Open Reading Frame 1a Research Articles

Effects of SARS-CoV-2 mutations on protein structures and intraviral protein-protein interactions.

Since 2019, severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) causing coronavirus disease 2019 (COVID‐19) has infected 10 millions of people across the globe, and massive mutations in virus genome have occurred during the rapid spread of this novel coronavirus. Variance in protein sequence might lead to a change in protein structure and interaction, then further affect the viral physiological characteristics, which could bring tremendous influence on the pandemic. In this study, we investigated 20 nonsynonymous mutations in the SARS‐CoV‐2 genome in which incidence rates were all ≥ 1% as of September 1st, 2020, and then modeled and analyzed the mutant protein structures. The results showed that four types of mutations caused dramatic changes in protein structures (RMSD ≥ 5.0 Å), which were Q57H and G251V in open‐reading frames 3a (ORF3a), S194L, and R203K/G204R in nucleocapsid (N). Next, we found that these mutations also affected the binding affinity of intraviral protein interactions. In addition, the hot spots within these docking mutant complexes were altered, among which the mutation Q57H was involved in both Orf3a–S and Orf3a–Orf8 protein interactions. Besides, these mutations were widely distributed all over the world, and their occurrences fluctuated as time went on. Notably, the incidences of R203K/G204R in N and Q57H in Orf3a were both over 50% in some countries. Overall, our findings suggest that SARS‐CoV‐2 mutations could change viral protein structure, binding affinity, and hot spots of the interface, thereby might have impacts on SARS‐CoV‐2 transmission, diagnosis, and treatment of COVID‐19.

Chicken Astrovirus (CAstV) Molecular Studies Reveal Evidence of Multiple Past Recombination Events in Sequences Originated from Clinical Samples of White Chick Syndrome (WCS) in Western Canada.

In this study, we aimed to molecularly characterize 14 whole genome sequences of chicken astrovirus (CAstV) isolated from samples obtained from white chick syndrome (WCS) outbreaks in Western Canada during the period of 2014–2019. Genome sequence comparisons showed all these sequences correspond to the novel Biv group from which no confirmed representatives were published in GenBank. Molecular recombination analyses using recombination detection software (i.e., RDP5 and SimPlot) and phylogenetic analyses suggest multiple past recombination events in open reading frame (ORF)1a, ORF1b, and ORF2. Our findings suggest that recombination events and the accumulation of point mutations may have contributed to the substantial genetic variation observed in CAstV and evidenced by the current seven antigenic sub-clusters hitherto described. This is the first paper that describes recombination events in CAstV following analysis of complete CAstV sequences originated in Canada.

FCoV Viral Sequences of Systemically Infected Healthy Cats Lack Gene Mutations Previously Linked to the Development of FIP.

Feline Infectious Peritonitis (FIP)—the deadliest infectious disease of young cats in shelters or catteries—is induced by highly virulent feline coronaviruses (FCoVs) emerging in infected hosts after mutations of less virulent FCoVs. Previous studies have shown that some mutations in the open reading frames (ORF) 3c and 7b and the spike (S) gene have implications for the development of FIP, but mainly indirectly, likely also due to their association with systemic spread. The aim of the present study was to determine whether FCoV detected in organs of experimentally FCoV infected healthy cats carry some of these mutations. Viral RNA isolated from different tissues of seven asymptomatic cats infected with the field strains FCoV Zu1 or FCoV Zu3 was sequenced. Deletions in the 3c gene and mutations in the 7b and S genes that have been shown to have implications for the development of FIP were not detected, suggesting that these are not essential for systemic viral dissemination. However, deletions and single nucleotide polymorphisms leading to truncations were detected in all nonstructural proteins. These were found across all analyzed ORFs, but with significantly higher frequency in ORF 7b than ORF 3a. Additionally, a previously unknown homologous recombination site was detected in FCoV Zu1.

Extensive genetic diversity and host range of rodent-borne coronaviruses.

