Full-length Genome Research Articles

Genetic characterization of HIV-1 viruses among cases with antiretroviral therapy failure in Suzhou City, China

BackgroundThis retrospective study aimed to characterize the distribution of HIV-1 genotypes and the prevalence of drug resistance mutations in people with antiretroviral treatment (ART) failure in Suzhou City, China.MethodsPol gene of HIV-1 viruses in blood samples of EDTA anticoagulants from 398 patients with failed antiviral treatment was successfully amplified by using an in-house assay. Drug resistance mutations were analyzed by using the Stanford HIV Drug Resistance Database system (https://hivdb.stanford.edu/hivdb/by-mutations/). HIV-1 genotypes were determined by the REGA HIV subtyping tool (version 3.46, https://www.genomedetective.com/app/typingtool/hiv). Near full-length genomes (NFLG) of HIV-1 viruses were obtained by next generation sequencing method.ResultsSequences analysis of the pol gene revealed that CRF 01_AE (57.29%, 228/398) was the dominant subtype circulating in Suzhou City, followed by CRF 07_BC (17.34%, 69/398), subtype B (7.54%, 30/398), CRF 08_BC (6.53%, 26/398), CRF 67_01B (3.02%, 12/398) and CRF55_01B (2.51%, 10/398). The overall prevalence of drug-resistant mutations in cases with ART failure was 64.57% (257/398), including 45.48% (181/398) for nucleotide reverse transcriptase inhibitors (NRTIs) mutations, 63.32% (252/398) for non-nucleoside reverse transcriptase inhibitors (NNRTIs) mutations, and 3.02% (12/398) for protease inhibitors (PIs) mutations. Ten near full-length genomes (NFLG) of HIV-1 viruses were identified, including six recombinants of CRF 01_AE and subtype B, two recombinants of CRF 01_AE, subtype B and subtype C sequences, one recombinant of CRF 01_AE and subtype C and one recombinant of CRF 01_AE, subtype A1 and subtype C.ConclusionsThe high prevalence of drug-resistant HIV-1 viruses was a serious challenge for HIV prevention and treatment of people with HIV infection. Treatment regimens for ART failure patients should be adjusted over time based on the outcome of drug resistance tests. NFLG sequencing facilitates the identification of new recombinants of HIV-1.

Identification and Characterization of Novel SPHINX/BMMF-like DNA Sequences Isolated from Non-Bovine Foods.

Sixteen novel circular rep-encoding DNA sequences with high sequence homologies to previously described SPHINX and BMMF sequences were isolated for the first time from non-bovine foods (pork, wild boar, chicken meat, Alaska pollock, pangasius, black tiger shrimp, apple, carrot, and sprouts from alfalfa, radish, and broccoli). The phylogenetic analysis of the full-length circular genomes grouped these together with previously described representatives of SPHINX/BMMF group 1 and 2 sequences (eight in each group). The characterization of genome lengths, genes present, and conserved structures confirmed their relationship to the known SPHINX/BMMF sequences. Further analysis of iteron-like tandem repeats of SPHINX/BMMF group 1-related genomes revealed a correlation with both full-length sequence tree branches as well as Rep protein sequence tree branches and was able to differentiate subtypes of SPHINX/BMMF group 1 members. For the SPHINX/BMMF group 2 members, a distinct grouping of sequences into two clades (A and B) with subgroups could be detected. A deeper investigation of potential functional regions upstream of the rep gene of the new SPHINX/BMMF group 2 sequences revealed homologies to the dso and sso regions of known plasmid groups that replicate via the rolling circle mechanism. Phylogenetic analyses were accomplished by a Rep protein sequence analysis of different ssDNA viruses, pCRESS, and plasmids with the known replication mechanism, as this yielded deeper insights into the relationship of SPHINX/BMMF group 1 and 2 Rep proteins. A clear relation of these proteins to the Rep proteins of plasmids could be confirmed. Interestingly, for SPHINX/BMMF group 2 members, the relationship to rolling circle replication plasmids could also be verified. Furthermore, a relationship of SPHINX/BMMF group 1 Rep proteins to theta-replicating plasmid Reps is discussed.

Genomic Characterization of a Novel HIV-1 Second-Generation Recombinant (CRF01_AE/CRF07_BC) Identified from an MSM Patient in Hebei, China.

