Remarkable mitochondrial genome heterogeneity in Meniocus linifolius (Brassicaceae).

KEY MESSAGE: Detailed analyses of 16 genomes identified a remarkable acceleration of mutation rate, hence mitochondrial sequence and structural heterogeneity, in Meniocus linifolius (Brassicaceae). The powerhouse, mitochondria, in plants feature high levels of structural variation, while the encoded genes are normally conserved. However, the substitution rates and spectra of mitochondria DNA within the Brassicaceae, a family with substantial scientific and economic importance, have not been adequately deciphered. Here, by analyzing three newly assembled and 13 known mitochondrial genomes (mitogenomes), we report the highly variable genome structure and mutation rates in Brassicaceae. The genome sizes and GC contents are 196,604 bp and 46.83%, 288,122 bp and 44.79%, and 287,054 bp and 44.93%, for Meniocus linifolius (Mli), Crucihimalaya lasiocarpa (Cla), and Lepidium sativum (Lsa), respectively. In total, 29, 33, and 34 protein-coding genes (PCGs) and 14, 18, and 18 tRNAs are annotated for Mli, Cla, and Lsa, respectively, while all mitogenomes contain one complete circular molecule with three rRNAs and abundant RNA editing sites. The Mli mitogenome features four conformations likely mediated by the two pairs of long repeats, while at the same time seems to have an unusual evolutionary history due to higher GC content, loss of more genes and sequences, but having more repeats and plastid DNA insertions. Corroborating with these, an ambiguous phylogenetic position with long branch length and elevated synonymous substitution rate in nearly all PCGs are observed for Mli. Taken together, our results reveal a high level of mitogenome heterogeneity at the family level and provide valuable resources for further understanding the evolutionary pattern of organelle genomes in Brassicaceae.

The mitochondrial genome of the diploid oat Avena longiglumis.

BACKGROUND: Avena longiglumis Durieu (2n = 2x = 14) is a wild relative of cultivated oat (Avena sativa, 2n = 6x = 42) with good agronomic and nutritional traits. The plant mitochondrial genome has a complex organization and carries genetic traits of value in exploiting genetic resources, not least male sterility alleles used to generate F1 hybrid seeds. Therefore, we aim to complement the chromosomal-level nuclear and chloroplast genome assemblies of A. longiglumis with the complete assembly of the mitochondrial genome (mitogenome) based on Illumina and ONT long reads, comparing its structure with Poaceae species. RESULTS: The complete mitochondrial genome of A. longiglumis can be represented by one master circular genome being 548,445 bp long with a GC content of 44.05%. It can be represented by linear or circular DNA molecules (isoforms or contigs), with multiple alternative configurations mediated by long (4,100-31,235 bp) and medium (144-792 bp) size repeats. Thirty-five unique protein-coding genes, three unique rRNA genes, and 11 unique tRNA genes are identified. The mitogenome is rich in duplications (up to 233 kb long) and multiple tandem or simple sequence repeats, together accounting for more than 42.5% of the total length. We identify homologous sequences between the mitochondrial, plastid and nuclear genomes, including the exchange of eight plastid-derived tRNA genes, and nuclear-derived retroelement fragments. At least 85% of the mitogenome is duplicated in the A. longiglumis nuclear genome. We identify 269 RNA editing sites in mitochondrial protein-coding genes including stop codons truncating ccmFC transcripts. CONCLUSIONS: Comparative analysis with Poaceae species reveals the dynamic and ongoing evolutionary changes in mitochondrial genome structure and gene content. The complete mitochondrial genome of A. longiglumis completes the last link of the oat reference genome and lays the foundation for oat breeding and exploiting the biodiversity in the genus.

The first complete mitochondrial genome of Carex (C. breviculmis): a significantly expanded genome with highly structural variations.

