viral reactivation rather than latency, suggesting the US28 ORF deletion impacts transcription of the surrounding genes. Here, we show an independently generated US28-stop mutant, like the US28 ORF deletion mutant, fails to maintain latency in hematopoietic cells. Further, we found US27 and US29 transcription in each of these mutants was comparable to their expression during wild type infection, suggesting neither US28 mutant alters mRNA levels of the surrounding genes. Finally, infection with a US28 ORF deletion virus expressed US27 protein comparable to its expression following wild type infection. In sum, our new data strongly support previous findings from our lab and others, detailing a requirement for US28 during HCMV latent infection.

SARS-CoV-2 genomic surveillance in Taiwan revealed novel ORF8-deletion mutant and clade possibly associated with infections in Middle East

ABSTRACT Taiwan experienced two waves of imported infections with Coronavirus Disease 2019 (COVID-19). This study aimed at investigating the genomic variation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in Taiwan and compared their evolutionary trajectories with the global strains. We performed culture and full-genome sequencing of SARS-CoV-2 strains followed by phylogenetic analysis. A 382-nucleotides deletion in open reading frame 8 (ORF8) was found in a Taiwanese strain isolated from a patient on February 4, 2020 who had a travel history to Wuhan. Patients in the first wave also included several sporadic, local transmission cases. Genomes of 5 strains sequenced from clustered infections were classified into a new clade with ORF1ab-V378I mutation, in addition to 3 dominant clades ORF8-L84S, ORF3a-G251V and S-D614G. This highlighted clade also included some strains isolated from patients who had a travel history to Turkey and Iran. The second wave mostly resulted from patients who had a travel history to Europe and Americas. All Taiwanese viruses were classified into various clades. Genomic surveillance of SARS-CoV-2 in Taiwan revealed a new ORF8-deletion mutant and a virus clade that may be associated with infections in the Middle East, which contributed to a better understanding of the global SARS-CoV-2 transmission dynamics.

Pathogenicity of an FAdV-4 isolate to chickens and its genomic analysis.

Fowl adenovirus serotype 4 (FAdV-4) strain SD1511 was isolated from chickens with severe inclusion body hepatitis and hydropericardium syndrome in Shandong Province, China. The isolate was cultured in primary chicken embryo kidney cells. A study of pathogenicity indicated that SD1511 readily infected 7-35-d-old chickens by intramuscular injection and intranasal and oral routes, causing 50%-100% mortality. The 35-d-old chickens suffered more severe infection than 7- and 21-d-old chickens with mortality highest in the intramuscular injection group. The serum from surviving chickens showed potent viral neutralizing capability. The complete genome of SD1511 was sequenced and analyzed. The strain was found to belong to the FAdV-4 cluster with more than 99% identity with the virulent FAdV-4 strains isolated in China in recent years except for some distinct variations, including deletions of open reading frame 27 (ORF27), ORF48, and part of ORF19. Our findings suggest that SD1511 might be used as a prototype strain for the study of pathogenesis and vaccine development.

Attenuation of replication by a 29 nucleotide deletion in SARS-coronavirus acquired during the early stages of human-to-human transmission

A 29 nucleotide deletion in open reading frame 8 (ORF8) is the most obvious genetic change in severe acute respiratory syndrome coronavirus (SARS-CoV) during its emergence in humans. In spite of intense study, it remains unclear whether the deletion actually reflects adaptation to humans. Here we engineered full, partially deleted (−29 nt), and fully deleted ORF8 into a SARS-CoV infectious cDNA clone, strain Frankfurt-1. Replication of the resulting viruses was compared in primate cell cultures as well as Rhinolophus bat cells made permissive for SARS-CoV replication by lentiviral transduction of the human angiotensin-converting enzyme 2 receptor. Cells from cotton rat, goat, and sheep provided control scenarios that represent host systems in which SARS-CoV is neither endemic nor epidemic. Independent of the cell system, the truncation of ORF8 (29 nt deletion) decreased replication up to 23-fold. The effect was independent of the type I interferon response. The 29 nt deletion in SARS-CoV is a deleterious mutation acquired along the initial human-to-human transmission chain. The resulting loss of fitness may be due to a founder effect, which has rarely been documented in processes of viral emergence. These results have important implications for the retrospective assessment of the threat posed by SARS.