Along with the co-circulation of dominant HIV-1 strains (CRF01_AE and CRF07_BC) in China, an increasing number of second-generation recombinants are being detected, especially among men who have sex with men (MSM). Here, we identified a unique CRF01_AE/CRF07_BC recombinant from a HIV-1-positive man (BDD015A) infected through homosexual transmission in Baoding city, Hebei Province. Analysis of the near full-length genome sequence of the recombinant revealed five segments divided by four breakpoints, with two regions of CRF07_BC inserted into the pol and env regions of the CRF01_AE backbone. Three CRF01_AE segments (I, III, and V) clustered within the cluster 4 lineage, which mainly circulated among MSM in China. This recombinant differed from previously reported CRF01_ AE and CRF07_BC recombinant forms. The continuous emergence of novel recombinants increases the genetic complexity of HIV-1 in Hebei. Further measures are needed to monitor the molecular epidemiological characteristics of HIV-1 to help control the spread of infections.

A novel VP2/VP3 recombinant of Senecavirus A isolated in northern China

Senecavirus A (SVA), previously called the Seneca Valley virus, is the only member of the genus Senecavirus within the family Picornaviridae. This virus was discovered as a serendipitous finding in 2002 and named Seneca Valley virus 001 (SVV-001). SVA is an emerging pathogen that can cause vesicular lesions and epidemic transient neonatal a sharp decline in swine. In this study, an SVA strain was isolated from a pig herd in Shandong Province in China and identified as SVA-CH-SDFX-2022. The full-length genome was 7282 nucleotides (nt) in length and contained a single open reading frame (ORF), excluding the poly (A) tails of the SVA isolates. Phylogenetic analysis showed that the isolate shares its genomic organization, resembling and sharing high nucleotide identities of 90.5% to 99.6%, with other previously reported SVA isolates. The strain was proved by in vitro characterization and the results demonstrate that the virus has robust growth ability in vitro. The recombination event of the SVA-CH-SDFX-2022 isolate was found and occurred between nts 1836 and 2710, which included the region of the VP2 (partial), and VP3 (partial) genes. It shows the importance of faster vaccine development and a better understanding of virus infection and spread because of increased infection rates and huge economic losses. This novel incursion has substantial implications for the regional control of vesicular transboundary diseases, and will be available for further study of the epidemiology of porcine SVA. Our findings provide useful data for studying SVA in pigs.

Evaluation of the genotype I Japanese encephalitis virus as a stable viral vector for foreign gene expression

Manipulation of the flavivirus genome to accommodate and express a heterologous gene of interest has become an attractive approach for gene delivery and the development of viral-vectored vaccines. However, due to the inherent genetic instability of the flavivirus genomes, the construction of recombinant viruses carrying a foreign gene could be problematic and heavily resistant. In this study, the possibility of the Japanese encephalitis virus (JEV) as a stable flavivirus vector for the expression of a foreign gene was assessed using reverse genetics. The full-length cDNA genome of genotype I (GI) JEV inherently possessed excellent stability and manipulability in a bacterial host, while mutations and deletions accumulated in the cDNA genomes of genotype Ⅲ (GⅢ) JEV strains. Using the GI JEV as backbones, we generate a panel of recombinant viruses expressing various foreign genes. All recombinant viruses exhibited excellent genetic stability and efficiently express foreign genes for at least ten serial passages in vitro. In application, a convenient, rapid and reliable image-based assay for neutralizing antibody testing and antiviral drug discovery was established with a mCherry-reporter recombinant virus (rBJ-mCherry). Meanwhile, the recombinant viruses expressing the antigens of the African swine fever virus (ASFV) or Classical swine fever virus (CSFV) could effectively induce antibody responses to the JEV vector and foreign antigens in a mouse vaccination model. Therefore, GI JEV strains could serve as viral vectors accommodating the expression of large foreign genes.

Comparative analysis of the open reading frames protein genes of genotype 4 Hepatitis E virus in swine and wild boar