To better understand the genetic diversity, host associations and evolution of coronaviruses (CoVs) in China we analyzed a total of 696 rodents encompassing 16 different species sampled from Zhejiang and Yunnan provinces. Based on reverse transcriptase PCR-based CoV screening of fecal samples and subsequent sequence analysis of the RNA-dependent RNA polymerase gene, we identified CoVs in diverse rodent species, comprising Apodemus agrarius, Apodemus chevrieri, Apodemus latronum, Bandicota indica, Eothenomys cachinus, Eothenomys miletus, Rattus andamanensis, Rattus norvegicus, and Rattus tanezumi. CoVs were particularly commonplace in A. chevrieri, with a detection rate of 12.44 per cent (24/193). Genetic and phylogenetic analysis revealed the presence of three groups of CoVs carried by a range of rodents that were closely related to the Lucheng Rn rat CoV (LRNV), China Rattus CoV HKU24 (ChRCoV_HKU24), and Longquan Rl rat CoV (LRLV) identified previously. One newly identified A. chevrieri-associated virus closely related to LRNV lacked an NS2 gene. This virus had a similar genetic organization to AcCoV-JC34, recently discovered in the same rodent species in Yunnan, suggesting that it represents a new viral subtype. Notably, additional variants of LRNV were identified that contained putative non-structural (NS)2b genes located downstream of the NS2 gene that were likely derived from the host genome. Recombination events were also identified in the open reading frame (ORF) 1a gene of Lijiang-71. In sum, these data reveal the substantial genetic diversity and genomic complexity of rodent-borne CoVs, and extend our knowledge of these major wildlife virus reservoirs.

Identification of nsp1 gene as the target of SARS-CoV-2 real-time RT-PCR using nanopore whole-genome sequencing.

Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) has caused the coronavirus disease 2019 (COVID‐19) pandemic. Accurate detection of SARS‐CoV‐2 using molecular assays is critical for patient management and the control of the COVID‐19 pandemic. However, there is an increasing number of SARS‐CoV‐2 viruses with mutations at the primer or probe binding sites, and these mutations may affect the sensitivity of currently available real‐time reverse transcription‐polymerase chain reaction (RT‐PCR) assays targeting the nucleocapsid (N), envelope (E), and open reading frame 1a or 1b genes. Using sequence‐independent single‐primer amplification and nanopore whole‐genome sequencing, we have found that the nonstructural protein 1 (nsp1) gene, located at the 5′ end of the SARS‐CoV‐2 genome, was highly expressed in the nasopharyngeal or saliva specimens of 9 COVID‐19 patients of different clinical severity. Based on this finding, we have developed a novel nsp1 real‐time RT‐PCR assay. The primers and probes are highly specific for SARS‐CoV‐2. Validation with 101 clinical specimens showed that our nsp1 RT‐PCR assay has a sensitivity of 93.1% (95% confidence interval [CI]: 86.2%‐97.2%), which was similar to those of N and E gene RT‐PCR assays. The diagnostic specificity was 100% (95% CI: 92.9%‐100%). The addition of nsp1 for multitarget detection of SARS‐CoV‐2 can avoid false‐negative results due to mutations at the primers/probes binding sites of currently available RT‐PCR assays.

The establishment of reference sequence for SARS-CoV-2 and variation analysis.

Starting around December 2019, an epidemic of pneumonia, which was named COVID‐19 by the World Health Organization, broke out in Wuhan, China, and is spreading throughout the world. A new coronavirus, named severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) by the Coronavirus Study Group of the International Committee on Taxonomy of Viruses was soon found to be the cause. At present, the sensitivity of clinical nucleic acid detection is limited, and it is still unclear whether it is related to genetic variation. In this study, we retrieved 95 full‐length genomic sequences of SARAS‐CoV‐2 strains from the National Center for Biotechnology Information and GISAID databases, established the reference sequence by conducting multiple sequence alignment and phylogenetic analyses, and analyzed sequence variations along the SARS‐CoV‐2 genome. The homology among all viral strains was generally high, among them, 99.99% (99.91%‐100%) at the nucleotide level and 99.99% (99.79%‐100%) at the amino acid level. Although overall variation in open‐reading frame (ORF) regions is low, 13 variation sites in 1a, 1b, S, 3a, M, 8, and N regions were identified, among which positions nt28144 in ORF 8 and nt8782 in ORF 1a showed mutation rate of 30.53% (29/95) and 29.47% (28/95), respectively. These findings suggested that there may be selective mutations in SARS‐COV‐2, and it is necessary to avoid certain regions when designing primers and probes. Establishment of the reference sequence for SARS‐CoV‐2 could benefit not only biological study of this virus but also diagnosis, clinical monitoring and intervention of SARS‐CoV‐2 infection in the future.

