Recombination Breakpoints Research Articles

Molecular epidemiology of coxsackievirus A6 circulating in Hong Kong reveals common neurological manifestations and emergence of novel recombinant groups

BackgroundCoxsackievirus A6 (CV-A6) represents the predominant enterovirus serotype in Hong Kong, but its epidemiology in our population was unknown. ObjectivesTo examine the clinical and molecular epidemiology of CV-A6 and detect emerging recombinant strains in Hong Kong. Study designNasopharyngeal aspirates (NPAs) from patients with febrile or respiratory illness were subject to RT-PCR for CV-A6 and sequencing of 5′-NCR and VP1. CV-A6-positive samples were further subject to 2C and 3D gene sequencing. Complete genome sequencing was performed on potential recombinant strains. ResultsThirty-six (0.35%) NPAs were positive for CV-A6 by 5′-NCR RT-PCR and sequencing, 28 of which confirmed by partial VP1 gene sequencing. Among the 28 patients (mainly young children) with CV-A6 infection, hand-foot-and-mouth disease (HFMD) (43%), herpangina (18%) and tonsillitis (11%) were the most common diagnoses. Seven (25%) patients had neurological manifestations, including febrile seizures, encephalitis and meningitis. VP1 gene analysis showed that 24 CV-A6 strains circulating in Hong Kong belonged to genotype D5, while 4 strains belonged to D4. Further 2C and 3D gene analysis revealed eight potential recombinant strains. Genome sequencing of five selected strains confirmed four recombinant strains: HK459455/2013 belonging to recombination group RJ arisen from CV-A6/CV-A4, HK458288/2015 and HK446377/2015 representing novel group RL arisen from CV-A6/CV-A4, and HK462069/2015 representing novel group RM arisen from CV-A6/EV-A71. Recombination breakpoints located at 3D were identified in the latter three recombinant strains, with HK462069/2015 (from a child with encephalitis) having acquired 3D region from EV-A71. ConclusionsWe identified novel recombinant CV-A6 strains in Hong Kong, with 3D being a common recombination site.

Efficient QTL detection of flowering date in a soybean RIL population using the novel restricted two-stage multi-locus GWAS procedure.

Eighty-six R1 QTLs accounting for 89.92% phenotypic variance in a soybean RIL population were identified using RTM-GWAS with SNPLDB marker which performed superior over CIM and MLM-GWAS with BIN/SNPLDB marker. A population (NJRIKY) composed of 427 recombinant inbred lines (RILs) derived from Kefeng-1 × NN1138-2 (MGII × MGV, MG maturity group) was applied for detecting flowering date (R1) quantitative trait locus (QTL) system in soybean. From a low-depth re-sequencing (~ 0.75 ×), 576,874 SNPs were detected and organized into 4737 BINs (recombination breakpoint determinations) and 3683 SNP linkage disequilibrium blocks (SNPLDBs), respectively. Using the association mapping procedures "Restricted Two-stage Multi-locus Genome-wide Association Study" (RTM-GWAS), "Mixed Linear Model Genome-wide Association Study" (MLM-GWAS) and the linkage mapping procedure "Composite Interval Mapping" (CIM), 67, 36 and 10 BIN-QTLs and 86, 14 and 23 SNPLDB-QTLs were detected with their phenotypic variance explained (PVE) 88.70-89.92% (within heritability 98.2%), 146.41-353.62% (overflowing) and 88.29-172.34% (overflowing), respectively. The RTM-GWAS with SNPLDBs which showed to be more efficient and reasonable than the others was used to identify the R1 QTL system in NJRIKY. The detected 86 SNPLDB-QTLs with their PVE from 0.02 to 30.66% in a total of 89.92% covered 51 out of 104 R1 QTLs in 18 crosses in SoyBase and 26 out of 139 QTLs in a nested association mapping population, while the rest 29 QTLs were novel ones. From the QTL system, 52 candidate genes were annotated, including the verified gene E1, E2, E9 and J, and grouped into 3 categories of biological processes, among which 24 genes were enriched into three protein-protein interaction networks, suggesting gene networks working together. Since NJRIKY involves only MGII and MGV, the QTL/gene system among MG000-MGX should be explored further.

