Identifying SARS-CoV-2-related coronaviruses in Malayan pangolins.

The ongoing outbreak of viral pneumonia in China and across the world is associated with a new coronavirus, SARS-CoV-21. This outbreak has been tentatively associated with a seafood market in Wuhan, China, where the sale of wild animals may be the source of zoonotic infection2. Although bats are probable reservoir hosts for SARS-CoV-2, the identity of any intermediate host that may have facilitated transfer to humans is unknown. Here we report the identification of SARS-CoV-2-related coronaviruses in Malayan pangolins (Manis javanica) seized in anti-smuggling operations in southern China. Metagenomic sequencing identified pangolin-associated coronaviruses that belong to two sub-lineages of SARS-CoV-2-related coronaviruses, including one that exhibits strong similarity in the receptor-binding domain to SARS-CoV-2. The discovery of multiple lineages of pangolin coronavirus and their similarity to SARS-CoV-2 suggests that pangolins should be considered as possible hosts in the emergence of new coronaviruses and should be removed from wet markets to prevent zoonotic transmission.

Fatal swine acute diarrhoea syndrome caused by an HKU2-related coronavirus of bat origin.

Cross-species transmission of viruses from wildlife animal reservoirs poses a marked threat to human and animal health 1 . Bats have been recognized as one of the most important reservoirs for emerging viruses and the transmission of a coronavirus that originated in bats to humans via intermediate hosts was responsible for the high-impact emerging zoonosis, severe acute respiratory syndrome (SARS) 2-10 . Here we provide virological, epidemiological, evolutionary and experimental evidence that a novel HKU2-related bat coronavirus, swine acute diarrhoea syndrome coronavirus (SADS-CoV), is the aetiological agent that was responsible for a large-scale outbreak of fatal disease in pigs in China that has caused the death of 24,693 piglets across four farms. Notably, the outbreak began in Guangdong province in the vicinity of the origin of the SARS pandemic. Furthermore, we identified SADS-related CoVs with 96-98% sequence identity in 9.8% (58 out of 591) of anal swabs collected from bats in Guangdong province during 2013-2016, predominantly in horseshoe bats (Rhinolophus spp.) that are known reservoirs of SARS-related CoVs. We found that there were striking similarities between the SADS and SARS outbreaks in geographical, temporal, ecological and aetiological settings. This study highlights the importance of identifying coronavirus diversity and distribution in bats to mitigate future outbreaks that could threaten livestock, public health and economic growth.

Progress on the quality control technology of next generation sequencing library.

As a key supporting technology in the fields of life sciences and medicine, high-throughput sequencing has developed rapidly and become increasingly mature. The workflow of this technology can be divided into nucleic acid extraction, library construction, sequencing, and data analysis. Among these, library construction is a pivotal step that bridges the previous and subsequent stages. The effectiveness of library construction is contingent on the quality of upstream samples and also impacts the data analysis following sequence data output. The selection and implementation of library construction quality control techniques are crucial for enhancing the reliability of results and reducing errors in sequencing data. This review provides an in-depth discussion of library construction quality control techniques, summarizing and evaluating their principles, advantages and disadvantages, and applicability. It also discusses the selection of relevant technologies in practical application scenarios. The aim is to offer theoretical foundations and references for researchers, disease prevention and control personnel, and others when choosing library quality control techniques, thereby promoting the quality and efficiency of high-throughput sequencing work.