MAIN CONCLUSION: The first complete mitochondrial genome of Carex (C. breviculmis) was sequenced and assembled, and its genomic signature was analyzed and the possible conformations of its mitochondrial genome were validated. Carex breviculmis is a very adaptable grass that is highly resistant to environmental stresses such as drought and low light. It is also admired as a landscape plant with high development prospects and scientific research value. In this study, the mitochondrial genome of C. breviculmis was assembled using Pacbio and Illumina sequencing data. We detected 267 pairs of repeats and found that three pairs of repeats could mediate the recombination of its mitochondrial genome and formed four possible conformations, of which we verified the two conformations mediated by the shortest pair of repeats using PCR amplification and Sanger sequencing. The major conformation of the C. breviculmis mitochondrial genome is a 1,414,795 bp long circular molecule with 33 annotated protein-coding genes, 15 tRNA genes, and three rRNA genes. We detected a total of 25 homologous sequences between the chloroplast and mitochondrial genomes, corresponding to 0.40% of the mitochondrial genome. Combined with the low GC content (41.24%), we conclude that the reduction in RNA editing sites in the C. breviculmis mitochondrial genome may be due to an accumulation of point mutations in C-to-T or retroprocessing events within the genome. The relatively low number of RNA editing sites in its mitochondrial genome could serve as important material for subsequent studies on the selection pressure of RNA editing in angiosperms. A maximum likelihood analysis based on 23 conserved mitochondrial genes from 28 species reflects an accurate evolutionary and taxonomic position of C. breviculmis. This research provided us with a comprehensive understanding of the mitochondrial genome of Carex and also provided important information for its molecular breeding.

Assembly of the complete mitochondrial genome of an endemic plant, Scutellaria tsinyunensis, revealed the existence of two conformations generated by a repeat-mediated recombination.

MAIN CONCLUSION: We assembled the complete mitochondrial genome of Scutellaria tsinyunensis in this study. Repeat-mediated recombination resulted in the formation of two conformations of the mitochondrial genome in S. tsinyunensis. Scutellaria tsinyunensis belongs to the family Lamiaceae, distributed only in the Jinyun Mountain, Chongqing, China. As a valuable endemic and small population species, it is regarded as a natural resource potentially with significant economic and ecological importance. In this study, we assembled a complete and gap-free mitochondrial genome of S. tsinyunensis. This genome had a length of 354,073 bp and the base composition of the genome was A (27.44%), T (27.30%), C (22.58%), and G (22.68%). This genome encodes 59 genes, including 32 protein-coding genes, 24 tRNA genes, and 3 rRNA genes. The Sanger sequencing and Oxford Nanopore sequencing confirmed a pair of direct repeats had mediated genome recombination, resulting in the formation of two conformations. The gene conversation between plastome and mitochondrial genome was also observed in S. tsinyunensis by detecting gene migration, including six tRNA genes (namely, trnW-CCA, trnI-CAU, trnH-UUU, trnD-GUC, trnN-GUU, and trnM-CAU), five protein-coding gene fragments, and the fragments from 2 rRNA genes. Moreover, the dN/dS analysis revealed the atp9 gene had undergone strong negative selection, and four genes (atp4, mttB, ccmFc, and ccmB) probably had undergone positive selection during evolution in Lamiales. This work reported the first mitochondrial genome of S. tsinyunensis, which could be used as a reference genome for the important medicinal plants of the genus Scutellaria, and also provide much-desired information for molecular breeding.

Genetic characterization of a novel recombinant echovirus 30 strain causing a regional epidemic of aseptic meningitis in Hokkaido, Japan, 2017.

A regional epidemic of aseptic meningitis caused by echovirus 30 (E30) occurred in Hokkaido, Japan, during the period of August-December 2017. To investigate their phylogenetic relationship to other human enteroviruses, we determined the complete genomic nucleotide sequences of isolates from this outbreak. Phylogenetic analysis of the viral capsid protein 1 gene showed that the strains were most closely related to E30 strains detected in Germany, France, and Russia in 2013. In contrast, the region encoding the viral protease and the RNA-dependent RNA polymerase had a close phylogenetic relationship to non-E30 enteroviruses detected in the United Kingdom and Switzerland in 2015-2017, suggesting that a recombination event had occurred.

A recombinant infectious bronchitis virus from a chicken with a spike gene closely related to that of a turkey coronavirus.