The goal of this study was to determine the molecular diversity of the open reading frames (ORFs) ORF1, ORF2, ORF3 protein genes from full-length genomes of genotype 4 hepatitis E virus (HEV) from pigs and wild boars at protein and gene level. Statistical technique Shannon entropy was used for mutational analysis of ORF1–ORF3 protein genes to identify amino acid substitutions in the HEV 4 sequences isolated from pigs and wild boars that were most susceptible to mutations. Gene selective pressure for genes was estimated using Tajima’s neutrality test. The ORF regions of 11 swine and 11 wild boar genotype 4 HEV isolates with complete genomes from the GenBank database were analyzed comparatively. The total number of polymorphic sites was determined. Nonsynonymous (amino acid changing) and synonymous (amino acid preserving) substitutions were identified in ORF1, ORF2, ORF3 in swine and wild boar HEV 4 isolates. No evidence of recombination was found for ORFs in 11 swine HEV 4 isolates, ORF2, ORF3 in 8 wild boar HEV 4 isolates. However, a recombination fragment with a length of 430 nucleotides was detected in the ORF1 gene of 3 wild boar HEV 4 isolates. Positive D Tajima factors were determined for ORF1, ORF2, ORF3 genes of swine HEV 4 and ORF1, ORF2 genes of wild boar HEV 4. While a negative value of D Tajima’s factor was determined for ORF3 gene of wild boar HEV 4. Molecular characteristics showing principal distinctions between the open-reading frames of swine and wild boar genotype 4 hepatitis E virus were obtained. Wild boar ORF1 is characterized by lower nucleotide diversity π value (0.144) and higher number of segregated sites S value (1,688) comparing with higher π value (0.159) and lower S value (1,602) of swine ORF1. Positive values of D Tajima’s factor for ORF1, ORF2 ORF3 genes of swine HEV 4 and ORF1, ORF2 genes of wild boar HEV 4 show on positive selection of these genes. Negative value of D Tajima’s factor for ORF3 gene of wild boar HEV 4 indicates onto purifying selection decreasing variability in ORF3 gene of wild boar HEV 4. The largest number of amino acid variation sites (19.2%) was found for wild boar HEV 4 ORF3 followed by swine HEV 4 ORF3 (15.7%) comparing with other swine and wild boars HEV 4 ORFs

First report of plumeria mosaic virus infecting Plumeria spp. in the United States.

Plumeria (Plumeria spp.) is a small ornamental tree grown in subtropical and tropical areas, providing shade and attractive and fragrant flowers in public and private landscapes. In Hawaii, plumeria is also commercially grown, with flowers harvested for cut flower and lei production. In 2019, a total of 10 cut flower plumeria farms in Hawaii sold 7,356,702 blossoms, valued at $395,791 (USDA/NASS). In July 2021, a commercial plumeria farm in Maui County, Hawaii reported trees with flowers exhibiting color breaking, and leaves with mosaic and vein banding. To determine if a viral pathogen was present, leaf tissue from a symptomatic plant was processed using a Double-RNA Viral dsRNA Extraction Kit for plant tissue (iNtRON Biotechnology, Korea), followed by random amplification (Melzer et al. 2010). The resulting products underwent paired-end sequencing on a MiSeq platform (Illumina, CA) at the Advanced Studies in Genomics, Proteomics, and Bioinformatics laboratory at the University of Hawaii. Of the 2,913,913 paired-end 300-bp reads generated, Geneious Prime (Biomatters, New Zealand) mapped 350,686 to plumeria mosaic virus (PluMV; KJ395757.1) and 293,911 to frangipani mosaic virus (FrMV; JN555602.2), with mean coverage depths of 10,318 and 7,348 respectively. A 6,648 nt contig representing the near full-length genome of PluMV-HI (OP342599) was found to be >97% identical to PluMV isolates PluMV-DR_TW (KX881422.2) and PluMV-Plu-Ind-1 (KJ395757.1). Similarly, a 6,631 nt contig representing the near full-length genome of FrMV-HI (OP342600) was found to be >98% identical to FrMV isolates FrMV-P (Lim et al. 2010; HM026454.1) and FrMV-Fr-Ind-1 (Kumar et al. 2015; JN555602.2). Bioinformatic analyses (Olmedo-Velarde et al. 2019) of the reads that did not map to PluMV and FrMV suggested the presence of a novel ampelovirus and reovirus in the tissue. To confirm the presence of PluMV and FrMV in Hawaii, leaves were collected in June 2022 from 5 plumeria trees on the Maui County farm and 16 ornamental plumeria trees from Honolulu County. Total RNA extracted as described by Li et al. (2008) as well as non-template (water) and positive (HTS tissue sample) controls underwent RT-PCR using random primers for cDNA synthesis, followed by virus-specific primers for PluMV (1133: 5'-TGGGCAAATAATCCGGCTATAC-3'/1134: 5'CCGGAGAGAGCATCAAACAA-3') and FrMV (1129: 5'-TGAGTTTAGGTCGCAGTTGATAG-3'/1130: 5-'AAAGACCAGAACCTCCAGAAAG-3') in singleplex reactions. The results indicated that all 5 plumeria samples from Maui County were positive for both PluMV and FrMV, whereas 6 out of 16 plumeria samples collected from Honolulu County tested positive for PluMV only. Both PluMV and FrMV are tobamoviruses (Virgaviridae) that can be transmitted mechanically and spread through the introduction and/or movement of cuttings, the primary method of plumeria propagation. Both PluMV and FrMV cause leaf mosaic (Kumar et al. 2015; Lim et al. 2010; Lin et al. 2020) and FrMV may also cause color break or "splash" in flowers, which was observed at the Maui County farm. This represents the first report of PluMV in the USA. FrMV was detected in Florida (Dey et al. 2020), but this is the first report of the virus in Hawaii. Additional surveys are required to determine the incidence of both viruses in other counties of Hawaii. The putative discovery of a novel ampelovirus and reovirus in plumeria warrants further research to characterize these agents and determine their distribution and impact on plumeria health.

