A novel virus in the family Marnaviridae as a potential pathogen of Penaeus vannamei glass post-larvae disease.

As an aquatic animal of great commercial relevance, Penaeus vannamei is currently the dominant species of cultured shrimp in China and many other countries worldwide. In recent years, the outbreak of glass post-larvae disease (GPD), which accounts for more than 90% of the mortality of shrimp seedlings in serious cases, in many regions of China has caused significant losses and threatened the sustainability of the aquaculture industry and the economy. It is extremely urgent to determine the infectious agent of GPD in P. vannamei. In this work, we performed metagenomic sequencing of glass post-larvae collected from diseased prawns in Tangshan Hebei, where GPD broke out recently. An evolutionary tree was constructed by MEGA 7 to understand the evolutionary history and relationship of the pathogen genome. A novel virus in the family Marnaviridae was first identified in P. vannamei suffering from GPD, and we tentatively named this virus Baishivirus (GenBank: ON550424). The identified pathogen was validated according to Koch's rule with a pathogenic challenge assay and reverse transcription-polymerase chain reaction. There was only 8% query coverage with 64.96% identity in the Baishivirus genome when compared with its most closely related genome sequence of Wenzhou picorna-like virus 21 reported in 2016. Baishivirus genomic RNA is 9.895 kb in length and encodes three potential open reading frames (ORFs). The identification of Baishivirus in P. vannamei enriches the family Marnaviridae and potentially provides a new candidate to study and prevent GPD in the aquaculture industry.

Novel Aeromonas Phage Ahy-Yong1 and Its Protective Effects against Aeromonas hydrophila in Brocade Carp (Cyprinus aka Koi).

Aeromonas hydrophila is a zoonotic pathogen and an important fish pathogen. A new lytic phage, Ahy-yong1, against multi-antibiotic-resistant pathogen A. hydrophila was isolated, identified, and tentatively used in therapy. Ahy-yong1 possesses a head of approximately 66 nm in diameter and a short tail of approximately 26 nm in length and 32 nm in width. Its complete dsDNA genome is 43,374 bp with a G + C content of 59.4%, containing 52 predicted opening reading frames (ORFs). Taxonomic analysis indicated Ahy-yong1 as a new species of the Ahphunavirus genus of the Autographiviridae family of the Caudoviricetes class. Ahy-yong1 was active only against its indicator host strain among the 35 strains tested. It is stable at 30-40 °C and at pH 2-12. Aeromonas phage Ahy-yong1 revealed an effective biofilm removal capacity and an obvious protective effect in brocade carp (Cyprinus aka Koi). The average cumulative mortality for the brocade carp in the blank groups intraperitoneally injected with PBS was 1.7% ± 2.4%;for the control groups treated with A. hydrophila (108 CFU/fish) via intraperitoneal injection, it was 100.00%;and for the test group I, successively treated with A. hydrophila (108 CFU/fish) and Aeromonas phage Ahy-yong1 (107 PFU/fish) via intraperitoneal injection witha time interval of 2 hours, it was only 43.4% ± 4.7%. Furthermore, the cumulative mortality of the test group II, successively treated with Aeromonas phage Ahy-yong1 (107 PFU/fish) and A. hydrophila (108 CFU/fish), was only 20.0% ± 8.2%, and that of the test group III, simultaneously treated with Aeromonas phage Ahy-yong1 (107 PFU/fish) and A. hydrophila (108 CFU/fish), was only 30.0% ± 8.2%. The results demonstrated that phage Ahy-yong1 was very effective in the therapies against A. hydrophila A18, prophylaxis was more effective than rescue, and earlier treatment was better for the reduction of mortality. This study enriches knowledge about Aeromonas phages.

A Novel Freshwater Cyanophage Mae-Yong1326-1 Infecting Bloom-Forming Cyanobacterium Microcystis aeruginosa.