Competitive fitness and homologous recombination of SARS-CoV-2 variants of concern.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants continue to emerge and cocirculate in humans and wild animals. The factors driving the emergence and replacement of novel variants and recombinants remain incompletely understood. Herein, we comprehensively characterized the competitive fitness of SARS-CoV-2 wild type (WT) and three variants of concern (VOCs), Alpha, Beta and Delta, by coinfection and serial passaging assays in different susceptible cells. Deep sequencing analyses revealed cell-specific competitive fitness: the Beta variant showed enhanced replication fitness during serial passage in Caco-2 cells, whereas the WT and Alpha variant showed elevated fitness in Vero E6 cells. Interestingly, a high level of neutralizing antibody sped up competition and completely reshaped the fitness advantages of different variants. More importantly, single clone purification identified a significant proportion of homologous recombinants that emerged during the passage history, and immune pressure reduced the frequency of recombination. Interestingly, a recombination hot region located between nucleotide sites 22,995 and 28,866 of the viral genomes could be identified in most of the detected recombinants. Our study not only profiled the variable competitive fitness of SARS-CoV-2 under different conditions, but also provided direct experimental evidence of homologous recombination between SARS-CoV-2 viruses, as well as a model for investigating SARS-CoV-2 recombination.

Characterization and Genomic Analysis of Bacteriophage vB_KpnM_IME346 Targeting Clinical Klebsiella pneumoniae Strain of the K63 Capsular Type.

A Klebsiella pneumoniae bacteriophage (vB_KpnM_IME346) was isolated from a hospital sewage sample. This bacteriophage specifically infects a clinical K. pneumoniae strain with a K63 capsular polysaccharide structure. The phage genome was evaluated by next-generation sequencing, which revealed a linear double-stranded DNA genome consisting of 49,482 base pairs with a G+C content of 49.1%. The latent period of vB_KpnM_IME346 was shown to be 20 min, and the burst size was 25-30 pfu (plaque-forming units)/infected cell. Transmission electron microscopy and phylogenetic analysis showed that the JD001-like phage belongs to the genus Jedunavirus of the family Myoviridae. The newly isolated vB_KpnM_IME346 shows infectivity in the clinical host K. pneumoniae KP576 strain, indicating that it is a promising alternative to antibacterial agents for removing K. pneumoniae from patients.

Characteristics and complete genome sequence of the virulent Vibrio alginolyticus phage VAP7, isolated in Hainan, China.

A novel Vibrio alginolyticus phage, VAP7, was isolated from seawater collected from Sanya, Hainan province, China. Whole-genome sequencing analysis revealed that phage VAP7 has a linear, double-stranded DNA genome of 144,685 bp with an average G+C content of 41.9% and a high degree of sequence similarity to Vibrio phage VP-1. Annotation results identified 193 open reading frames and one transfer RNA-encoding gene in the phage genome. The morphology and the results of phylogenetic analysis suggest that VAP7 should be classified as a new member of the family Ackermannviridae. Moreover, phage VAP7 grew over a wide pH (5.0-10.0) and temperature (4-40 °C) range. Host-range experiments revealed that VAP7 could infect 31 Vibrio alginolyticus strains. Thus, VAP7 infecting Vibrio alginolyticus strains represents a potential new candidate for use in phage therapy.

A novel freshwater cyanophage vB_MelS-Me-ZS1 infecting bloom-forming cyanobacterium Microcystis elabens.

Blooms of cyanobacteria cause enormous losses in both the economy and environment. Cyanophages are of great potential for fighting blooming cyanobacteria. Research report on cyanophage of bloom-forming cyanobacterium, Microcystis elabens is deficient. vB_MelS-Me-ZS1 (abbreviated as Me-ZS1) was isolated from fresh water by double-layer agar plate method using M. elabens. TEM exhibited that cyanosiphovirus Me-ZS1 has an icosahedral head about 60 nm in diameter, and a noncontractile tail approximately 260 nm. Experimental infection against 15 cyanobacterial strains showed that Me-ZS1 can infect 12 strains across taxonomic orders (Chroococcales, Nostocales and Oscillatoriales). High-throughput sequencing and bioinformatics analysis revealed that Me-ZS1 has a double-stranded DNA genome of 49,665 bp, with a G + C content of 58.22%, and 73 predicted open reading frames (ORFs). BLASTn and ORF comparisons showed that Me-ZS1 shares very low homology with the public sequences, and the phylogenetic tree based on TerL indicated that Me-ZS1 may delegate a novel and genetically distinct clade of Siphoviridae phages. In microcosm experiment, Me-ZS1 represented apparent effect on reducing relative abundance of cyanobacteria, increasing relative abundance of Saprospiraceae and protecting brocade carp (Carassius auratus) in cyanobacterial bloom water. This study isolated and characterized a novel broad-host-range Microcystis phage Me-ZS1 presenting a genetically distinct clade of freshwater cyanophage. The features of cyanophage Me-ZS1 provide a potential solution to the loss caused by cyanobacterial bloom.