First report of Tomato bushy stunt virus naturally infecting sugar beet in the United States.

Sugar beet (Beta vulgaris L.) is an important crop grown for its sucrose content used in sugar production around the world. Tomato bushy stunt virus (TBSV) is an RNA virus that belongs to the Tombusvirus genus of the family Tombusviridae (Hearne et al., 1990). The virus was first isolated from tomato, and it is known to infect a wide range of plants (Smith, 1935; Martelli et al., 1988; Hafez et al., 2010). In 1980, a natural infection of TBSV was reported in sugar beet leaves with chlorotic and necrotic ring spots and line pattern symptoms based on serological affinity to TBSV anti-sera in Czechoslovakia (Novak and Lanzova, 1980). In March 2021, sugarbeet plants showing stunted and bushy growth with yellowing and necrotic leaves were observed in a production field in the Imperial Valley of California. Harvested roots exhibited stunted and abnormal growth compared to roots from healthy plants (sFig. 1A). These symptoms prompted a screen for potential infection by TBSV. Root-tissue harvested from the symptomatic sugar beet was initially screened using a TBSV double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA; Agdia, Inc., Elkhart, IN), which reacted positive for TBSV. To obtain the full-length sequence of TBSV and potentially other viruses in the sample, total RNA isolated using the RNeasy Plant Mini Kit (Qiagen, Valencia, CA) from the root-tissue was subjected to high-throughput sequencing (HTS). Libraries were prepared using the TruSeq Stranded Total RNA Library Prep kit (Illumina, San Diego, CA) and sequenced using Illumina NovoSeq 6000 paired-end platform (Novogene, Sacramento, CA). A total of 52 million reads were obtained after removing the adapters and reads mapping to the host genome. These high-quality reads were de novo assembled into 75,891 contigs that are larger than 500 base pairs using the SPAdes assembler (Bankevitch et al., 2012; Prjibelski et al., 2020). The resulting contigs were searched for matching sequences to known viruses using the NCBI non-redundant database. A single contig of 4770 nts representing the full-length genome of TBSV was generated (Accession number OP477335), which showed 100% coverage to previously reported TBSV isolates 'statice' (AJ249740.1) and 'nipplefruit' (AY579432.1) with 92.19% and 91.25% nucleotide sequence identities, respectively, and thus confirming the presence of TBSV in sugar beet root-tissue. However, it showed 74% coverage with only 87% nucleotide identity to a previously reported Lettuce necrotic stunt virus (LNSV) from sugar beet, a tombusvirus that was re-classified as Moroccan pepper virus (MPV) due to high degree (>97%) of sequence identity (Obermeier et al., 2001; Wintermantel and Anchieta, 2012; Wintermantel and Hladky, 2013). The coat protein is conserved within species in tombusvirus, and it plays a significant role by providing serological relationships to tombusvirus taxonomy. The coat protein of TBSV-isolate of this study shared 98.45% and 96.91% identities at amino acid level with TBSV 'nipplefruit' (AY579432.1) and TBSV 'statice' (AJ249740.1) isolates, respectively. In contrast, it showed only 61.56% identity with the coat protein of MPV as shown in the phylogenetic tree indicating that the TBSV-isolate reported here is different from MPV (sFig. 2). To confirm the presence of TBSV, reverse-transcription (RT)-PCR was performed using the total RNA isolated from the root-tissue with primers (VR306: 5'-CGCTCACGAGCCCAGCATCCTTGA-3' and VR297: 5'-ACACCGCCACAGGAGCCATGATTG-3') designed based on the HTS data to amplify a portion of the TBSV genome. Sequencing of the RT-PCR product confirmed the presence of TBSV sequence with 99.1% identity to the TBSV-isolate identified in this study. Further, mechanical inoculation of total RNA isolated from the symptomatic sugar beet roots produced local lesions and systemic necrosis symptoms on the leaves of Chenopodium quinoa (sFig. 1B). Sequencing of the amplicon obtained using RT-PCR with primers VR306 and VR297 confirmed the presence of TBSV in C. quinoa. In addition to TBSV, several viral contigs representing Beet necrotic yellow vein virus were identified in the root-tissue indicating mixed infection in the field. To our knowledge, this is the first report that documents the occurrence of TBSV in sugar beet in the United States. Since TBSV is a soil-borne virus, our findings indicate the need for further studies focused on the frequency and coexistence of the TBSV with BNYVV in sugar beet production fields to understand the disease complexity resulting from potential mixed infections.