New Isolates of Sweet potato feathery mottle virus and Sweet potato virus C: Biological and Molecular Properties, and Recombination Analysis Based on Complete Genomes.

Sweet potato feathery mottle virus (SPFMV) and Sweet potato virus C (SPVC) isolates were obtained from sweetpotato shoot or tuberous root samples from three widely separated locations in Australia's tropical north (Cairns, Darwin, and Kununurra). The samples were planted in the glasshouse and scions obtained from the plants were graft inoculated to Ipomoea setosa plants. Virus symptoms were recorded in the field in Kununurra and in glasshouse-grown sweetpotato and I. setosa plants. RNA extracts from I. setosa leaf samples were subjected to high-throughput sequencing. New complete SPFMV (n = 17) and SPVC (n = 6) genomic sequences were obtained and compared with 47 sequences from GenBank. Phylogenetic analysis revealed that the 17 new SPFMV genomes all fitted within either major phylogroup A, minor phylogroup II, formerly O; or major phylogroup B, formerly RC. Major phylogroup A's minor phylogroup I, formerly EA, only appeared when recombinants were included. Numbers of SPVC genomes were insufficient to subdivide it into phylogroups. Within phylogroup A's minor phylogroup II, the closest genetic match between an Australian and a Southeast Asian SPFMV sequence was the 97.4% nucleotide identity with an East Timorese sequence. Recombination analysis of the 43 SPFMV and 27 SPVC sequences revealed evidence of 44 recombination events, 16 of which involved interspecies sequence transfers between SPFMV and SPVC and 28 intraspecies transfers, 17 in SPFMV and 11 in SPVC. Within SPFMV, 11 intraspecies recombination events were between different major phylogroups and 6 were between members of the same major phylogroup. Phylogenetic analysis accounting for the detected recombination events within SPFMV sequences yielded evidence of minor phylogroup II and phylogroup B but the five sequences from minor phylogroup I were distributed in two separate groups among the sequences of minor phylogroup II. For the SPVC sequences, phylogenetic analysis accounting for the detected recombination events revealed three major phylogroups (A, B, and C), with major phylogroup A being further subdivided into two minor phylogroups. Within the recombinant genomes of both viruses, their PI, NIa-Pro, NIb, and CP genes contained the highest numbers of recombination breakpoints. The high frequency of interspecies and interphylogroup recombination events reflects the widespread occurrence of mixed SPVC and SPFMV infections within sweetpotato plants. The prevalence of infection in northern Australian sweetpotato samples reinforces the need for improved virus testing in healthy sweetpotato stock programs. Furthermore, evidence of genetic connectivity between Australian and East Timorese SPFMV genomes emphasizes the need for improved biosecurity measures to protect against potentially damaging international virus movements.

Genetic characterization of a novel HIV-1 CRF01_AE/CRF07_BC recombinant form among men who have sex with men in Guangdong, China.

Genome recombination is a major strategy employed by HIV to generate new variants for the benefit of escaping immune surveillance. Near full-length genome phylogenic analysis was utilized to characterize HIV diversity in a malepatient in Guangdong, China. The result showed a unique recombinant form (URF) composed of two circulating recombinant forms, CRF01_AE (92%) and CRF07_BC (8%), using six recombinant breakpoints, nt 2794, 3092, 4482, 5988, 7021, and 7722. The emergence of this URF indicates that HIV-1 co-infection or super-infections are common. The increasing genetic complexity of the HIV-1 epidemic in China warrants continued investigation.

Phylogenomic relationship and evolutionary insights of sweet potato viruses from the western highlands of Kenya.