Isolation and Characterization of a Novel Bacteriophage Infecting Carbapenem-Resistant Klebsiella pneumoniae.

A novel virulent phage, vB_KpnP_IME337, isolated from a hospital sewage in Beijing, China, that infects carbapenem-resistant Klebsiella pneumoniae KN2 capsular type was identified and characterized. Next-generation sequencing and genome analysis revealed that vB_KpnP_IME337 had a linear double-stranded genome with a length of 44,266 base pairs and G+C content of 53.7%. Fifty-two putative open reading frames were identified, and no transfer RNA-encoding genes were detected. BLASTn analysis revealed that phage vB_KpnP_IME337 had the highest sequence similarity with Klebsiella phage phiBO1E, with genome coverage of 79%. Based on morphology, phage vB_KpnP_IME337 was determined to belong to the family Podoviridae of the order Caudovirales. It was shown that phage vB_KpnP_IME337 had an infection duration of ~ 90 min and 10 min latent period, and a highly specific to host strain. In conclusion, phage vB_KpnP_IME337 may be a promising alternative candidate to antibiotic treatment for controlling diseases caused by drug-resistant K. pneumoniae.

Features of Ebola Virus Disease at the Late Outbreak Stage in Sierra Leone: Clinical, Virological, Immunological, and Evolutionary Analyses.

We performed Ebola virus disease diagnosis and viral load estimation for Ebola cases in Sierra Leone during the late stage of the 2014-2015 outbreak (January-March 2015) and analyzed antibody and cytokine levels and the viral genome sequences. Ebola virus disease was confirmed in 86 of 1001 (9.7%) patients, with an overall case fatality rate of 46.8%. Fatal cases exhibited significantly higher levels of viral loads, cytokines, and chemokines at late stages of infection versus early stage compared with survivors. The viruses converged in a new clade within sublineage 3.2.4, which had a significantly lower case fatality rate.

Complete genome sequence of a novel, virulent Ahjdlikevirus bacteriophage that infects Enterococcus faecium.

A novel virulent bacteriophage named vB_EfaP_IME199 that specifically infects Enterococcus faecium was isolated and characterized. Its optimal multiplicity of infection was 0.01, and it had a 30 minute outbreak period. High-throughput sequencing revealed that the phage has a dsDNA genome of 18,838 bp with 22 open reading frames. The genome has very low homology to all other bacteriophage sequences in the GenBank database. Run-off sequencing experiments confirmed that vB_EfaP_IME199 has short inverted terminal repeats. Phylogenetic analysis indicated that vB_EfaP_IME199 can be taxonomically classified as a new member of the genus Ahjdlikevirus of family Podoviridae.

Complete genome sequence of Menghai flavivirus, a novel insect-specific flavivirus from China.

Menghai flavivirus (MFV) was isolated from Aedes albopictus in Menghai county of Yunnan Province, China, during an arboviruses screening program in August 2010. Whole genome sequencing of MFV was performed using an Ion PGM™ Sequencer. The complete genome of MFV was 10897 nucleotides in length and encoded a polyprotein and fairly interesting flavivirus orf (FIFO). The polyprotein contained three flavivirus structural proteins (C, prM/M and E) and seven nonstructural proteins. Nucleotide BLAST analysis revealed that the MFV genome showed highest similarity to Xishuangbanna Aedes flavivirus, a novel insect-specific flavivirus recently isolated from the same area. These species shared a query cover of 99%, but only 71% identity, while FIFO showed no similarity with any of the published sequences. Genomic and phylogenetic analyses suggested that MFV was a novel species of the genus Flavivirus. Our findings enrich our understanding of the genetics and prevalence of the family Flaviviridae.