Development of Label-Free Colorimetric Assay for MERS-CoV Using Gold Nanoparticles.

Worldwide outbreaks of infectious diseases necessitate the development of rapid and accurate diagnostic methods. Colorimetric assays are a representative tool to simply identify the target molecules in specimens through color changes of an indicator (e.g., nanosized metallic particle, and dye molecules). The detection method is used to confirm the presence of biomarkers visually and measure absorbance of the colored compounds at a specific wavelength. In this study, we propose a colorimetric assay based on an extended form of double-stranded DNA (dsDNA) self-assembly shielded gold nanoparticles (AuNPs) under positive electrolyte (e.g., 0.1 M MgCl2) for detection of Middle East respiratory syndrome coronavirus (MERS-CoV). This platform is able to verify the existence of viral molecules through a localized surface plasmon resonance (LSPR) shift and color changes of AuNPs in the UV–vis wavelength range. We designed a pair of thiol-modified probes at either the 5′ end or 3′ end to organize complementary base pairs with upstream of the E protein gene (upE) and open reading frames (ORF) 1a on MERS-CoV. The dsDNA of the target and probes forms a disulfide-induced long self-assembled complex, which protects AuNPs from salt-induced aggregation and transition of optical properties. This colorimetric assay could discriminate down to 1 pmol/μL of 30 bp MERS-CoV and further be adapted for convenient on-site detection of other infectious diseases, especially in resource-limited settings.

Genome analysis of newly emerging goose-origin nephrotic astrovirus in China reveals it belongs to a novel genetically distinct astrovirus

Since 2017, a new type of goose-origin astrovirus (GoAstV) disease occurred in China. This disease can cause joint swelling of sick geese, and the anatomy shows a clear urate precipitation in the viscera. The rate of death or amputation can reach more than 30%, revealing its severe pathogenicity. One novel goose-origin astrovirus strain, designated as CXZ18, was isolated from diseased geese with a fatal infection characterized by visceral urate deposition. Similar clinical anatomy symptoms were partially reproduced by attacking infection of healthy geese. The CXZ18 has no hemagglutination with chicken erythrocyte, only reproduced in goose embryos, not in SPF chicken or duck embryos. The complete genome-encoded three open reading frames (ORFs) of CXZ18 were 7252 nt in length. BLAST-based homology analysis of viral complete genome showed that CXZ18 has only 53.0%–61.8% with other classic avian astrovirus from various hosts. Further analysis of ORF 1a, ORF 1b, and ORF 2 genes revealed that the isolate was genetically distinct from known astroviruses and belonged to a distinctive branch of avian astroviruses. To conclude, a naturally occurring novel nephrotic astrovirus, distinguished with all previously reported avian astroviruses, was derived from goose

Molecular characterization and detection of a new closterovirus identified from blackcurrant by high-throughput sequencing.

Two large contigs with high sequence similarities to several closteroviruses were identified by high-throughput sequencing from a blackcurrant plant. The complete genome of this new virus was determined to be 17,320 nucleotides. Its genome contains ten open reading frames (ORF) that include, in the 5'-3' direction, a large ORF encoding a putative viral polyprotein (ORF 1a) and nine ORFs that encode RNA-dependent RNA polymerase (RdRp, ORF 1b), p6 (ORF 2), heat shock protein 70-like protein (Hsp70h, ORF 3), Hsp-90-like protein (p61, ORF 4), CP minor (ORF 5), CP (ORF 6), p17 (ORF 7), p11 (ORF 8), and p26 (ORF 9), respectively. BCCV-1 shares nucleotide sequence identities of 43-45% with other 9 closteroviruses at genome sequences. The amino acid sequence identities between BCCV-1 and the closteroviruses were 49-55% (RdRp), 37-41% (Hsp70h), 19-33% (p61), 26-38% (CPm), and 19-28% (CP), respectively. Phylogenetic analysis of Hsp70h sequences placed the new virus with members of genus Closterovirus in the same group. The results indicate that this new virus, which is provisionally named as Blackcurrant closterovirus 1, should represent a new species of the genus Closterovirus. A RT-PCR was developed and used to detect BCCV-1 in more germplasm accessions of Ribes spp.

