A Toxicity Screening Approach to Identify Bacteriophage-Encoded Anti-Microbial Proteins

The rapid emergence of antibiotic resistance among many pathogenic bacteria has created a profound need to discover new alternatives to antibiotics. Bacteriophages, the viruses of microbes, express special proteins to overtake the metabolism of the bacterial host they infect, the best known of which are involved in bacterial lysis. However, the functions of majority of bacteriophage encoded gene products are not known, i.e., they represent the hypothetical proteins of unknown function (HPUFs). In the current study we present a phage genomics-based screening approach to identify phage HPUFs with antibacterial activity with a long-term goal to use them as leads to find unknown targets to develop novel antibacterial compounds. The screening assay is based on the inhibition of bacterial growth when a toxic gene is expression-cloned into a plasmid vector. It utilizes an optimized plating assay producing a significant difference in the number of transformants after ligation of the toxic and non-toxic genes into a cloning vector. The screening assay was first tested and optimized using several known toxic and non-toxic genes. Then, it was applied to screen 94 HPUFs of bacteriophage φR1-RT, and identified four HPUFs that were toxic to Escherichia coli. This optimized assay is in principle useful in the search for bactericidal proteins of any phage, and also opens new possibilities to understanding the strategies bacteriophages use to overtake bacterial hosts

Human Protoparvovirus DNA and IgG in Children and Adults with and without Respiratory or Gastrointestinal Infections

Abstract: Three human protoparvoviruses, bufavirus (BuV), tusavirus (TuV) and cutavirus (CuV), have recently been discovered in diarrheal stool. BuV has been associated with diarrhea and CuV with cutaneous T-cell lymphoma, but there are hardly any data for TuV or CuV in stool or respiratory samples. Hence, using qPCR and IgG enzyme immunoassays, we analyzed 1072 stool, 316 respiratory and 445 serum or plasma samples from 1098 patients with and without gastroenteritis (GE) or respiratory-tract infections (RTI) from Finland, Latvia and Malawi. The overall CuV-DNA prevalences in stool samples ranged between 0–6.1% among our six patient cohorts. In Finland, CuV DNA was significantly more prevalent in GE patients above rather than below 60 years of age (5.1% vs 0.2%). CuV DNA was more prevalent in stools among Latvian and Malawian children compared with Finnish children. In 10/11 CuV DNA-positive adults and 4/6 CuV DNA-positive children with GE, no known causal pathogens were detected. Interestingly, for the first time, CuV DNA was observed in two nasopharyngeal aspirates from children with RTI and the rare TuV in diarrheal stools of two adults. Our results provide new insights on the occurrence of human protoparvoviruses in GE and RTI in different countries.Peer reviewe

Human Protoparvovirus DNA and IgG in Children and Adults with and without Respiratory or Gastrointestinal Infections

Abstract: Three human protoparvoviruses, bufavirus (BuV), tusavirus (TuV) and cutavirus (CuV), have recently been discovered in diarrheal stool. BuV has been associated with diarrhea and CuV with cutaneous T-cell lymphoma, but there are hardly any data for TuV or CuV in stool or respiratory samples. Hence, using qPCR and IgG enzyme immunoassays, we analyzed 1072 stool, 316 respiratory and 445 serum or plasma samples from 1098 patients with and without gastroenteritis (GE) or respiratory-tract infections (RTI) from Finland, Latvia and Malawi. The overall CuV-DNA prevalences in stool samples ranged between 0–6.1% among our six patient cohorts. In Finland, CuV DNA was significantly more prevalent in GE patients above rather than below 60 years of age (5.1% vs 0.2%). CuV DNA was more prevalent in stools among Latvian and Malawian children compared with Finnish children. In 10/11 CuV DNA-positive adults and 4/6 CuV DNA-positive children with GE, no known causal pathogens were detected. Interestingly, for the first time, CuV DNA was observed in two nasopharyngeal aspirates from children with RTI and the rare TuV in diarrheal stools of two adults. Our results provide new insights on the occurrence of human protoparvoviruses in GE and RTI in different countries.Peer reviewe

Global Distribution of Human Protoparvoviruses

Development of next-generation sequencing and metagenomics has revolutionized detection of novel viruses. Among these viruses are 3 human protoparvoviruses: bufavirus, tusavirus, and cutavirus. These viruses have been detected in feces of children with diarrhea. In addition, cutavirus has been detected in skin biopsy specimens of cutaneous T-cell lymphoma patients in France and in 1 melanoma patient in Denmark. We studied seroprevalences of IgG against bufavirus, tusavirus, and cutavirus in various populations (n = 840), and found a striking geographic difference in prevalence of bufavirus IgG. Although prevalence was low in adult populations in Finland (1.9%) and the United States (3.6%), bufavirus IgG was highly prevalent in populations in Iraq (84.8%), Iran (56.1%), and Kenya (72.3%). Conversely, cutavirus IgG showed evenly low prevalences (0%-5.6%) in all cohorts, and tusavirus IgG was not detected. These results provide new insights on the global distribution and endemic areas of protoparvoviruses.Peer reviewe