Complete genome sequence of Menghai rhabdovirus, a novel mosquito-borne rhabdovirus from China.

Menghai rhabdovirus (MRV) was isolated from Aedes albopictus in Menghai county of Yunnan Province, China, in August 2010. Whole-genome sequencing of MRV was performed using an Ion PGM™ Sequencer. We found that MRV is a single-stranded, negative-sense RNA virus. The complete genome of MRV has 10,744 nt, with short inverted repeat termini, encoding five typical rhabdovirus proteins (N, P, M, G, and L) and an additional small hypothetical protein. Nucleotide BLAST analysis using the BLASTn method showed that the genome sequence most similar to that of MRV is that of Arboretum virus (NC_025393.1), with a Max score of 322, query coverage of 14%, and 66% identity. Genomic and phylogenetic analyses both demonstrated that MRV should be considered a member of a novel species of the family Rhabdoviridae.

Characterization and complete genome sequence analysis of a novel virulent Siphoviridae phage against Staphylococcus aureus isolated from bovine mastitis in Xinjiang, China.

Bovine mastitis is one of the most costly diseases in dairy cows worldwide. It can be caused by over 150 different microorganisms, where Staphylococcus aureus is the most frequently isolated and a major pathogen responsible for heavy economic losses in dairy industry. Although antibiotic therapy is most widely used, alternative treatments are necessary due to the increasing antibiotic resistance. Using phage for pathogen control is a promising tool in the fight against antibiotic resistance. Mainly using high-throughput sequencing, bioinformatics and our proposed phage termini identification method, we have isolated and characterized a novel virulent phage, designated as vB_SauS_IMEP5, from manure collected from dairy farms in Shihezi, Xinjiang, China, for use as a biocontrol agent against Staphylococcus aureus infections. Its latent period was about 30 min and its burst size was approximately 272PFU/cell. Phage vB_SauS_IMEP5 survives in a wide pH range between 3 and 12. A treatment at 70 °C for 20 min can inactive the phage. Morphological analysis of vB_SauS_IMEP5 revealed that phage vB_SauS_IMEP5 morphologically resembles phages in the family Siphoviridae. Among our tested multiplicity of infections (MOIs), the optimal multiplicity of infection (MOI) of this phage was determined to be 0.001, suggesting that phage vB_SauS_IMEP5 has high bacteriolytic potential and good efficiency for reducing bacterial growth. The complete genome of IME-P5 is a 44,677-bp, linear, double-stranded DNA, with a G+C content of 34.26%, containing 69 putative ORFs. The termini of genome were determined with next-generation sequencing data using our previously proposed termini identification method, which suggests that this phage has non-redundant termini with 9nt 3' protruding cohesive ends. The genomic and proteomic characteristics of IMEP5 demonstrate that this phage does not belong to any of the previously recognized Siphoviridae Staphylococcus phage groups, suggesting the creation of a new lineage, thus adding to the knowledge on the diversity of Staphylococcus phages. An N-acetylmuramoyl-L-alanine amidase gene and several conserved genes were predicted, while no virulence or antibiotic resistance genes were identified. This study isolated and characterized a novel S. aureus phage vB_SauS_IMEP5, and our findings suggest that this phage may be potentially utilized as a therapeutic or prophylactic candidate against S.aureus infections.

Complete genome sequence of Xishuangbanna flavivirus, a novel mosquito-specific flavivirus from China.

A new flavivirus, Xishuangbanna flavivirus (XFV), infecting Aedes albopictus mosquitoes in Yunnan Province, China, was isolated and sequenced. The single-stranded RNA genome of 10,884 nt contained two open reading frames (ORFs) encoding the polyprotein and FIFO. The genome had a maximum nucleotide sequence identity of 65 % to Parramatta River virus with coverage of only 27 %. Phylogenetic analysis suggested that this virus is most closely related to recognized classical insect-specific flaviviruses (cISF) and most likely has a similar host range. Sequence comparisons and phylogenetic analysis demonstrated that XFV is a new member of the genus Flavivirus.