Cellular localisation of the proteins of region 3 of feline enteric coronavirus.

Feline enteric coronaviruses have three open reading frames (ORFs) in region 3 (3a, 3b, and 3c). All three ORFs were expressed with C-terminal eGFP and 3xFLAG tags in different cell lines and their localisation was determined. ORF 3a is predicted to contain DNA-binding and transcription activator domains, and it is localised in the nucleus and in the cytoplasm. ORF 3b is also predicted to contain DNA-binding and activator domains, and was found to localise in the mitochondrion. Besides that, in some of the non-infected and FIPV-infected cells nucleolar, perinuclear or nuclear membrane accumulation of the eGFP-tagged 3b was observed. The exact compartmental localisation of ORF 3c is yet to be determined. However, based on our co-localisation studies 3c does not seem to be localised in the ER-Golgi network, ERGIC or peroxisomes. The expression of 3c-eGFP is clearly cell type dependent, it is more stable in MARC 145 cells than in Fcwf-4 or CrFK cells, which might reflect in vivo stability differences of 3c in natural target cells (enterocytes vs. monocytes/macrophages).

No molecular evidence of MERS-CoV circulation in Jeddah, Saudi Arabia between 2010-2012: a single-center retrospective study.

Middle East respiratory syndrome coronavirus (MERS-CoV) is an emerging zoonotic viral pathogen and a serious public health concern. The virus was first reported in Saudi Arabia in 2012 and continues to be endemic in the region. Most of the initial MERS-CoV cases in 2012 and early 2013 were sporadic, and it remains unclear whether MERS-CoV was circulating before 2012 or not. Therefore, we tried here to find any molecular evidence of MERS-CoV circulation in humans before or during 2012 in the city of Jeddah, Saudi Arabia. We examined 349 archived respiratory samples collected between January 2010 and December 2012 from patients with acute respiratory illnesses from the city of Jeddah in Western Saudi Arabia. All samples were screened for MERS-CoV by real-time RT-PCR targeting the upstream E-gene (UpE) and the open reading frame 1 a (ORF1a). All tested samples which were originally found negative for influenza A H1N1 virus were also found to be negative for MERS-CoV. These results suggest that circulation of MERS-CoV was uncommon among patients with acute respiratory symptoms in Western Saudi Arabia between 2010 and 2012.

Complete Genome Sequence of Human Coronavirus NL63 CN0601/14, First Isolated in South Korea.

ABSTRACTWe report here the complete genome sequence of the human coronavirus NL63 CN0601/14 strain, first isolated from South Korea. It contains 18-nucleotide discontinuous deletions of the open reading frame 1a (ORF1a) and spike regions. This study will aid in our understanding of the complete genome sequences of isolated coronaviruses in South Korea.

Complete genome sequence of a lettuce chlorosis virus isolate from China and genome recombination/rearrangement analysis.

We determined the complete genome sequence of a lettuce chlorosis crinivirus (LCV) from China (LCV-NJ). The bipartite genome of LCV-NJ consists of RNA1 and RNA2 which are 8165 and 8454 nucleotides (nt) in length, respectively. The genomic structure of LCV-NJ RNA1 resembles that of LCV-California, an isolate with four open reading frames (ORFs) in RNA1. Although the amino acid sequences of ORF 1a and 1b have 92 and 99% identity between LCV-NJ and LCV-California, ORF 2 and ORF3 of LCV-NJ share only 63 and 71% identity with those of LCV-California, respectively. In addition LCV-NJ RNA2 contains 9 ORFs, compared to 10 ORFs in LCV-California. ORF10 was missing due to the deletion of a 173-nt sequence within the 3'-terminal region of LCV-NJ RNA2. Insertion or deletion of sequences of varying lengths was also observed in RNA1 and other regions of RNA2. Based on these findings, we propose that LCV-NJ/LCV-California may have undergone genome recombination and/or rearrangement in RNA1 and RNA2.