Fowl Adenovirus 4 (FAdV-4)-Based Infectious Clone for Vaccine Vector Development and Viral Gene Function Studies.

Fowl adenovirus 4 (FAdV-4) is associated with economically important poultry diseases. Recent studies of fully sequenced genomes of FAdV-4 isolates suggest potential genomic regions associated with virulence and amenable for manipulation and vector development. Direct manipulation of viral genomes is cumbersome, as opposed to that of infectious clones—viral genomes cloned into plasmid or cosmid vectors. In this work, we generated an infectious clone, pFAdV-4 ON1, containing the entire viral genome of a nonpathogenic FAdV-4 (ON1 isolate). pFAdV-4 ON1 was used for targeted deletion of open reading frames (ORFs) 16 and 17 and replacement with the enhanced green fluorescence protein (EGFP) expression cassette to generate recombinant viruses. These viruses were viable, and EGFP was expressed in infected cells. Their replication, however, was significantly reduced with respect to that of the wild-type virus. These observations suggest the potential utility of FAdV-4 as a vaccine vector and the importance of ORFs 16 and 17 for virus replication at wild-type levels. To our knowledge, this is the first report of an infectious clone based on the FAdV-4 genome, and our results demonstrate its utility for studies of virulence determinants and as a platform for either vaccine or gene delivery vectors.

Targeted Genome Sequencing Reveals Varicella-Zoster Virus Open Reading Frame 12 Deletion.

The neurotropic herpesvirus varicella-zoster virus (VZV) establishes a lifelong latent infection in humans following primary infection. The low abundance of VZV nucleic acids in human neurons has hindered an understanding of the mechanisms that regulate viral gene transcription during latency. To overcome this critical barrier, we optimized a targeted capture protocol to enrich VZV DNA and cDNA prior to whole-genome/transcriptome sequence analysis. Since the VZV genome is remarkably stable, it was surprising to detect that VZV32, a VZV laboratory strain with no discernible growth defect in tissue culture, contained a 2,158-bp deletion in open reading frame (ORF) 12. Consequently, ORF 12 and 13 protein expression was abolished and Akt phosphorylation was inhibited. The discovery of the ORF 12 deletion, revealed through targeted genome sequencing analysis, points to the need to authenticate the VZV genome when the virus is propagated in tissue culture.IMPORTANCE Viruses isolated from clinical samples often undergo genetic modifications when cultured in the laboratory. Historically, VZV is among the most genetically stable herpesviruses, a notion supported by more than 60 complete genome sequences from multiple isolates and following multiple in vitro passages. However, application of enrichment protocols to targeted genome sequencing revealed the unexpected deletion of a significant portion of VZV ORF 12 following propagation in cultured human fibroblast cells. While the enrichment protocol did not introduce bias in either the virus genome or transcriptome, the findings indicate the need for authentication of VZV by sequencing when the virus is propagated in tissue culture.

An Inactivated Novel Genotype Fowl Adenovirus 4 Protects Chickens against the Hydropericardium Syndrome That Recently Emerged in China.

Since 2015, China has experienced outbreaks of severe hydropericardium syndrome (HPS), associated with a novel genotype and hypervirulent fowl adenovirus serotype 4 (FAdV-4) infection, with a prevalence in various provinces of the country. This has resulted in huge economic losses in the poultry industry. The novel FAdV-4 showed new genome characters, such as the natural deletion of open reading frame (ORF) 19 and ORF 27 (1966 bp), and high pathogenicity toward chickens. These are coupled with severe hydropericardium, inclusion body hepatitis, and mortality rates ranging from 30% to 90%. Although several inactivated and subunit vaccines against the traditional FAdV-4 have been developed, no commercial vaccine against the emerged disease caused by the novel strain has been available until now. The potential risks of infection with this novel hypervirulent FAdV-4 urgently require an effective vaccine. Thus, an inactivated oil-emulsion FAdV-4 vaccine formulated with the novel genotype virus was developed in this study. The vaccine provided a high level of antibody, preferential T helper 2 (Th2) (interleukin-4 secretion) not Th1 (interferon-γ secretion) response, and full protection against a lethal dose of the novel hypervirulent FAdV-4. Therefore, the novel genotype FAdV-4 vaccine is proposed as an attractive candidate to prevent and reduce the spread of HPS in the poultry industry of China.