Sweet potato is a major food security crop within sub-Saharan Africa where 90% of Africa production occurs. One of the major limitations of sweet potato production are viral infections. In this study, we used a combination of whole genome sequences from a field isolate obtained from Kenya and those available in GenBank. Sequences of four sweet potato viruses: Sweet potato feathery mottle virus (SPFMV), Sweet potato virus C (SPVC), Sweet potato chlorotic stunt virus (SPCSV), Sweet potato chlorotic fleck virus (SPCFV) were obtained from the Kenyan sample. SPFMV sequences both from this study and from GenBank were found to be recombinant. Recombination breakpoints were found within the Nla-Pro, coat protein and P1 genes. The SPCSV, SPVC, and SPCFV viruses from this study were non-recombinant. Bayesian phylogenomic relationships across whole genome trees showed variation in the number of well-supported clades; within SPCSV (RNA1 and RNA2) and SPFMV two well-supported clades (I and II) were resolved. The SPCFV tree resolved three well-supported clades (I–III) while four well-supported clades were resolved in SPVC (I–IV). Similar clades were resolved within the coalescent species trees. However, there were disagreements between the clades resolved in the gene trees compared to those from the whole genome tree and coalescent species trees. However the coat protein gene tree of SPCSV and SPCFV resolved similar clades to the genome and coalescent species tree while this was not the case in SPFMV and SPVC. In addition, we report variation in selective pressure within sites of individual genes across all four viruses; overall all viruses were under purifying selection. We report the first complete genomes of SPFMV, SPVC, SPCFV, and a partial SPCSV from Kenya as a mixed infection in one sample. Our findings provide a snap shot on the evolutionary relationship of sweet potato viruses (SPFMV, SPVC, SPCFV, and SPCSV) from Kenya as well as assessing whether selection pressure has an effect on their evolution.

Molecular Characterization of Begomoviruses DNA-A and Associated Beta Satellites with New Host Ocimum sanctum in India

Ocimum sanctum (Tulsi) is a native Indian subcontinent plant with important medicinal values. Leaves with mosaic and curling observed on O. sanctum in Rajasthan province of India were severely stunted having symptoms of typical Begomovirus infection. The authors report on a comprehensive study of sequence variations, phylogeny and recombination breakpoints for Tomato leaf curl virus, prevailing in the plant O. sanctum. To confirm Begomovirus infection, PCR and whole genome amplification via rolling circle experiments were exhibited by means of complete nucleic acid (DNA) isolated from symptomatic plants leaves. Sequence analysis established the association of betasatellite molecule along with DNA-A genome and contemplated as isolate of Tomato leaf curl virus as it showed 98% similarity with ToLCV. The present study includes phylogenetic, recombination and pathogenicity analysis of ToLCV infecting O. sanctum.

Discovery of Enterovirus A71-like nonstructural genomes in recent circulating viruses of the Enterovirus A species

Enterovirus A71 (EV-A71) is an important nonpolio enterovirus that causes severe neurological complications. In 1998, Taiwan experienced an EV-A71 outbreak that caused 78 deaths. Since then, periodic epidemics of EV-A71 associated with newly emerging strains have occurred. Several of these strains are known to be recombinant; however, how these strains arose within such a short period of time remains unknown. Here, we sequenced 64 full-length genomes from clinical isolates collected from 2005 to 2016 and incorporated all 91 Taiwanese genomes downloaded from the Virus Pathogen Resource to extensively analyze EV-A71 recombination in Taiwan. We found that the B3 subgenotype was a potential recombinant parent of the EV-A71 C2-like and C4 strains by intratypic recombination. Such B3-similar regions were also found in many cocirculating coxsackieviruses belonging to Enterovirus A species (EV-A) through a series of intertypic recombinations. Therefore, locally enriched outbreaks of cocirculating viruses from different genotypes/serotypes may facilitate recombination. Most recombination breakpoints we found had nonrandom distributions and were located within the region spanning from the boundary of P1 (structural gene) and P2 (nonstructural) to the cis-acting replication element at P2, indicating that specific genome reassembly of structural and nonstructural genes may be subject to natural selection. Through intensive recombination, 11 EV-A71-like signatures (including one in 3A, two in 3C, and eight in 3D) were found to be present in a variety of recently cocirculating EV-A viruses worldwide, suggesting that these viruses may be targets for wide-spectrum antiviral development.

Norovirus recombinants: recurrent in the field, recalcitrant in the lab - a scoping review of recombination and recombinant types of noroviruses.