One-Step Reverse Transcription-Loop-Mediated Isothermal Amplification (RT-LAMP) Assay for the Detection of Canna Yellow Streak Virus.

Canna yellow streak virus (CaYSV) is a potyvirus that causes severe damage to the ornamental plant canna in the United Kingdom and Brazil. Here we identified CaYSV in China by isolating total RNA from infected plant, amplifying the virus genome segments, cloning and sequencing the amplicons. After assembly, the full-length genome of the virus was obtained and uploaded to the NCBI database. Phylogenetic analysis results showed that the Guizhou isolate (OL546222) was most closley related to the KS isolate (MG545919.1). Virus detection is essential for virus disease control, but the subclinical infection of CaYSV on canna in its early development increases the difficulty of CaYSV diagnosis. The goal of this study was to develop an efficient method for detection of CaYSV. We designed the primers, optimized the reaction conditions, and finally established a one-step reverse-transcription loop-mediated isothermal amplification (RT-LAMP) method. The product of RT-LAMP can be analyzed by both agarose gel electrophoresis and visible color change. The established one-step RT-LAMP assay showed high specificity and sensitivity in detecting CaYSV. This RT-LAMP method was also applied in analysis of 61 field samples collected from Guizhou and Jiangsu provinces. The results showed that the infection rates of CaYSV on canna samples from these two provinces were very high (63% and 96% respectively).

Deep Sequencing and Molecular Characterisation of BK Virus and JC Virus WHO International Reference Materials for Clinical Diagnostic Use.

Reactivation of JC and BK polyomaviruses during immunosuppression can lead to adverse clinical outcomes. In renal transplant recipients, BKV-associated nephropathy can result in graft loss, while in patients with autoimmune disorders, prolonged immunomodulatory drug use can cause rare onset of progressive multifocal leukoencephalopathy due to JCV reactivation. In such patients, accurate BK and JC viral load determinations by molecular technologies are important for diagnosis and clinical management; however, comparability across centres requires effective standardisation of diagnostic molecular detection systems. In October 2015, the WHO Expert Committee for Biological Standardisation (ECBS) established the 1st WHO International Standards (ISs) for use as primary-order calibrants for BKV and JCV nucleic acid detection. Two multi-centre collaborative studies confirmed their utility in harmonising agreement across the wide range of BKV and JCV assays, respectively. Previous Illumina-based deep sequence analysis of these standards, however, identified deletions in different regions, including the large T-antigen coding region. Hence, further detailed characterization was warranted. Comprehensive sequence characterisation of each preparation using short- and long-read next-generation sequencing technologies was performed with additional corroborative independent digital PCR (dPCR) determinations. Potential error rates associated with long-read sequencing were minimised by applying rolling circle amplification (RCA) protocols for viral DNA (circular dsDNA), generating a full validation of sequence identity and composition and delineating the integrity of full-length BK and JC genomes. The analysed genomes displayed subpopulations frequently characterised by complex gene re-arrangements, duplications and deletions. Despite the recognition of such polymorphisms using high-resolution sequencing methodologies, the ability of these reference materials to act to enhance assay harmonisation did not appear significantly impacted, based on data generated by the 2015 WHO collaborative studies, but highlights cautionary aspects of IS generation and commutability for clinical molecular diagnostic application.

Identification of Nocardioides marmotae sp. nov. and Nocardioides faecalis sp. nov., two new members of the genus Nocardioides.