Using viral metagenomics, the complete genome sequence of an infectious bronchitis virus (IBV) strain (named ahysx-1) from a fecal sample from a healthy chicken in Anhui province, China, was determined. The genome sequence of ahysx-1 was found to be very similar to that of IBV strain ck/CH/LLN/131040 (KX252787), except for the spike gene region, which is similar to that of a turkey coronavirus strain (EU022526), suggesting that ahysx-1 is a recombinant. Recombination analysis and phylogenetic analysis based on the genomic sequences of ahysx-1 and other related strains confirmed that ahysx-1 appears to be a recombinant resulting from a recombination event that occurred between a chicken coronavirus and a turkey coronavirus. Further studies need to be performed to determine whether this recombinant IBV strain is pathogenic and whether it is transmitted between chickens and turkeys.

Human norovirus genogroup II recombinants in Thailand, 2009-2014.

Norovirus (NoV) is a major cause of nonbacterial acute gastroenteritis worldwide. New strains emerge partly due to viral recombination. In Thailand, there is a lack of data on NoV recombinants among clinical isolates. We screened stool samples from pediatric diarrheal patients for norovirus by RT-PCR and found GII.4 to be the most prevalent genotype. Phylogenetic and SimPlot analyses detected seven intra-genogroup recombinant strains: three GII.21/GII.3, two GII.12/GII.3, and two GII.12/GII.1 recombinants. Maximum chi-square analysis indicated that all had similar breakpoints near the ORF1/ORF2 junction (p < 0.001), either slightly upstream within the C-terminus of RdRp or downstream within the N-terminal domain of VP1.

Prevalence and genetic diversity of Parechovirus.

Human parechovirus (HPeV) is a common virus that can cause severe infections in newborns. Due to the limited knowledge of the prevalence of HPeV in different cities in China and the unknown association between HPeV infection and clinical characteristics of newborns, this research investigated the epidemiological and clinical characteristics of HPeV infection in hospitalized neonates in Changsha. From August to October 2023, 145 anal swab samples from 96 newborns and 38 pharyngeal swab samples from 33 newborns in the neonatal intensive care unit (NICU) were collected. The prevalence of HPeV was detected by reverse transcription-polymerase chain reaction (RT-PCR). The genomes of HPeV were sequenced and the viral protein 1 (VP1) region was used for genotyping. Phylogenetic analysis and recombination analysis of HPeV genome were performed. Finally, HPeV was detected in 10 out of 44 patients in October, all of them were HPeV-1. The sequenced 4 genomes of HPeV showed high genetic diversity with known strains. Additionally, a HPeV-1 recombinant strain was detected. Compared with HPeV negative patients, HPeV patients had higher prevalence of abdominal pain and diarrhea, intracranial hemorrhage, and purulent meningitis. Compared with HPeV negative patients, HPeV patients had higher peripheral blood lymphocytes, albumin, globulin, pH and lower peripheral blood neutrophils and hemoglobin. HPeV is an important viral cause of newborn infections and appears to be increasing in prevalence in recent years. Characteristic clinical pictures exist in HPeV infections, and further research is needed to accumulate more cases to obtain a comprehensive understanding of HPeV infections.

Molecular characteristic, evolution, and pathogenicity analysis of avian infectious bronchitis virus isolates associated with QX type in China.

Infectious bronchitis virus (IBV) is one of the major avian pathogens plaguing the global poultry industry. Although vaccination is the primary preventive measure for IBV infection, the emergence of virus variants with mutations and recombination has resulted in IBV circulating globally, presenting a challenge for IB control. Here, we isolated 3 IBV strains (CZ200515, CZ210840, and CZ211063) from suspected sick chickens vaccinated with IBV live attenuated vaccines (H120, 4/91, or QXL87). Phylogenetic analysis of the S1 gene sequence of the spike (S) revealed that the 3 isolates belonged to the QX-type (GI-19 lineage). Whole genome sequencing and recombination analysis indicated that CZ200515 and CZ210840 contained genetic material from 4/91 and Scyz3 (QX-type), possibly due to recombination between the circulating strain and the 4/91 vaccine strain, while no evidence of recombination was found in CZ211063. Pathogenicity analysis in 1-day-old specific pathogen-free (SPF) chickens demonstrated that all 3 isolates caused severe tissue damage and varying degrees of mortality. Virus cross-neutralization assay revealed decreased antigen relatedness between the isolates and the QX-type vaccine strain (QXL87). Amino acid sequence homology analysis of S1 revealed 5%-6.5% variances between the isolates and QXL87. Analysis of the S1 subunit structure revealed that mutations of amino acid residues in the hypervariable region (HVR) and the neutralizing epitope region resulted in antigenic variation in isolates by changing the antigen conformation. Our data indicate antigenicity variances between QX isolates and QXL87 vaccine strains, potentially resulting in immune evasion occurrences. Overall, these results offer crucial insights into the epidemiology and pathogenicity of QX-type IBV, facilitating improved selection and formulation of vaccines for disease management.