A novel termini analysis theory using HTS data alone for the identification of Enterococcus phage EF4-like genome termini.

BACKGROUND: Enterococcus faecalis and Enterococcus faecium are typical enterococcal bacterial pathogens. Antibiotic resistance means that the identification of novel E. faecalis and E. faecium phages against antibiotic-resistant Enterococcus have an important impact on public health. In this study, the E. faecalis phage IME-EF4, E. faecium phage IME-EFm1, and both their hosts were antibiotic resistant. To characterize the genome termini of these two phages, a termini analysis theory was developed to provide a wealth of terminal sequence information directly, using only high-throughput sequencing (HTS) read frequency statistics. RESULTS: The complete genome sequences of phages IME-EF4 and IME-EFm1 were determined, and our termini analysis theory was used to determine the genome termini of these two phages. Results showed 9 bp 3' protruding cohesive ends in both IME-EF4 and IME-EFm1 genomes by analyzing frequencies of HTS reads. For the positive strands of their genomes, the 9 nt 3' protruding cohesive ends are 5'-TCATCACCG-3' (IME-EF4) and 5'-GGGTCAGCG-3' (IME-EFm1). Further experiments confirmed these results. These experiments included mega-primer polymerase chain reaction sequencing, terminal run-off sequencing, and adaptor ligation followed by run-off sequencing. CONCLUSION: Using this termini analysis theory, the termini of two newly isolated antibiotic-resistant Enterococcus phages, IME-EF4 and IME-EFm1, were identified as the byproduct of HTS. Molecular biology experiments confirmed the identification. Because it does not require time-consuming wet lab termini analysis experiments, the termini analysis theory is a fast and easy means of identifying phage DNA genome termini using HTS read frequency statistics alone. It may aid understanding of phage DNA packaging.

Occurrence and reassortment of avian influenza A (H7N9) viruses derived from coinfected birds in China.

UNLABELLED: Over the course of two waves of infection, H7N9 avian influenza A virus has caused 436 human infections and claimed 170 lives in China as of July 2014. To investigate the prevalence and genetic diversity of H7N9, we surveyed avian influenza viruses in poultry in Jiangsu province within the outbreak epicenter. We found frequent occurrence of H7N9/H9N2 coinfection in chickens. Molecular clock phylogenetic analysis confirms coinfection by H7N9/H9N2 viruses and also reveals that the identity of the H7N9 outbreak lineage is confounded by ongoing reassortment between outbreak viruses and diverse H9N2 viruses in domestic birds. Experimental inoculation of a coinfected sample in cell culture yielded two reassortant H7N9 strains with polymerase segments from the original H9N2 strain. Ongoing reassortment between the H7N9 outbreak lineage and diverse H9N2 viruses may generate new strains with the potential to infect humans, highlighting the need for continued viral surveillance in poultry and humans. IMPORTANCE: We found frequent occurrence of H7N9/H9N2 coinfection in chickens. The H7N9 outbreak lineage is confounded by ongoing reassortment between H7N9 and H9N2 viruses. The importance of H9N2 viruses as the source of novel avian influenza virus infections in humans requires continuous attention.

Characterization and Genomic Analysis of Quinolone-Resistant Delftia sp. 670 Isolated from a Patient Who Died from Severe Pneumonia.

Antibiotic-resistant opportunistic pathogens have become a serious concern in recent decades, as they are increasingly responsible for hospital-acquired infections. Here, we describe quinolone-resistant Delftia sp. strain 670, isolated from the sputum of a patient who died from severe pulmonary infection. The draft genome sequence of this strain was obtained by whole-genome shotgun sequencing, and was subjected to comparative genome analysis. Genome analysis revealed that one critical mutation (Ser83Ile in gyrA) might play a decisive role in quinolone resistance. The genome of Delftia sp. strain 670 contains both type II and type VI secretion systems, which were predicted to contribute to the virulence of the strain. Phylogenetic analysis, assimilation tests, and comparative genome analysis indicated that strain 670 differed from the four known Delftia species, suggesting this strain could represent a novel species. Although the study could not determine the strain 670 as the pathogen led to mortality, our findings also presented the pathogenic potential of Delftia species, and the increasing severity of antibiotic resistance among emerging opportunistic pathogens. The whole genome sequencing and comparative analysis improved our understanding of genome evolution in the genus Delftia, and provides the foundation for further study on drug resistance and virulence of Delftia strains.