A polyphasic taxonomic characterization of two novel strain pairs (designated zg-579T/zg-578 and zg-536T/zg-ZUI104) isolated from the faeces of Marmota himalayana was conducted based on phylogenetic analysis of the nearly full-length 16S rRNA gene and genome, digital DNA-DNA hybridization, ortho-average nucleotide identity (Ortho-ANI), and phenotypic and chemotaxonomic traits. Comparative analysis of the nearly full-length 16S rRNA gene sequences showed that strain zg-579T was most closely related to Nocardioides dokdonensis FR1436T (97.57 %) and Nocardioides deserti SC8A-24T (97.36 %), whereas strain zg-536T had the highest similarity to Nocardioides caeni MN8T (98.33 %), Nocardioides convexus W2-2-3T (98.26 %) and Nocardioides daeguensis 2C1-5T (98.19 %). Low levels of DNA-DNA relatedness and Ortho-ANI values (19.8-31.0 %/78.6-88.2 %, zg-579T; 19.9-31.3 %/78.8-86.2 %, zg-536T) between the two new type strains and previously known species within the genus Nocardioides support the hypothesis that the four newly characterized strains could be considered to represent two novel species within this genus. The dominant cellular fatty acids found in strain pair zg-536T/zg-ZUI104 were iso-C16 : 0 and C18 : 1 ω9c, whereas C17 : 1 ω8c was major component in zg-579T/zg-578. Galactose and ribose were the main cell-wall sugars in these two new strain pairs. Diphosphatidylglycerol (DPG), phosphatidylcholine, phosphatidylglycerol (PG) and phosphatidylinositol (PI) were the major polar lipids in zg-579T, whereas DPG, PG and PI predominated in zg-536T. Both strain pairs had MK8(H4) as the major respiratory quinone and ll-diaminopimelic acid as the major cell-wall peptidoglycan. The optimal growth conditions for the two novel strain pairs were 30 °C, pH 7.0 and 0.5 % NaCl (w/v). Based on these polyphasic characterizations, two novel species within the genus Nocardioides are proposed, i.e. Nocardioides marmotae sp. nov. and Nocardioides faecalis sp. nov., with zg-579T (=CGMCC 4.7663T=JCM 33892T) and zg-536T (=CGMCC 4.7662T=JCM 33891T) as the type strains.

Hepatitis B Virus Genotype E/A Recombinants from Blood Donors in Beira, Mozambique.

Hepatitis B virus (HBV) is endemic in many parts of sub-Saharan Africa. Here, we present 5 full-length HBV recombinant genomes from blood donors in Beira, Mozambique. The genomes are recombinants between genotypes E and A and are distantly related to one another, based on their genetic distances.

Rapid construction of infectious clones for distinct Newcastle disease virus genotypes.

The reverse genetics system of the Newcastle disease virus (NDV) has provided investigators with a powerful approach to understand viral molecular biology and vaccine development. It has been impressively improved with modified strategies since its first report, but it still poses some challenges. Most noteworthy, the genome complexity and length made full-length error-free cDNA assembly the most challenging and time-consuming step of NDV rescue. In the present study, we report a rapid full-length NDV genome construction with only a two-step ligation-independent cloning (LIC) strategy, which could be applied to distinct genotypes. In this approach, the genome of NDV was divided into two segments, and the cDNA clones were generated by RT-PCR followed by LIC. Subsequently, the infectious NDVs were rescued by co-transfection of the full-length cDNA clones and supporting plasmids expressing the NP, P, and L proteins of NDV in BHK-21 cells. Compared with the conventional cloning approaches, the two-step cloning method drastically reduced the number of cloning steps and saved researchers a substantial amount of time for constructing NDV infectious clones, thus enabling a rapid rescue of different genotypes of NDVs in a matter of weeks. Therefore, this two-step LIC cloning strategy may have an application to the rapid development of NDV-vectored vaccines against emerging animal diseases and the generation of different genotypes of recombinant NDVs for cancer therapy.

Mutation rate, selection, andepistasis inferred from RNA virus haplotypes via neural posterior estimation.

RNA viruses are particularly notorious for their high levels of genetic diversity, which is generated through the forces of mutation and natural selection. However, disentangling these two forces is a considerable challenge, and this may lead to widely divergent estimates of viral mutation rates, as well as difficulties in inferring the fitness effects of mutations. Here, we develop, test, and apply an approach aimed at inferring the mutation rate and key parameters that govern natural selection, from haplotype sequences covering full-length genomes of an evolving virus population. Our approach employs neural posterior estimation, a computational technique that applies simulation-based inference with neural networks to jointly infer multiple model parameters. We first tested our approach on synthetic data simulated using different mutation rates and selection parameters while accounting for sequencing errors. Reassuringly, the inferred parameter estimates were accurate and unbiased. We then applied our approach to haplotype sequencing data from a serial passaging experiment with the MS2 bacteriophage, a virus that parasites Escherichia coli. We estimated that the mutation rate of this phage is around 0.2 mutations per genome per replication cycle (95% highest density interval: 0.051-0.56). We validated this finding with two different approaches based on single-locus models that gave similar estimates but with much broader posterior distributions. Furthermore, we found evidence for reciprocal sign epistasis between four strongly beneficial mutations that all reside in an RNA stem loop that controls the expression of the viral lysis protein, responsible for lysing host cells and viral egress. We surmise that there is a fine balance between over- and underexpression of lysis that leads to this pattern of epistasis. To recap, we have developed an approach for joint inference of the mutation rate and selection parameters from full haplotype data with sequencing errors and used it to reveal features governing MS2 evolution.