Genome Recombination between the Delta and Alpha Variants of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2).

Prominent genomic recombination has been observed between the Delta and Alpha variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), isolated from clinical specimens in Japan. Interestingly, the recombination variant detected in this study carries a spike protein identical to that in the domestic Delta variant, thereby suggesting that further risks would not be associated with infectivity and immune escape. The recombinant was classified as an XC lineage in the PANGOLIN database. It is necessary to intensively study such marked genetic variations and characterize emerging variants after careful verification of their lineage and clade assignment.

Epidemiology and evolution of novel deltacoronaviruses in birds in central China.

The variety and widespread of coronavirus in natural reservoir animals is likely to cause epidemics via interspecific transmission, which has attracted much attention due to frequent coronavirus epidemics in recent decades. Birds are natural reservoir of various viruses, but the existence of coronaviruses in wild birds in central China has been barely studied. Some bird coronaviruses belong to the genus of Deltacoronavirus. To explore the diversity of bird deltacoronaviruses in central China, we tested faecal samples from 415 wild birds in Hunan Province, China. By RT-PCR detection, we identified eight samples positive for deltacoronaviruses which were all from common magpies, and in four of them, we successfully amplified complete deltacoronavirus genomes distinct from currently known deltacoronavirus, indicating four novel deltacoronavirus stains (HNU1-1, HNU1-2, HNU2 and HNU3). Comparative analysis on the four genomic sequences showed that these novel magpie deltacoronaviruses shared three different S genes among which the S genes of HNU1-1 and HNU1-2 showed 93.8% amino acid (aa) identity to that of thrush coronavirus HKU12, HNU2 S showed 71.9% aa identity to that of White-eye coronavirus HKU16, and HNU3 S showed 72.4% aa identity to that of sparrow coronavirus HKU17. Recombination analysis showed that frequent recombination events of the S genes occurred among these deltacoronavirus strains. Two novel putative cleavage sites separating the non-structural proteins in the HNU coronaviruses were found. Bayesian phylogeographic analysis showed that the south coast of China might be a potential origin of bird deltacoronaviruses existing in inland China. In summary, these results suggest that common magpie in China carries diverse deltacoronaviruses with novel genomic features, indicating an important source of environmental coronaviruses closed to human communities, which may provide key information for prevention and control of future coronavirus epidemics.

Complex Physical Structure of Complete Mitochondrial Genome of Quercus acutissima (Fagaceae): A Significant Energy Plant.

Quercus acutissima Carruth. is a Chinese important energy plant with high ecological and economic values. While the species chloroplast genome has been reported, its mitochondrial genome (mitogenome) is still unexplored. Here, we assembled and annotated the Q. acutissima mitogenome, and we compared its characteristic differences with several closely related species. The Q. acutissima mitogenome's main structure is branched with three distinguished contigs (linear molecule 1, circular molecule 2, and circular molecule 3) with 448,982 bp total length and 45.72% GC content. The mitogenome contained 51 genes, including 32 protein-coding, 16 tRNA and 3 rRNA genes. We examined codon usage, repeated sequences, genome recombination, chloroplast to mitochondrion DNA transformation, RNA editing, and synteny in the Q. acutissima mitogenome. Phylogenetic trees based on 29 species mitogenomes clarified the species classification. Our results provided comprehensive information of Q. acutissima mitogenome, and they are expected to provide valuable information for Fagaceae evolutionary biology and to promote the species germplasm utilization.