Noroviruses are recognized as the major global cause of sporadic and epidemic non-bacterial gastroenteritis in humans. Molecular mechanisms driving norovirus evolution are the accumulation of point mutations and recombination. Intragenotypic recombination has long been postulated to be a driving force of GII.4 noroviruses, the predominant genotype circulating in humans for over two decades. Increasingly, emergence and re-emergence of different intragenotype recombinants have been reported. The number and types of norovirus recombinants remained undefined until the 2007 Journal of General Virology research article 'Norovirus recombination' reported an assembly of 20 hitherto unclassified intergenotypic norovirus recombinant types. In the intervening decade, a host of novel recombinants has been analysed. New recombination breakpoints have been described, in vitro and in vivo studies supplement in silico analyses, and advances have been made in analysing factors driving norovirus recombination. This work presents a timely overview of these data and focuses on important aspects of norovirus recombination and its role in norovirus molecular evolution. An overview of intergenogroup, intergenotype, intragenotype and 'obligatory' norovirus recombinants as detected via in silico methods in the field is provided, enlarging the scope of intergenotypic recombinant types to 80 in total, and notably including three intergenogroup recombinants. A recap of advances made studying norovirus recombination in the laboratory is given. Putative drivers and constraints of norovirus recombination are discussed and the potential link between recombination and norovirus zoonosis risk is examined.

Identification of a recombinant isolate of ungulate copiparvovirus.

To evaluate the status of parvovirus infection in free-range cows in a region of northeast China, nine serum samples were collected and analysed by sequencing and polymerase chain reaction. A new bovine parvovirus-2 (BPV2) was identified and named QQHE16. The genome of the virus is 5759 nucleotides long and retains two ORFs that are typical of the Parvovirinae family. Compared with reference BPV2 strains, BPV2 QQHE16 appeared to have a close relationship with strain BSRI isolated in the USA in 2013. A putative recombination breakpoint located at nucleotide position 2121 and in the interval between the non-structural gene and the VP gene was identified. From our analysis, we propose that strain QQHE16 originates from the natural recombination of strains ujs2665 and BSRI.

Novel canine circovirus strains from Thailand: Evidence for genetic recombination

Canine circoviruses (CanineCV’s), belonging to the genus Circovirus of the Circoviridae family, were detected by next generation sequencing in samples from Thai dogs with respiratory symptoms. Genetic characterization and phylogenetic analysis of nearly complete CanineCV genomes suggested that natural recombination had occurred among different lineages of CanineCV’s. Similarity plot and bootscaning analyses indicated that American and Chinese viruses had served as major and minor parental viruses, respectively. Positions of recombination breakpoints were estimated using maximum-likelihood frameworks with statistical significant testing. The putative recombination event was located in the Replicase gene, intersecting with open reading frame-3. Analysis of nucleotide changes confirmed the origin of the recombination event. This is the first description of naturally occurring recombinant CanineCV’s that have resulted in the circulation of newly emerging CanineCV lineages.

Characterization of a New HIV-1 CRF01_AE/CRF07_BC Recombinant Virus Form Among Men Who Have Sex with Men in Beijing, China.

Recent studies have revealed that three major viral strains (CRF07_BC, CRF01_AE, and subtype B) have been cocirculating among men who have sex with men in Beijing. Cocirculation of multiple subtypes in the same population contributes significantly to the emergence of recombinant viruses. This study reports a near full-length genome of a novel, second-generation HIV-1 recombinant (BJ2015EU16) between CRF07_BC and CRF01_AE. The analysis of BJ2015EU16 shows that the genome comprises three interlaced segments, including two CRF07_BC and one CRF01_AE segments, with CRF07_BC as the backbone. There were two recombinant breakpoints in the vpu and env genes of BJ2015EU16. This is different from a previously identified CRF01_AE/07_BC recombinant strain from Beijing. The new emergence of the unique recombinant form increases the complexity of the HIV epidemic in the Beijing homosexual group, and the ongoing generation of recombinant viruses may provide an important basis for future analysis of the recombination mechanism of HIV-1.

Within-Host Recombination in the Foot-and-Mouth Disease Virus Genome.