Identification of a natural recombinant transmissible gastroenteritis virus between Purdue and Miller clusters in China

Transmissible gastroenteritis virus (TGEV) is an infective coronavirus (CoV) that causes diarrhea-related morbidity and mortality in piglets. For the first time, a natural recombination strain of a TGEV Anhui Hefei (AHHF) virus between the Purdue and the Miller clusters was isolated from the small intestine content of piglets in China. A phylogenetic tree based on a complete genome sequence placed the TGEV AHHF strain between the Purdue and the Miller clusters. The results of a computational analysis of recombination showed that the TGEV AHHF strain is a natural recombinant strain between these clusters. Two breakpoints located in the open reading frame 1a (ORF1a) and spike (S) genes were identified. The pathogenicity of the TGEV AHHF strain was evaluated in piglets, and the results show that TGEV AHHF is an enteric pathogenic strain. These results provide valuable information about the recombination and evolution of CoVs and will facilitate future investigations of the molecular pathogenesis of TGEV.Emerging Microbes & Infections (2017) 6, e74; doi:10.1038/emi.2017.62; published online 23 August 2017

The sample of choice for detecting Middle East respiratory syndrome coronavirus in asymptomatic dromedary camels using real-time reversetranscription polymerase chain reaction

The newly identified Middle East respiratory syndrome coronavirus (MERS-CoV), which causes severe respiratory disease, particularly in people with comorbidities, requires further investigation. Studies in Qatar and elsewhere have provided evidence that dromedary camels are a reservoir for the virus, but the exact modes of transmission of MERS-CoV to humans remain unclear. In February 2014, an assessment was made of the suitability and sensitivity of different types of sample for the detection of MERSCoV by real-time reverse-transcription polymerase chain reaction (RT-PCR) for three gene targets: UpE (upstream of the E gene), the N (nucleocapsid) gene and open reading frame (ORF) 1a. Fifty-three animals presented for slaughter were sampled. A high percentage of the sampled camels (79% [95% confidence interval 66.9-91.5%, standard error 0.0625]; 42 out of 53) were shown to be shedding MERS-CoV at the time of slaughter, yet all the animals were apparently healthy. Among the virus-positive animals, nasal swabs were most often positive (97.6%). Oral swabs were the second most frequently positive (35.7%), followed by rectal swabs (28.5%). In addition, the highest viral load, expressed as a cycle threshold (Ct) value of 11.27, was obtained from a nasal swab. These findings lead to the conclusion that nasal swabs are the candidate sample of choice for detecting MERS-CoV using RT-PCR technology in apparently healthy camels.

Open reading frames 1a and 1b of the porcine reproductive and respiratory syndrome virus (PRRSV) collaboratively initiate viral minus-strand RNA synthesis

The porcine reproductive and respiratory syndrome virus (PRRSV) causes a persistent threat to the swine industry, especially when highly pathogenic PRRSV (HP-PRRSV) emerges. Previous studies have indicated that PRRSV RNA synthesis was correlated with HP-PRRSV virulence. PRRSV RNA synthesis includes genomic RNA and sub-genomic mRNA, and these processes require minus-strand RNA as a template. However, the mechanisms involved in PRRSV minus-strand RNA synthesis are not fully understood. A mini-genome system can be used to assess viral replication mechanisms and to evaluate the effects of potential antiviral drugs on viral replicase activities. In this study, we developed a mini-genome system that uses firefly luciferase as a reporter. Based on this system, we found that PRRSV RNA-dependent RNA polymerase nsp9 alone failed to activate virus minus-strand RNA synthesis. We also demonstrated that combinations of open reading frames 1a (ORF1a) and ORF1b are necessary for viral minus-strand RNA synthesis.

Epidemiological investigation of Middle East respiratory syndrome coronavirus in dromedary camel farms linked with human infection in Abu Dhabi Emirate, United Arab Emirates.