Characterizing Complex Populations of Endogenous Adeno-Associated Viruses by Single-Genome Amplification.

The isolation of adeno-associated virus (AAV) genomes from biomaterials at the molecular level has traditionally relied on polymerase chain reaction-based and cloning-based techniques. However, when applied to samples containing multiple species, traditional techniques for isolating viral genomes can amplify artificial recombinants and introduce polymerase misincorporation errors. In this study, we describe AAV single-genome amplification (AAV-SGA): a powerful technique to isolate, amplify, and sequence single AAV genomes from mammalian genomic DNA, which can then be used to construct vectors for gene therapy. We used AAV-SGA to precisely isolate 15 novel AAV genomes belonging to AAV clades A, D, and E and the Fringe outgroup. This technique also enables investigations of AAV population dynamics and recombination events to provide insights into virus-host interactions and virus biology. Using AAV-SGA, we identified regional heterogeneity within AAV populations from different lobes of the liver of a rhesus macaque and found evidence of frequent genomic recombination between AAV populations. This study highlights the strengths of AAV-SGA and demonstrates its capability to provide valuable insights into the biology and diversity of AAVs.

Decoding genome recombination and sex reversal.

Over the past 440 years since the discovery of the medicinal value of swamp eels, much progress has been made in the study of their biology. The fish is emerging as an important model animal in sexual development, in addition to economic and pharmaceutical implications. Tracing genomic history that shapes speciation of the fish has led to discovery of the whole genome-wide chromosome fission/fusion events. Natural intersex differentiation is a compelling feature for sexual development research. Notably, identification of progenitors of germline stem cells that have bipotential to differentiate into either male or female germline stem cells provides new insight into sex reversal. Here, we review these advances that have propelled the field forwards and present unsolved issues that will guide future investigations to finally elucidate vertebrate sexual development using the new model.

Molecular Characteristics and Pathogenicity of a Novel Recombinant Porcine Reproductive and Respiratory Syndrome Virus Strain from NADC30-, NADC34-, and JXA1-Like Strains That Emerged in China.

Recently, the emergence of a NADC34-like porcine reproductive and respiratory syndrome virus (PRRSV), which causes a large number of abortions in swine herds, has raised great concern in China. In this study, a PRRSV variant strain, PRRSV/CN/FJGD01/2021, evolved from recombination between NADC30-like, NADC34-like, and JXA1-like viruses was isolated in Fujian province in 2021, and its pathogenicity in piglets was examined. Animal experiments demonstrated that PRRSV/CN/FJGD01/2021 infection could induce 100% morbidity and cause higher viremia, a persistently higher fever (>40°C for 14 consecutive days), significant weight loss, and severe histopathological lung lesions compared to the NADC30-like FJZ03 strain and NADC34-like FJ0908 strain in piglets. The PRRSV/CN/FJGD01/2021 strain displayed higher pathogenicity than the FJZ03 and FJ0908 strains, but lower pathogenicity than the Chinese highly pathogenic (HP)-PRRSVs in piglets. Moreover, the Ingelvac PRRS modified live vaccine (MLV) provides incomplete cross-protection against heterologous PRRSV/CN/FJGD01/2021 in piglets. Our findings contribute to the understanding of the current epidemic situation of NADC34-like PRRSV in China. IMPORTANCE The pathogenicity of NADC34-like PRRSV has broad variations in virulence. Importantly, NADC34-like PRRSV has undergone complex recombination with local strains since it first emerged in 2017 in China. However, the pathogenicity of the recombinant NADC34-like virus was rarely experimentally evaluated in pigs. In this study, a novel PRRSV strain, PRRSV/CN/FJGD01/2021, was isolated from sows enduring a high-abortion-rate (20%) period in China in 2021. Notably, phylogenetic and recombination analyses revealed that PRRSV/CN/FJGD01/2021 is a recombinant virus from NADC30-, NADC34-, and JXA1-like isolates. PRRSV/CN/FJGD01/2021 was shown to cause higher virus load, persistent fever, significant weight loss, moderate respiratory clinical signs, and severe histopathological lung lesions in piglets. PRRSV/CN/FJGD01/2021 exhibited higher pathogenicity than NADC30-like FJZ03 and NADC34-like FJ0908, but lower than Chinese HP-PRRSVs for piglets. These data indicated that PRRSV/CN/FJGD01/2021 has intermediate virulence for piglets. Furthermore, the Ingelvac PRRS MLV could partly provide protective efficacy against PRRSV/CN/FJGD01/2021 challenge in piglets.

