Due to the emergence of multidrug-resistant bacteria, commonly known as “superbugs”, phage therapy for the control of bacterial diseases rose in popularity. In this context, the use of phages for the management of many important bacterial diseases in the aquaculture environment is auspicious. Vibrio harveyi, a well-known and serious bacterial pathogen, is responsible for many disease outbreaks in aquaculture, resulting in huge economic and production losses. We isolated and fully characterized a novel bacteriophage, Vibrio phage Virtus, infecting V. harveyi strain VH2. Vibrio phage Virtus can infect a wide spectrum of Vibrio spp., including strains of V. harveyi, V. owensii, V. campbellii, V. parahaemolyticus, and V. mediterranei. It has a latent period of 40 min with an unusually high burst size of 3200 PFU/cell. Vibrio phage Virtus has a double-stranded DNA of 82,960 base pairs with 127 predicted open reading frames (ORFs). No virulence, antibiotic resistance, or integrase-encoding genes were detected. In vivo phage therapy trials in gilthead seabream, Sparus aurata, larvae demonstrated that Vibrio phage Virtus was able to significantly improve the survival of larvae for five days at a multiplicity of infection (MOI) of 10, which suggests that it can be an excellent candidate for phage therapy.

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Genomic and Biological Profile of a Novel Bacteriophage, Vibrio phage Virtus, Which Improves Survival of Sparus aurata Larvae Challenged with Vibrio harveyi

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

Vibrio alginolyticus is an important pathogen of marine animals and has been the target of phage therapy applications in marine aquaculture for many years. Here, we report the isolation and partial characterization of a novel species of the Siphoviridae family, the Vibrio phage Artemius. The novel phage was species-specific and could only infect strains of V. alginolyticus. It could efficiently reduce the growth of the host bacterium at various multiplicities of infection as assessed by an in vitro lysis assay. It had a genome length of 43,349 base pairs. The complete genome has double-stranded DNA with a G + C content of 43.61%. In total, 57 ORFs were identified, of which 19 were assigned a predicted function. A genomic analysis indicated that Vibrio phage Artemius is lytic and does not harbor genes encoding toxins and antibiotic resistance determinants.

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Vibrio Phage Artemius, a Novel Phage Infecting Vibrio alginolyticus

In the present study, a novel lytic Vibrio parahaemolyticus phage, vB_VpaP_DE10, was isolated from sewage samples collected in Guangzhou city, China. Transmission electron microscopy revealed that phage vB_VpaP_DE10 has an icosahedral head (52.4 ± 2.5 nm) and a short non-contracted tail (21.9 ± 1.0 nm). Phage vB_VpaP_DE10 lysed approximately 31% (8/26) of the antibiotic-resistant V. parahaemolyticus strains tested. A one-step growth curve showed that phage vB_VpaP_DE10 has a relatively long latency time of 25 min and a burst size of ~19 PFU per cell. The genome of phage vB_VpaP_DE10 is a 42,871-bp-long dsDNA molecule with a G + C content of 49.19% and is predicted to contain 46 open reading frames, 26 of which are predicted to be related to functions such as phage structure, packaging, host lysis, and DNA metabolism. Sequence comparisons suggested that vB_VpaP_DE10 is a member of the genus Maculvirus within the family Autographiviridae. Morphological and genomic analysis indicated that vB_VpaP_DE10 is a novel V. parahaemolyticus phage.

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Characterization and Genomic Analysis of Novel Vibrio parahaemolyticus Phage vB_VpaP_DE10

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.

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

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.

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

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.

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

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.

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Fei Wang

Papers

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

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

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

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

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