Identification of a TuMV isolate (TuMV-ZR) from Pseudostellaria heterophylla and its development into a viral expression vector

Pseudostellaria heterophylla (P. heterophylla) is a popular Chinese medicinal herb that is cultivated widely in China. Viral infection is commonly encountered during the production of P. heterophylla. To identify viruses causing P. heterophylla disease, sRNA and mRNA libraries were built for 2 sets of P. heterophylla plants, one set that was planted only once (FGP) and one that was planted three consecutive three times (TGP) in a field, using virus-free tuberous roots as reproductive materials. A comprehensive procedure, including assembling virus-derived sRNA (vsRNA), assessing and cloning the full-length viral genome, building an infectious cloning vector and constructing a virus-based expression vector, was performed to identify viruses infecting P. heterophylla. Ultimately, 48 contig-related viruses were mined from 6 sRNA and 6 mRNA P. heterophylla libraries. A 9762-bp fragment was predicted to be the complete genome of TuMV virus. This sequence was cloned from P. heterophylla, and its infectivity was evaluated using the virus-infection model plant Nicotiana benthamiana (N. benthamiana) and host plant P. heterophylla. The resulting 9839-bp viral genome was successfully obtained from P. heterophylla and identified as a new P. heterophylla TuMV-ZR isolate. Simultaneously, TuMV-ZR infectious clones were shown to effectively infect P. heterophylla. Furthermore, TuMV-ZR expression vectors were developed, and the ability of a TuMV-ZR-based vector to express foreign genes was determined by analysis with the reporter gene EGFP. TuMV-ZR-based vectors were found to continuously express foreign genes in different organs of P. heterophylla throughout the whole vegetative period. In addition, TuMV-ZR vectors carrying EGFP accumulated in the tuberous roots of P. heterophylla, confirming that tuberous roots are key targets for viral infection and transmission. This study revealed the core pathogenicity of P. heterophylla mosaic virus and developed a new TuMV-ZR-based expression tool that led to long-term protein expression in P. heterophylla, laying the foundation for the identification of the mechanisms of P. heterophylla infection with mosaic viruses and developing tools to express value proteins in the tuberous roots of the medicinal plant P. heterophylla.

The whole genomic analysis of the Orf virus strains ORFV-SC and ORFV-SC1 from the Sichuan province and their weak pathological response in rabbits

The Orf virus (ORFV) is a member of the Parapoxvirus genus of the Poxviridae family and can cause contagious diseases in sheep, goats, and wild ungulates. In the present study, two ORFV isolates (ORFV-SC isolated from Sichuan province and ORFV-SC1 produced by 60 passages of ORFV-SC in cells) were sequenced and compared to multiple ORFVs. The two ORFV sequences had entire genome sizes of 14,0707 bp and 141,154 bp, respectively, containing 130 and 131 genes, with a G + C content of 63% for the ORFV-SC sequence and 63.9% for the ORFV-SC1 sequence. Alignment of ORFV-SC and ORFV-SC1 with five other ORFV isolates revealed that ORFV-SC, ORFV-SC1, and NA1/11 shared > 95% nucleotide identity with 109 genes. Five genes (ORF007, ORF20, ORF080, ORF112, ORF116) have low amino acids identity between ORFV-SC and ORFV-SC1. Mutations in amino acids result in changes in the secondary and tertiary structure of ORF007, ORF020, and ORF112 proteins. The phylogenetic tree based on the complete genome sequence and 37 single genes revealed that the two ORFV isolates originated from sheep. Finally, animal experiments demonstrated that ORFV-SC1 is less harmful to rabbits than ORFV-SC. The exploration of two full-length viral genome sequences provides valuable information in ORFV biology and epidemiology research. Furthermore, ORFV-SC1 demonstrated an acceptable safety profile following animal vaccination, indicating its potential as a live ORFV vaccine.

Genetic Characterization and Evolution of Porcine Deltacoronavirus Isolated in the Republic of Korea in 2022.

Porcine deltacoronavirus (PDCoV) is an emerging coronavirus that causes diarrhea in nursing piglets. Since its first outbreak in the United States in 2014, this novel porcine coronavirus has been detected worldwide, including in Korea. However, no PDCoV case has been reported since the last report in 2016 in Korea. In June 2022, the Korean PDCoV strain KPDCoV-2201 was detected on a farm where sows and piglets had black tarry and watery diarrhea, respectively. We isolated the KPDCoV-2201 strain from the intestinal samples of piglets and sequenced the viral genome. Genetically, the full-length genome and spike gene of KPDCoV-2201 shared 96.9-99.2% and 95.8-98.8% nucleotide identity with other global PDCoV strains, respectively. Phylogenetic analysis suggested that KPDCoV-2201 belongs to G1b. Notably, the molecular evolutionary analysis indicated that KPDCoV-2201 evolved from a clade different from that of previously reported Korean PDCoV strains and is closely related to the emergent Peruvian and Taiwanese PDCoV strains. Furthermore, KPDCoV-2201 had one unique and two Taiwanese strain-like amino acid substitutions in the receptor-binding domain of the S1 region. Our findings suggest the possibility of transboundary transmission of the virus and expand our knowledge about the genetic diversity and evolution of PDCoV in Korea.