Isolation and characterization of a novel invertebrate iridovirus from adult Anopheles minimus (AMIV) in China.

An invertebrate iridovirus (designated AMIV) was isolated from adult wild-captured Anopheles minimus mosquitoes in China. AMIV was pathologically and morphologically characterized and sequenced using the Ion Torrent™ sequencing platform. Phylogenetic analysis based on both the major capsid protein and core genes revealed that AMIV differs from all the members of the family Iridoviridae. The AMIV negatively strained virion has a diameter of about 130nm. AMIV contains a linear DNA molecule of 163,023bp, with 39% G+C content and 148 coding sequences. The genome analysis revealed that AMIV genome encodes a high content of replication associated genes including BRO-like genes. This is the ninth complete genome of IIV reported.

Characterization and complete genome sequence analysis of novel bacteriophage IME-EFm1 infecting Enterococcus faecium.

We isolated and characterized a novel virulent bacteriophage, IME-EFm1, specifically infecting multidrug-resistant Enterococcus faecium. IME-EFm1 is morphologically similar to members of the family Siphoviridae. It was found capable of lysing a wide range of our E. faecium collections, including two strains resistant to vancomycin. One-step growth tests revealed the host lysis activity of phage IME-EFm1, with a latent time of 30 min and a large burst size of 116 p.f.u. per cell. These biological characteristics suggested that IME-EFm1 has the potential to be used as a therapeutic agent. The complete genome of IME-EFm1 was 42 597 bp, and was linear, with terminally non-redundant dsDNA and a G+C content of 35.2 mol%. The termini of the phage genome were determined with next-generation sequencing and were further confirmed by nuclease digestion analysis. To our knowledge, this is the first report of a complete genome sequence of a bacteriophage infecting E. faecium. IME-EFm1 exhibited a low similarity to other phages in terms of genome organization and structural protein amino acid sequences. The coding region corresponded to 90.7 % of the genome; 70 putative ORFs were deduced and, of these, 29 could be functionally identified based on their homology to previously characterized proteins. A predicted metallo-ß-lactamase gene was detected in the genome sequence. The identification of an antibiotic resistance gene emphasizes the necessity for complete genome sequencing of a phage to ensure it is free of any undesirable genes before use as a therapeutic agent against bacterial pathogens.

Characterization of the morphology and genome of an Escherichia coli podovirus.

Escherichia coli is an important opportunistic pathogen. It can cause sepsis and severe infection. The application of lytic bacteriophages to treat infectious diseases is an alternative to antibiotics. A lytic Escherichia coli phage, designated IME-EC2, was isolated from hospital sewage. Transmission electron microscopy revealed that IME-EC2 to be a member of the family Podoviridae. It had a 60-nm head and a 15-nm tail. Here, we present the complete genome sequence of this phage, which consists of 41,510 bp with an overall G+C content of 59.2 %. A total of 60 coding sequences (CDS) were identified, and the phage genome does not contain any tRNA genes. Forty percent of the unknown CDSs are unique to IME-EC2. This phage does not show significant similarity to other phages at the DNA level, which suggests that IME-EC2 could be a novel phage. One of the unique features identified in the IME-EC2 genome was a gene coding for a putative colanic-acid-degrading protein, which could allow the phage to degrade bacterial capsule and biofilms. Another unique feature is that IME-EC2 does not contain a terminase small subunit, which suggests that this phage may have a unique packaging mechanism. The present work provides novel information on phages and shows that this lytic phage or its products could be exploited to destroy bacterial biofilms and pathogenic E. coli.