Recombination is one of the determinants of genetic diversity in the foot-and-mouth disease virus (FMDV). FMDV sequences have a mosaic structure caused by extensive intra- and inter-serotype recombination, with the exception of the capsid-encoding region. While these genome-wide patterns of broad-scale recombination are well studied, not much is known about the patterns of recombination that may exist within infected hosts. In addition, detection of recombination among viruses evolving at the within-host level is challenging due to the similarity of the sequences and the limitations in differentiating recombination from point mutations. Here, we present the first analysis of recombination events between closely related FMDV sequences occurring within buffalo hosts. The detection of these events was made possible by the occurrence of co-infection of two viral swarms with about 1% nucleotide divergence. We found more than 15 recombination events, unequally distributed across eight samples from different animals. The distribution of these events along the FMDV genome was neither uniform nor related to the phylogenetic distribution of recombination breakpoints, suggesting a mismatch between within-host evolutionary pressures and long-term selection for infectivity and transmissibility.

A traditional evolutionary history of foot-and-mouth disease viruses in Southeast Asia challenged by analyses of non-structural protein coding sequences

Recombination of rapidly evolving RNA-viruses provides an important mechanism for diversification, spread, and emergence of new variants with enhanced fitness. Foot-and-mouth disease virus (FMDV) causes an important transboundary disease of livestock that is endemic to most countries in Asia and Africa. Maintenance and spread of FMDV are driven by periods of dominance of specific viral lineages. Current understanding of the molecular epidemiology of FMDV lineages is generally based on the phylogenetic relationship of the capsid-encoding genes, with less attention to the process of recombination and evolution of non-structural proteins. In this study, the putative recombination breakpoints of FMDVs endemic to Southeast Asia were determined using full-open reading frame sequences. Subsequently, the lineages’ divergence times of recombination-free genome regions were estimated. These analyses revealed a close relationship between two of the earliest endemic viral lineages that appear unrelated when only considering the phylogeny of their capsid proteins. Contrastingly, one lineage, named O/CATHAY, known for having a particular host predilection (pigs) has evolved independently. Additionally, intra-lineage recombination occurred at different breakpoints compared to the inter-lineage process. These results provide new insights about FMDV recombination patterns and the evolutionary interdependence of FMDV serotypes and lineages.

A Survey of Virus Recombination Uncovers Canonical Features of Artificial Chimeras Generated During Deep Sequencing Library Preparation.

Chimeric reads can be generated by in vitro recombination during the preparation of high-throughput sequencing libraries. Our attempt to detect biological recombination between the genomes of dengue virus (DENV; +ssRNA genome) and its mosquito host using the Illumina Nextera sequencing library preparation kit revealed that most, if not all, detected host–virus chimeras were artificial. Indeed, these chimeras were not more frequent than with control RNA from another species (a pillbug), which was never in contact with DENV RNA prior to the library preparation. The proportion of chimera types merely reflected those of the three species among sequencing reads. Chimeras were frequently characterized by the presence of 1-20 bp microhomology between recombining fragments. Within-species chimeras mostly involved fragments in opposite orientations and located less than 100 bp from each other in the parental genome. We found similar features in published datasets using two other viruses: Ebola virus (EBOV; -ssRNA genome) and a herpesvirus (dsDNA genome), both produced with the Illumina Nextera protocol. These canonical features suggest that artificial chimeras are generated by intra-molecular template switching of the DNA polymerase during the PCR step of the Nextera protocol. Finally, a published Illumina dataset using the Flock House virus (FHV; +ssRNA genome) generated with a protocol preventing artificial recombination revealed the presence of 1-10 bp microhomology motifs in FHV–FHV chimeras, but very few recombining fragments were in opposite orientations. Our analysis uncovered sequence features characterizing recombination breakpoints in short-read sequencing datasets, which can be helpful to evaluate the presence and extent of artificial recombination.

Surging HIV-1 CRF07_BC epidemic among recently infected men who have sex with men in Fujian, China.