Assembly of the Complete Mitochondrial Genome of Chinese Plum (Prunus salicina): Characterization of Genome Recombination and RNA Editing Sites.

Despite the significant progress that has been made in the genome sequencing of Prunus, this area of research has been lacking a systematic description of the mitochondrial genome of this genus for a long time. In this study, we assembled the mitochondrial genome of the Chinese plum (Prunus salicina) using Illumina and Oxford Nanopore sequencing data. The mitochondrial genome size of P. salicina was found to be 508,035 base pair (bp), which is the largest reported in the Rosaceae family to date, and P. salicina was shown to be 63,453 bp longer than sweet cherry (P. avium). The P. salicina mitochondrial genome contained 37 protein-coding genes (PCGs), 3 ribosomal RNA (rRNA) genes, and 16 transfer RNA (tRNA) genes. Two plastid-derived tRNA were identified. We also found two short repeats that captured the nad3 and nad6 genes and resulted in two copies. In addition, nine pairs of repeat sequences were identified as being involved in the mediation of genome recombination. This is crucial for the formation of subgenomic configurations. To characterize RNA editing sites, transcriptome data were used, and we identified 480 RNA editing sites in protein-coding sequences. Among them, the initiation codon of the nad1 gene confirmed that an RNA editing event occurred, and the genomic encoded ACG was edited as AUG in the transcript. Combined with previous reports on the chloroplast genome, our data complemented our understanding of the last part of the organelle genome of plum, which will facilitate our understanding of the evolution of organelle genomes.

Identification and Characterization of a Novel Recombinant Porcine Astrovirus from Pigs in Anhui, China.

Porcine astroviruses (PAstVs) have wide distribution in swine herds worldwide. At present, five porcine astrovirus genotypes have been identified. In this study, using viral metagenomics, a novel PAstV strain (designated as Ahast) was identified in fecal samples from pigs in Anhui of China, and the complete genomic sequence of Ahast was obtained by assembling and PCR amplification. Genomic structural analysis indicated that Ahast had a typical ribosomal frameshifting signal, and some conserve amino acid motifs were also found in virally encoded proteins. Phylogenetic analysis and sequence comparison indicated that this virus belonged to porcine astrovirus genotype 4 (PAstV4), which formed a clade clustered with other PAstV4. Multiple recombinant events were confirmed by recombination analysis and indicated that Ahast was a potential recombinant. Epidemiological investigation indicated that PAstV4 has a 10.7% prevalence in this pig farm. The new recombinant identified in this study will be beneficial to comprehend the origin, genetic diversity, and evolution of porcine astroviruses in Anhui of China.

Insights into the evolutionary history and epidemiological characteristics of the emerging lineage 1 porcine reproductive and respiratory syndrome viruses in China.