Microcystis aeruginosa is a major harmful cyanobacterium causing water bloom worldwide. Cyanophage has been proposed as a promising tool for cyanobacterial bloom. In this study, M. aeruginosa FACHB-1326 was used as an indicator host to isolate cyanophage from Lake Taihu. The isolated Microcystis cyanophage Mae-Yong1326-1 has an elliptical head of about 47 nm in diameter and a slender flexible tail of about 340 nm in length. Mae-Yong1326-1 could lyse cyanobacterial strains across three orders (Chroococcales, Nostocales, and Oscillatoriales) in the host range experiments. Mae-Yong1326-1 was stable in stability tests, maintaining high titers at 0-40 °C and at a wide pH range of 3-12. Mae-Yong 1326-1 has a burst size of 329 PFU/cell, which is much larger than the reported Microcystis cyanophages so far. The complete genome of Mae-Yong1326-1 is a double-stranded DNA of 48, 822 bp, with a G + C content of 71.80% and long direct terminal repeats (DTR) of 366 bp, containing 57 predicted ORFs. No Mae-Yong1326-1 ORF was found to be associated with virulence factor or antibiotic resistance. PASC scanning illustrated that the highest nucleotide sequence similarity between Mae-Yong1326-1 and all known phages in databases was only 17.75%, less than 70% (the threshold to define a genus), which indicates that Mae-Yong1326-1 belongs to an unknown new genus. In the proteomic tree based on genome-wide sequence similarities, Mae-Yong1326-1 distantly clusters with three unclassified Microcystis cyanophages (MinS1, Mwe-Yong1112-1, and Mwes-Yong2). These four Microcystis cyanophages form a monophyletic clade, which separates at a node from the other clade formed by two independent families (Zierdtviridae and Orlajensenviridae) of Caudoviricetes class. We propose to establish a new family to harbor the Microcystis cyanophages Mae-Yong1326-1, MinS1, Mwe-Yong1112-1, and Mwes-Yong2. This study enriched the understanding of freshwater cyanophages.

Genome sequence of the novel freshwater Microcystis cyanophage Mwe-Yong1112-1.

The freshwater cyanophage Mwe-Yong1112-1 was isolated using Microcystis wesenbergii as a host and found to have an icosahedral head, about 45 nm in diameter, and a flexible tail, approximately 133 nm in length and 4.5 nm in width. The complete genome of the cyanophage is 39,679 bp in length with a G+C content of 66.6%. Mwe-Yong1112-1 shared the highest pairwise average nucleotide identity (ANI) value of 67.7% (below the ≥95% boundary to define a species) and the highest nucleotide sequence similarity of 17.48% (below the >70% boundary to define a genus) with the most closely related phage. In a proteomic tree, Mwe-Yong1112-1 and three unclassified phages formed a monophyletic clade between the families Saparoviridae and Pyrstoviridae, but Mwe-Yong1112-1 occupied a separate branch from the other three phages, suggesting that it represents a new evolutionary lineage. This study enriches the available information about freshwater cyanophages.

Genomic analysis of a novel active prophage of Hafnia paralvei.

Little is known about the prophages in Hafniaceae bacteria. A novel Hafnia phage, yong2, was induced from Hafnia paralvei by treatment with mitomycin C. The phage has an elliptical head with dimensions of approximately 45 × 38 nm and a long noncontractile tail of approximately 157 × 4 nm. The complete genome of Hafnia phage yong2 is a 39,546-bp double-stranded DNA with a G+C content of 49.9%, containing 59 open reading frames (ORFs) and having at least one fixed terminus (GGGGCAGCGACA). In phylogenetic analysis, Hafnia phage yong2 clustered with four predicted Hafnia prophages and one predicted Enterobacteriaceae prophage. These prophages and members of the family Drexlerviridae together formed two distinct subclades nested within a clade, suggesting the existence of a novel class of prophages with conserved sequences and a unique evolutionary status not yet studied before in Hafniaceae and Enterobacteriaceae bacteria.

A Novel Freshwater Cyanophage, Mae-Yong924-1, Reveals a New Family.

Cyanobacterial blooms are a worldwide ecological issue. Cyanophages are aquatic viruses specifically infecting cyanobacteria. Little is known about freshwater cyanophages. In this study, a freshwater cyanophage, Mae-Yong924-1, was isolated by the double-layer agar plate method using Microcystis aeruginosa FACHB-924 as an indicator host. Mae-Yong924-1 has several unusual characteristics: a unique shape, cross-taxonomic order infectivity and a very unique genome sequence. Mae-Yong924-1 contains a nearly spherical head of about 100 nm in diameter. The tail or tail-like structure (approximately 40 nm in length) is like the tassel of a round Chinese lantern. It could lyse six diverse cyanobacteria strains across three orders including Chroococcales, Nostocales and Oscillatoriales. The genome of the cyanophage is 40,325 bp in length, with a G + C content of 48.32%, and 59 predicted open reading frames (ORFs), only 12 (20%) of which were functionally annotated. Both BLASTn and BLASTx scanning resulted in "No significant similarity found", i.e., the Mae-Yong924-1 genome shared extremely low homology with sequences in NCBI databases. Mae-Yong924-1 formed a root node alone and monopolized a root branch in the proteomic tree based on genome-wide sequence similarities. The results suggest that Mae-Yong924-1 may reveal a new unknown family apparently distinct from other viruses.

Induction and Genomic Analysis of a Lysogenic Phage of Hafnia paralvei.