A rapidly increasing number of HIV-1 infections have been identified among men who have sex with men (MSM) in Fujian province of China since 2010. We aimed to investigate the causative factors underlying this surging epidemic. Using immunoassays for HIV-1 diagnosis and phylogenetic analysis for viral genotyping, we found that the number of MSM infections doubled from 171 in 2011 to 340 in 2013 with a significantly increased prevalent rate from 4.1% to 5.2%. Majority of these increased infections took place in Fuzhou, Xiamen, and Quanzhou, three large cities in Fujian, mainly among youth, unemployed, business, and well-educated MSMs. Phylogenetic analysis revealed three major HIV-1 genotypes including CRF01_AE, CRF07_BC, and B/B' yet the surging MSM infections were primarily associated with the rapid sexual spread of CRF07_BC in addition to CRF01_AE. In particular, there was a significant proportional expansion of CRF07_BC infections among recently infected MSMs from 19% in 2012 to 41.9% in 2013. This increase was accompanied by emergence of complex patterns of viral recombination including multiple hybrid variants derived from CRF01_AE and CRF07_BC. Full-genome analysis indicated that CRF07_BC in Fujian was likely originated from similar strains previously found among IDUs in Yunnan province but with unique recombination break points. Our findings indicated that HIV-1 CRF07_BC has adapted for rapid sexual transmission, resulting in the surging HIV-1 epidemic and the emergence of new recombinant strains among MSMs in Fujian. Our findings have implications to vaccine and passive immunization trials in Fujian with emphasis on the induction of cross-subtype protective immunity.

Identification of a Novel Recombinant Type 2 Porcine Reproductive and Respiratory Syndrome Virus in China.

Since the emergence of NADC30-like porcine reproductive and respiratory syndrome virus (PRRSV) in China in 2013, PRRSVs have undergone rapid evolution. In this study, a novel variant of PRRSV strain (designated SCcd17) was successfully isolated from piglets with clinical signs in Sichuan Province in China in 2017, and the complete genomic sequence was determined. The genome of this new isolate was 15,015 nucleotides (nt) long, and comparative analysis revealed that SCcd17 exhibited 90.2%, 85.2%, 84.9%, and 84.0% nucleotide similarity to PRRSVs NADC30, JXA1, CH-1a, and VR-2332, respectively. Phylogenetic analysis indicated that the SCcd17 strain was classified into the NADC30-like sub-genotype, in which all the strains contained the unique discontinuous 131-amino acid deletion in nonstructural protein 2 (nsp2) when compared to VR-2332-like viruses. Notably, extensive amino acid substitutions were observed in nsp2 and a unique single amino acid deletion at position 33 of the GP5 is being described for the first time. Strikingly, recombination analysis revealed that SCcd17 was the result of recombination between the NADC30-like, JXA1-like, and VR-2332-like strains at five recombination breakpoints: nsp1α (nt 641), nsp3 (nt 5141), nsp10 (nt 9521), open reading frame 3 (ORF3) (nt 12,581), and ORF4 (nt 13,021). The genomic data of SCcd17 will be helpful for understanding the role of genomic recombination in the evolution of PRRSV.

A new hybrid approach for MHC genotyping: high-throughput NGS and long read MinION nanopore sequencing, with application to the non-model vertebrate Alpine chamois (Rupicapra rupicapra)

The major histocompatibility complex (MHC) acts as an interface between the immune system and infectious diseases. Accurate characterization and genotyping of the extremely variable MHC loci are challenging especially without a reference sequence. We designed a combination of long-range PCR, Illumina short-reads, and Oxford Nanopore MinION long-reads approaches to capture the genetic variation of the MHC II DRB locus in an Italian population of the Alpine chamois (Rupicapra rupicapra). We utilized long-range PCR to generate a 9 Kb fragment of the DRB locus. Amplicons from six different individuals were fragmented, tagged, and simultaneously sequenced with Illumina MiSeq. One of these amplicons was sequenced with the MinION device, which produced long reads covering the entire amplified fragment. A pipeline that combines short and long reads resolved several short tandem repeats and homopolymers and produced a de novo reference, which was then used to map and genotype the short reads from all individuals. The assembled DRB locus showed a high level of polymorphism and the presence of a recombination breakpoint. Our results suggest that an amplicon-based NGS approach coupled with single-molecule MinION nanopore sequencing can efficiently achieve both the assembly and the genotyping of complex genomic regions in multiple individuals in the absence of a reference sequence.