The newly emerged lineage 1 porcine reproductive and respiratory syndrome viruses (PRRSVs) (especially the NADC30-like and NADC34-like viruses) have posed a direct threat to the Chinese pig industry since 2013. The phylogenetic, epidemic, and recombinant properties of these viruses have not yet systematically analysed in China. This report presents regular surveillance and field epidemiological studies for PRRSV across China from 2007 to 2019. From over 4,000 detected clinical samples, 70 open reading frame five sequences and four complete genomes of lineage 1 viruses were successfully obtained. Combined with global data, we conducted an extensive and systematic molecular phylogeny analysis using a maximum likelihood tree. The Chinese lineage 1 viruses were clustered, and their temporal and spatial distribution was further explored. Multiple viral introductions of lineage 1 virus from the United States to China were detected, and some became endemic in China. There are three sub-lineage 1 clusters: lineage 1.5 (NADC34-like), lineage 1.6 and New Intro cluster (NADC30-like). These viruses show high genetic diversity and a wide distribution in China, with Henan Province showing the highest diversity. Moreover, Chinese lineage 1 viruses have developed an endemic NADC30-like cluster. The demographic feature of this cluster showed a more or less constant population expansion history with a recent decreasing trend. Moreover, the genome recombination of Chinese lineage 1 with two dominant clusters (Chinese HP-PRRSVs: lineage 8.7 and VR2332-like: lineage 5.1) was frequently detected, both of which have commercial vaccine strains available. Furthermore, recombination hotspots were discovered near NSP9 and ORF2-4 regions of the genome. Overall, these findings provide important insights into the evolution and geographical diversity of Chinese lineage 1 PRRSV. These results will facilitate the development of programmes for the control and prevention of the emerging lineage 1 viruses in China.

Phage-delivered sensitisation with subsequent antibiotic treatment reveals sustained effect against antimicrobial resistant bacteria.

Temperate phages integrated with clustered regularly interspaced short palindromic repeat (CRISPR)/Cas systems have been gaining attention as potential strategies for combating bacteria resistant to antimicrobials. To further advance this technology, phage recombination procedure should be improved, and the bactericidal effect should be examined in detail and compared with conventional lytic phage strategy. The possibility of the emergence of mutational resistance, a phenomenon commonly observed with lytic phage therapy, should be illustrated. Methods: Here, we developed a novel one-step cloning method to fulfil the recombination of CRISPR/Cas9 system within the genome of a new isolated lysogenic Escherichia coli phage. Then, we proposed and developed a phage-delivered resistance eradication with subsequent antibiotic treatment (PRESA) strategy. The removal efficiency and antimicrobial effect of the plasmids were analysed. Long-term antimicrobial effect was evaluated by continued OD600 monitoring for 240 hours to illustrate the potential mutational resistance, compared with the lytic phage strategy. The treatment effect of PRESA was evaluated in vivo by determining bacterial loads in the skin and intestine of infected mice, in contrast with lytic phage therapy. Genome sequencing was performed to identify mutations in bacterial cells treated with phage strategies. Results: Phage-delivered CRISPR targeting efficiently eradicated and blocked the transfer of the antibiotic resistance plasmid. PRESA decreased the bacterial load by over 6- and 5-logs in vitro and in vivo, respectively. Importantly, while lytic phages induced mutational phage resistance at 24 h in vitro and 48 hours in vivo, PRESA demonstrated a constant effect and revealed no resistant mutants. Genes involved in DNA mismatch repair were upregulated in cells undergoing Cas9-based plasmid cleavage, which may reduce the development of mutations. Conclusion: The PRESA strategy for eradicating resistant bacteria showed high bactericidal efficacy and a sustained inhibition effect against resistant bacteria. By restoring the efficacy of low-cost antibiotics, PRESA could be developed as an efficient and economical therapy for infections of antibiotic resistant bacteria.

A Survey of Recent Adenoviral Respiratory Pathogens in Hong Kong Reveals Emergent and Recombinant Human Adenovirus Type 4 (HAdV-E4) Circulating in Civilian Populations.

Human adenovirus type 4 (HAdV-E4), which is intriguingly limited to military populations, causes acute respiratory disease with demonstrated morbidity and mortality implications. This respiratory pathogen contains genome identity with chimpanzee adenoviruses, indicating zoonotic origins. A signature of these "old" HAdV-E4 is the absence of a critical replication motif, NF-I, which is found in all HAdV respiratory pathogens and most HAdVs. However, our recent survey of flu-like disease in children in Hong Kong reveals that the emergent HAdV-E4 pathogens circulating in civilian populations contain NF-I, indicating recombination and reflecting host-adaptation that enables the "new" HAdV-E4 to replicate more efficiently in human cells and foretells more potential HAdV-E4 outbreaks in immune-naïve civilian populations. Special attention should be paid by clinicians to this emergent and recombinant HAdV-E4 circulating in civilian populations.