Description of an Equine Hepacivirus Cluster in a Horse Stable in Italy

Equine hepacivirus (EqHV), also known as Hepacivirus A, represents the most closely related genetic homologue of human hepatitis C virus (HCV). Although detected worldwide, limited information on the clinical features of this infection is available and on the mechanisms by which EqHV is transmitted. In this study, we describe a spread of infection of EqHV that occurred in a small stable of horses in southern Italy. The RNA of EqHV was detected in 6/13 (46.2%) sera of the horses introduced into the herd, at different times, over a period of approximately one year. Based on the sequencing analyses of genomic portions located in the NS5B, 5′UTR, and NS3 genes, the viruses detected in the animals were genetically highly related (100% nt identity) to each other. The nearly full-length genome of the virus identified from two horses was generated. For one animal with a profile of chronic infection, the genome sequence was determined with a 7-month interval, revealing 26nt changes resulting in 11 nonsynonymous intrahost nucleotide variations. Overall, based on the epidemiological information, we support the hypothesis that horizontal transmission occurred in the herd.

Characterization of two novel HIV-1 second-generation recombinants (CRF01_AE/CRF07_BC) identified in Hebei Province, China.

The unique recombinant forms (URFs) of HIV-1 consist of a mixture of subtypes, and each URF has a unique breakpoint. In this study, we identified the near fulllength genome (NFLG) sequences of two novel HIV-1 URFs (Sample ID: BDD034A and BDL060) isolated during HIV-1 molecular surveillance in 2022 in Baoding city, Hebei Province, China. The two sequences were aligned with subtype reference sequences and CRFs from China using MAFFT v7.0, and the alignments were adjusted manually using BioEdit (v7.2.5.0). Phylogenetic and subregion trees were constructed using MEGA11 with the neighbor-joining (N-J) method. Recombination breakpoints were identified by SimPlot (v3.5.1) based on Bootscan analyses. Recombinant breakpoint analysis revealed that the NFLGs of BDD034A and BDL060 were composed of CRF01_AE and CRF07_BC, containing seven segments, respectively. For BDD034A, three CRF01_AE fragments were inserted into the CRF07_BC main framework, whereas for BDL060, three CRF07_BC fragments were inserted into the CRF01_AE main framework. The emergence of the CRF01_AE/CRF07_BC recombinant strains indicates that HIV-1 co-infection is common. The increasing genetic complexity of the HIV-1 epidemic in China warrants continued investigation.

Characteristics of the Complete Genome of Ageratum Yellow Vein China Virus Infecting Sonchus oleraceus.

Sonchus (Sonchus oleraceus) originated from Europe and is now cultivated worldwide. The wild resources of sonchus are very abundant, and it has rich nutritional and medicinal value. In this study, 15 sonchus samples with typical symptoms showing leaf curling, vein thickening, and enations were collected from Guigang and Baise City of Guangxi, China. Diseased sonchus were identified by PCR detection, whole genome sequence amplification, and phylogenetic and recombination analysis. The results showed that all the samples were confirmed infected by begomoviruses, and three full-length viral genomes were obtained from 15 sonchus, named GG7-13, GG8-6, and BS63-5. The full genome lengths were 2,584, 2,735, and 2,746 nt, respectively. The nucleotide identities among the three isolates ranged from 92.67 to 99.93%. All of them shared the highest identities (greater than 91.69%) with other isolates of ageratum yellow vein China virus (AYVCNV) (available on GenBank). According to the guidelines of classification of begomoviruses, the virus isolates obtained in this study are different isolates of AYVCNV; a phylogenetic tree analysis showed that these isolates formed a large branch with three other Guangxi isolates of AYVCNV, indicating their close evolution. The genome structures of GG8-6 and BS63-5 are consistent with the monopartite genome virus of the begomoviruses, and both have six open reading frames (ORFs), while GG7-13 has a 151-nt deletion between C2 and C3, resulting in a mutant strain of only five ORFs. This study is the first report on S. oleraceus infected by ageratum yellow vein China virus.