A natural recombinant PRRSV between HP-PRRSV JXA1-like and NADC30-like strains.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a major economically significant pathogen that has adversely affected China's swine industry. Currently, a novel type 2 PRRSV, called the NADC30-like strain, is epidemic in numerous provinces of China, and commercial vaccines provide limited protection for infected animals. The extensive recombination phenomenon among NADC30-like PRRSVs is identified as a unique molecular characteristic of the virus. However, our understanding of how recombination influences NADC30-like PRRSVs is largely inadequate. In this study, we analysed the genetic characteristics of a recombinant NADC30-like PRRSV (SC-d) and examined its pathogenicity compared with a non-recombinant NADC30-like PRRSV (SD-A19) and a highly pathogenic PRRSV (HuN4). SC-d has three discontinuous deletions in nsp2, consistent with NADC30 isolated from the United States in 2008. Furthermore, we identified four recombination breakpoints in the SC-d genome, which separated the SC-d genome into four regions (regions A, B, C and D). Regions A and C are closely related to the JXA1-like strain, one of the earliest Chinese HP-PRRSV strains, and regions B and D are closely related to the NADC30 strain. Moreover, SC-d inoculated piglets exhibited a persistent fever, moderate weight loss, mild thymus atrophy and obvious microscopic lung lesions. In summary, the recombinant NADC30-like PRRSV SC-d strain displayed a higher pathogenicity than the non-recombinant NADC30-like PRRSV SD-A19 strain; however, the pathogenicity of the NADC30-like PRRSV SC-d was lower compared with the HP-PRRSV HuN4 strain in piglets. Our findings demonstrate that recombination is responsible for the enormous genetic diversity and pathogenicity variance of the NADC30-like PRRSV in China. This study provides a theoretical basis for developing a more reasonable PRRSV control and prevention strategy.

Delimiting Coalescence Genes (C-Genes) in Phylogenomic Data Sets.

Summary coalescence methods have emerged as a popular alternative for inferring species trees with large genomic datasets, because these methods explicitly account for incomplete lineage sorting. However, statistical consistency of summary coalescence methods is not guaranteed unless several model assumptions are true, including the critical assumption that recombination occurs freely among but not within coalescence genes (c-genes), which are the fundamental units of analysis for these methods. Each c-gene has a single branching history, and large sets of these independent gene histories should be the input for genome-scale coalescence estimates of phylogeny. By contrast, numerous studies have reported the results of coalescence analyses in which complete protein-coding sequences are treated as c-genes even though exons for these loci can span more than a megabase of DNA. Empirical estimates of recombination breakpoints suggest that c-genes may be much shorter, especially when large clades with many species are the focus of analysis. Although this idea has been challenged recently in the literature, the inverse relationship between c-gene size and increased taxon sampling in a dataset—the ‘recombination ratchet’—is a fundamental property of c-genes. For taxonomic groups characterized by genes with long intron sequences, complete protein-coding sequences are likely not valid c-genes and are inappropriate units of analysis for summary coalescence methods unless they occur in recombination deserts that are devoid of incomplete lineage sorting (ILS). Finally, it has been argued that coalescence methods are robust when the no-recombination within loci assumption is violated, but recombination must matter at some scale because ILS, a by-product of recombination, is the raison d’etre for coalescence methods. That is, extensive recombination is required to yield the large number of independently segregating c-genes used to infer a species tree. If coalescent methods are powerful enough to infer the correct species tree for difficult phylogenetic problems in the anomaly zone, where concatenation is expected to fail because of ILS, then there should be a decreasing probability of inferring the correct species tree using longer loci with many intralocus recombination breakpoints (i.e., increased levels of concatenation).

Recombination in the rabies virus and other lyssaviruses

Recombination is a common event in RNA viruses; however, in the rabies virus there have been only a few reports of isolated recombination events. Comprehensive analysis found traces of recent recombination events within Arctic, Arctic-like and Africa 1b rabies virus groups, as well as recombination between distinct lyssaviruses. Recombination breakpoints were not linked to gene boundaries and could be detected all over the genome. However, there was no evidence that recombination is an important factor in the genetic variability of the rabies virus. It is therefore likely that recombination in the rabies virus is limited by ecological factors (e.g., rare co-circulation of distinguishable lineages and a narrow window for productive coinfection in most carnivore hosts), rather than molecular barriers (e.g., incompatibility of genome fragments).