The objective of this research was to investigate the prevalence of Middle East respiratory syndrome coronavirus (MERS-CoV) infection primarily in dromedary camel farms and the relationship of those infections with infections in humans in the Emirate of Abu Dhabi. Nasal swabs from 1113 dromedary camels (39 farms) and 34 sheep (1 farm) and sputum samples from 2 MERS-CoV-infected camel farm owners and 1 MERS-CoV-infected sheep farm owner were collected. Samples from camels and humans underwent real-time reverse-transcription quantitative PCR screening to detect MERS-CoV. In addition, sequencing and phylogenetic analysis of partially characterized MERS-CoV genome fragments obtained from camels were performed. Among the 40 farms, 6 camel farms were positive for MERS-CoV; the virus was not detected in the single sheep farm. The maximum duration of viral shedding from infected camels was 2 weeks after the first positive test result as detected in nasal swabs and in rectal swabs obtained from infected calves. Three partial camel sequences characterized in this study (open reading frames 1a and 1ab, Spike1, Spike2, and ORF4b) together with the corresponding regions of previously reported MERS-CoV sequence obtained from one farm owner were clustering together within the larger MERS-CoV sequences cluster containing human and camel isolates reported for the Arabian Peninsula. Data provided further evidence of the zoonotic potential of MERS-CoV infection and strongly suggested that camels may have a role in the transmission of the virus to humans.

Middle East respiratory syndrome coronavirus on inanimate surfaces: A risk for health care transmission

The Middle East Respiratory syndrome coronavirus (MERS-CoV) has been responsible for multiple health care–associated outbreaks. We investigated whether high-touch surfaces in 3 rooms of laboratory-confirmed MERS-CoV patients were contaminated with MERS-CoV RNA. We found 2 out of 51 surfaces were contaminated with MERS-CoV viral genetic material. Hence, environmental contamination may be a potential source of health care transmission and outbreaks. Meticulous environmental cleaning may be important in preventing transmission within the health care setting.

MERS-CoV diagnosis: An update.

SummaryDiagnosis of MERS-Cov still a major concern in most of daignostic laboratories. To date the Real-time Polymerase Chain reaction (RT-PCR) is the mainstay for diagnosis of MERS-CoV. RT-PCR has limitations, including a long turnaround time and lack of common measurements and correlations with Viral Load (VL). It is recommended to screen for MERS-CoV using RT-PCR of the upstream of envelope gene (upE) followed by confirmation of the presence of one of the following genes; open reading frame 1A, 1B genes or nucleocapsid (N) gene. Scientists are looking to implement viral sequencing on all negative samples by RT-PCR and they beleive that can be exposed to another level of testing using sequencing of the RNA-dependent RNA polymerase (RdRp) gene or N gene and in this case a positive result is diagnostic. It is also very important to maintain a contineous and random sequencing for MERS-Cov samples to be able to pick early viral mutations. Serological assays still not widely or routinely performed, and a lot of studies looking to implement such method in routine patient's testings.

Use of the Human Coronavirus 2012 (MERS) GeneSig kit for MERS-CoV detection

IntroductionMortality due to MERS-CoV infection is common especially among immunocompromised patients. The pathogenesis and the transmission mode of this virus are still not well understood. The name of the virus is derived from the area of its appearance and the genomic sequence that was used in the development of qRT-PCR assays for MERS-CoV detection was retrieved from the first detected case isolate. The employed assays target various regions including the area upstream of the envelope gene (upE) that is used for screening and the open reading frames (ORF) 1a and 1b used for confirmation. AimThis study assesses the use of a MERS-CoV specific assay for screening of respiratory samples in anticipation of the possible spread of the virus in the region. Methods46 respiratory specimens were tested using the qualitative one-step qRT-PCR GeneSig Human Coronavirus 2012 (MERS) kit (PrimerDesign™). ResultsOut of the 46 tested samples, 45 were negative for MERS-CoV and one sample was found MERS-CoV positive. ConclusionThe GeneSig Human Coronavirus 2012 (MERS) kit is very useful for the screening of suspected respiratory cases in the Middle East area as well as other regions.