Increase in Middle East Respiratory Syndrome-Coronavirus Cases in Saudi Arabia Linked to Hospital Outbreak With Continued Circulation of Recombinant Virus, July 1-August 31, 2015.

During July–August 2015, the number of cases of Middle East respiratory syndrome (MERS) reported from Saudi Arabia increased dramatically. We reviewed the 143 confirmed cases from this period and classified each based upon likely transmission source. We found that the surge in cases resulted predominantly (90%) from secondary transmission largely attributable to an outbreak at a single healthcare facility in Riyadh. Genome sequencing of MERS coronavirus from 6 cases demonstrated continued circulation of the recently described recombinant virus. A single unique frameshift deletion in open reading frame 5 was detected in the viral sequence from 1 case.

Genome Sequence of a Fowl Adenovirus Serotype 4 Strain Lethal to Chickens, Isolated from China.

We report here the complete genome sequence of virulent fowl adenovirus serotype 4 strain HB1510, isolated from a diseased chicken with hydropericardium-hepatitis syndrome in Hubei, China, in 2015. The viral genome is 43,721 bp long, and sequence analysis showed an 11-amino-acid deletion in open reading frame 29 (ORF29).

Human cytomegalovirus UL49 encodes an early, virion-associated protein essential for virus growth in human foreskin fibroblasts.

Despite recent results of deletion experiments showing that open reading frame (ORF) UL49 of human cytomegalovirus (HCMV) is essential, the expression, function and functional location of its encoded protein remain unknown. We generated an antibody specific for pUL49 to investigate the protein product encoded by the UL49 ORF and identified its function in HCMV-infected host foreskin fibroblasts. A bacterial artificial chromosome (BAC) of HCMV strain Towne (pRV-Towne) and the UL49-deleted mutant pRV-delUL49Towne were used to observe virus growth by plaque assay. Using a UL49-protein-binding antibody, we located pUL49 in the fibroblast cytoplasm. pUL49 exhibited expression kinetics resembling those of the class β-2 proteins and was detected in the virion tegument. Following deletion of UL49 ORF, the virus failed to replicate, but it could be recovered by addition of pUL49 from pCDNA3.1 (+)-UL49. Our findings indicate that UL49 ORF is essential for HCMV replication in host foreskin fibroblasts.

S gene mutants occurrence among hepatitis B carriers in malaysia.

Background:The S gene region of the hepatitis B virus (HBV) codes for surface antigen (HBs Ag) and is responsible for classification of HBV strains.Objectives:The current study aimed to identify important mutations in the S gene in Hepatitis B virus (HBV) isolated from Malaysian HBV carriers.Materials and Methods:Isolated HBV DNAs were subjected for PCR amplification and sequencing of HBV full genome.Results:A total of 76 HBV full genome and 17 partial genome sequences were obtained from the 93 sequenced sera samples Genotyping of the full genome sequences by HEPSEQ software revealed a distribution of 49.46%, 48.39% and 2.15% of genotypes C, B, and D, respectively; whereas phylogenetic and jumping profile Hidden Markov Model (jpHMM) analysis identified six (7.89%) recombinant B/C strains. The distribution of sub-genotypes were B2 (78.79%) and B3 (21.21%) for genotype B, sub genotype D2 (100%) for genotype D and sub genotype C1 (75.76%), C2 (15.15%), C3 (6.06%) and C5 (3.13%) for genotype C. Mutation analysis in the S gene demonstrated two significant mutations which were W182 stop codon and deletion at open reading frame (ORF) of pre-S1 with the frequency occurrence of 2.2% (2/93) and 5.4% (5/93), respectively. The two patients with W182 stop codon were both male, infected with HBV genotype C and one showed progression of liver disease to hepatocellular carcinoma (HCC).Conclusions:Association with sex, genotype and clinical symptoms revealed that the pre-S1 ORF deletion occurred in 40% , 40%,and 20% of genotypes B,C, and D respectively, and 80% of the female population, of which all but one were diagnosed with chronic hepatitis B. Additionally, several mutations were found in the BCP region with the following incidence rate; C1653 T (8.6%), A1752 G (10.8%),1762 AGG--TGA 1764 (26.9%), C1766T(2.2%),T1768 A (10.8%), C1858 T (64.5%), G1896 A (25.8%).