Hafnia paralvei is a bacterium that can cause zoonoses. No research has been reported on H. paralvei prophage. In this study, a Hafnia phage yong1 was induced from pathogenic H. paralvei LY-23 by mitomycin C. The phage showed a Myoviridae-like morphology having a hexagonal head of approximately 65 nm in diameter and a contractile tail of approximately 95 nm in length and 17 nm in width. Its genome was sequenced by using the Illumina Miseq platform. The complete genome of Hafnia phage yong1 is 43,329 bp with a G + C content of 47.65%. BLASTn analysis revealed that Hafnia phage yong1 had the highest sequence similarity with the predicted prophages of Enterobacter chengduensis strain WCHECl-C4 = WCHECh050004 recovered from a human blood sample and Escherichia coli strain L103-2 recovered from a goose farm in China. Hafnia phage yong1 contains a tRNA gene and 76 predicted open reading frames, 33 of which were annotated. Gene strings similar to the bacteriophage λ cro-cI-rexA-rexB operon conferring Imm and Rex to lysogenic cells were found in Hafnia phage yong1 genome. Hafnia phage yong1 is the first Myoviridae-like phage found to contain such contiguous genes. Hafnia phage yong1 formed an independent branch between two families, Chaseviridae and Drexlerviridae, in the Proteomic tree.

Isolation, characterization and biocontrol efficacy of a T4-like phage virulent to multidrug-resistant Enterobacter hormaechei.

Enterobacter hormaechei is an important emerging pathogen, often exhibiting resistance to multiple clinically important antibiotics. In this study, E. hormaechei was found, for the first time, to be lethal to fish. Bacteriophages are considered potential treatments for bacterial infections. The lytic phage vB_EhoM-IME523 (abbreviated 'IME523') infecting multidrug-resistant E. hormaechei was isolated from hospital sewage. IME523 exhibits T4-like morphology, including a prolate icosahedral head 110 ± 1.89 nm (mean ± SD) long and 82 ± 0.75 nm wide, and a contractile tail of ca. 110 ± 0.91 nm in length. The complete genome length of phage IME523 is 172763 bp, with a G + C content of 39.97%. The whole genome sequence of IME523 has a 93.10% average nucleotide identity (ANI) and a 53.3% in silico DNA-DNA hybridization (isDDH) value with the closest-related Enterobacter phage vB_EclM_CIP9 ('CIP9'). ANI and isDDH values between IME523 and other phages were lower than 78 and 22%, respectively. IME523 and CIP9 formed a monophyletic branch in a phylogenetic tree based on the terminase large subunit, DNA polymerase protein and whole genome phylogenetic analysis. Results suggest that IME523 is a novel species in the subfamily Tevenvirinae and forms a novel genus together with CIP9. No IME523 open reading frame was found to be associated with virulence factors or antibiotic resistance genes. IME523 showed promising protection to zebrafish and brocade carp against E. hormaechei challenge.

First Characterization of a Hafnia Phage Reveals Extraordinarily Large Burst Size and Unusual Plaque Polymorphism.

A unique lytic phage infecting Hafnia paralvei was isolated and identified. Hafnia phage Ca belongs to the family Autographiviridae, possessing an icosahedral head with a diameter of 55 nm and a short non-contractile tail. Unusually, the burst size of Hafnia phage Ca of 10,292 ± 1,097 plaque-forming units (PFUs)/cell is much larger than other dsDNA phages reported before. Compared to the genome of the related phage, Hafnia phage Ca genome contains extra genes including DNA mimic ocr, dGTP triphosphohydrolase inhibitor, endonuclease, endonuclease VII, and HNH homing endonuclease gene. Extraordinarily, the phage developed different sizes of plaques when a single plaque was picked out and inoculated on a double-layer Luria broth agar plate with its host. Furthermore, varied packaging tightness for the tails of Hafnia phage Ca was observed (tail length: 4.35-45.92 nm). Most of the tails appeared to be like a cone with appendages, some were dot-like, bun-like, table tennis racket handle-like, and ponytail-like. Although the complete genome of Hafnia phage Ca is 40,286 bp, an incomplete genome with a deletion of a 397-bp fragment, containing one ORF predicted as HNH homing endonuclease gene (HEG), was also found by high throughput sequencing. Most of the genome of the virus particles in large plaques is complete (>98%), while most of the genome of the virus particles in small plaques is incomplete (>98%), and the abundance of both of them in medium-sized plaques is similar (complete, 40%; incomplete, 60%). In an experiment to see if the phage could be protective to brocade carps intramuscularly injected with H. paralvei LY-23 and phage Ca, the protection rate of Hafnia phage Ca to brocade carp (Cyprinus aka Koi) against H. paralvei was 33.38% (0.01 < p < 0.05). This study highlights some new insights into the peculiar biological and genomic characteristics of phage.