Natural antisense transcripts of UL123 packaged in human cytomegalovirus virions

In this study, we demonstrated that antisense transcripts of human cytomegalovirus (HCMV) UL123, UL21.5 and cellular GAPDH genes were present in highly purified virions. These virion RNAs were delivered into the host cells upon infection, and de novo synthesized ones appeared in the infected cell at the immediate early stage. Although the sequence of UL123 antisense transcripts in virions is uncertain, we found that these transcripts in Towne-infected human fibroblasts had novel transcriptional start sites (TSSs) with various 5'-terminal deletions of open reading frame (ORF) 59. These findings not only provide new insight into the composition of HCMV virions but also reveal a possible viral strategy for initiating latent infection and switching between latent and productive infections.

Cis preferential replication of Lettuce infectious yellows virus (LIYV) RNA 1: The initial step in the asynchronous replication of the LIYV genomic RNAs

A series of Lettuce infectious yellows virus (LIYV) RNA 1 mutants was created to evaluate their ability to replicate in tobacco protoplasts. Mutants ΔEcoRI, ΔE-LINK, and Δ1B, having deletions in open reading frames (ORFs) 1A and 1B, did not replicate when individually inoculated to protoplasts or when co-inoculated with wild-type RNA1 as a helper virus. A fragment of the green fluorescent protein (GFP) gene was inserted into the RNA 1 ORF 2 (P34) in order to provide a unique sequence tag. This mutant, P34-GFP TAG, was capable of independent replication in protoplasts. Mutants derived from P34-GFP TAG having frameshift mutations in the ORF 1A or 1B were unable to replicate in protoplasts alone or in trans when co-inoculated with wild-type RNA1 as a helper virus. Taken together, these data strongly suggest that LIYV RNA 1 replication is cis-preferential.

The genetics of ageing: insight from genome‐wide approaches in invertebrate model organisms

Intense effort has been directed at understanding pathways modulating ageing in invertebrate model organisms. Prior to this decade, several longevity genes had been identified in flies, worms and yeast. More recently, with the development of RNAi libraries in worms and the yeast open reading frame (ORF) deletion collection, it has become routine to perform genome-wide screens for phenotypes of interest. A number of worm screens have now been performed to identify genes whose reduced expression leads to longer lifespan, and two ORF deletion longevity screens have been performed in yeast. Interestingly, these screens have linked previously unidentified cellular pathways to invertebrate ageing. More surprising, however, is the sheer number of longevity genes in worms and yeast. In this review, I discuss data from genome-wide screens in the context of evolutionary theories of ageing and raise issues regarding the increasing complexity associated with the genetics of longevity.

Deletion in Open Reading Frame 49 of Varicella-Zoster Virus Reduces Virus Growth in Human Malignant Melanoma Cells but Not in Human Embryonic Fibroblasts

The ORF49 gene product (ORF49p) of the varicella-zoster virus (VZV) is likely a myristylated tegument protein, and its homologs are conserved across the herpesvirus subfamilies. The UL11 gene of herpes simplex virus type 1 and of pseudorabies virus and the UL99 gene of human cytomegalovirus are the homologs of ORF49 and have been well characterized by using mutant viruses; however, little research on the VZV ORF49 gene has been reported. Here we report on VZV ORF49p expression, subcellular localization, and effect on viral spread in vitro. ORF49p was expressed during the late phase of infection and located in the juxtanuclear region of the cytoplasm, where it colocalized mainly with the trans-Golgi network-associated protein. ORF49p was incorporated into virions and showed a molecular mass of 13 kDa in VZV-infected cells and virions. To elucidate the role of the ORF49 gene, we constructed a mutant virus that lacked a functional ORF49. No differences in plaque size or cell-cell spread were observed in human embryonic fibroblast cells, MRC-5 cells, infected with the wild-type or the mutant virus. However, the mutant virus showed diminished cell-cell infection in a human malignant melanoma cell line, MeWo cells. Therefore, VZV ORF49p is important for virus growth in MeWo cells, but not in MRC-5 cells. VZV may use different mechanisms for virus growth in MeWo and MRC-5 cells. If so, understanding the role of ORF49p should help elucidate how VZV accomplishes cell-cell infections in different cell types.