Global Distribution of Human Protoparvoviruses

Development of next-generation sequencing and metagenomics has revolutionized detection of novel viruses. Among these viruses are 3 human protoparvoviruses: bufavirus, tusavirus, and cutavirus. These viruses have been detected in feces of children with diarrhea. In addition, cutavirus has been detected in skin biopsy specimens of cutaneous T-cell lymphoma patients in France and in 1 melanoma patient in Denmark. We studied seroprevalences of IgG against bufavirus, tusavirus, and cutavirus in various populations (n = 840), and found a striking geographic difference in prevalence of bufavirus IgG. Although prevalence was low in adult populations in Finland (1.9%) and the United States (3.6%), bufavirus IgG was highly prevalent in populations in Iraq (84.8%), Iran (56.1%), and Kenya (72.3%). Conversely, cutavirus IgG showed evenly low prevalences (0%-5.6%) in all cohorts, and tusavirus IgG was not detected. These results provide new insights on the global distribution and endemic areas of protoparvoviruses

Human Protoparvovirus DNA and IgG in Children and Adults with and without Respiratory or Gastrointestinal Infections

Three human protoparvoviruses, bufavirus (BuV), tusavirus (TuV) and cutavirus (CuV), have recently been discovered in diarrheal stool. BuV has been associated with diarrhea and CuV with cutaneous T-cell lymphoma, but there are hardly any data for TuV or CuV in stool or respiratory samples. Hence, using qPCR and IgG enzyme immunoassays, we analyzed 1072 stool, 316 respiratory and 445 serum or plasma samples from 1098 patients with and without gastroenteritis (GE) or respiratory-tract infections (RTI) from Finland, Latvia and Malawi. The overall CuV-DNA prevalences in stool samples ranged between 0-6.1% among our six patient cohorts. In Finland, CuV DNA was significantly more prevalent in GE patients above rather than below 60 years of age (5.1% vs 0.2%). CuV DNA was more prevalent in stools among Latvian and Malawian children compared with Finnish children. In 10/11 CuV DNA-positive adults and 4/6 CuV DNA-positive children with GE, no known causal pathogens were detected. Interestingly, for the first time, CuV DNA was observed in two nasopharyngeal aspirates from children with RTI and the rare TuV in diarrheal stools of two adults. Our results provide new insights on the occurrence of human protoparvoviruses in GE and RTI in different countries

Identification of bacteriophage ϕR1-RT encoded toxic gene products as leads for new antibacterials

The rapid emergence of antibiotic resistance among many pathogenic bacteria has created a profound need to discover new alternatives to antibiotics. Bacteriophages are viruses which infect bacteria and are able to produce special proteins involved in bacterial lysis. However, for many bacteriophage-encoded gene products, the function is not known, i.e., hypothetical proteins of unknown function (HPUFs). Screening these proteins likely identifies a rich source of leads that will help in the development of novel antibacterial compounds. The current study presents two phage genomics-based screening approaches to identify phage HPUFs with antibacterial activity. Both screening assays are based on inhibition of bacterial growth when a toxic gene is expression cloned into a plasmid vector. The first approach was a luxAB/luxCDE -based luminescence screening assay. The luxCDE genes encoding the luciferase substrate producing enzymes were integrated into an Escherichia coli strain genome as a transcriptional fusion. Also, a vector carrying the luxAB genes, encoding the luciferase enzyme, and a cloning site for the phage HPUF genes, was constructed. Ligation of a toxic gene into the vector would result in few or rare transformants after electroporation while ligation of a non-toxic gene would result in large number of transformants, and the difference in number of transformants will be reflected in the amount of bioluminescence after electroporation. The proof of concept of the approach was verified using the control genes g150 (a structural, thus a non-toxic gene of phage R1-RT) and regB (a known toxic gene of phage T4). The results demonstrated a significant difference in Relative Luminescence Units (RLU) between the g150 and regB electroporation mixtures. The second screening approach was an optimized plating assay producing a significant difference in the number of transformants after ligation of the toxic and non-toxic genes into a cloning vector. This assay was tested and optimized with several known control toxic and non-toxic genes. Using the plating assay approach, in the current study, ninety-four R1-RT HPUFs were screened and ten of them showed toxicity in E. coli. In future, the identified toxic HPUFs of R1-RT could be purified and characterized to identify their bacterial targets. Further, both of these screening assays can be used to screen among HPUFs of other phages, and this should allow the discovery of a wide variety of putative inhibitors for the control of current and emerging bacterial pathogens

A Toxicity Screening Approach to Identify Bacteriophage-Encoded Anti-Microbial Proteins

The rapid emergence of antibiotic resistance among many pathogenic bacteria has created a profound need to discover new alternatives to antibiotics. Bacteriophages, the viruses of microbes, express special proteins to overtake the metabolism of the bacterial host they infect, the best known of which are involved in bacterial lysis. However, the functions of majority of bacteriophage encoded gene products are not known, i.e., they represent the hypothetical proteins of unknown function (HPUFs). In the current study we present a phage genomics-based screening approach to identify phage HPUFs with antibacterial activity with a long-term goal to use them as leads to find unknown targets to develop novel antibacterial compounds. The screening assay is based on the inhibition of bacterial growth when a toxic gene is expression-cloned into a plasmid vector. It utilizes an optimized plating assay producing a significant difference in the number of transformants after ligation of the toxic and non-toxic genes into a cloning vector. The screening assay was first tested and optimized using several known toxic and non-toxic genes. Then, it was applied to screen 94 HPUFs of bacteriophage φR1-RT, and identified four HPUFs that were toxic to Escherichia coli. This optimized assay is in principle useful in the search for bactericidal proteins of any phage, and also opens new possibilities to understanding the strategies bacteriophages use to overtake bacterial hosts

Significant association of cutavirus with parapsoriasis en plaques: high prevalence both in skin swab and biopsy samples

Cutavirus (CuV) is associated with cutaneous T-cell lymphoma (CTCL), of which parapsoriasis is a precursor. Our study reveals a significantly higher CuV-DNA prevalence in skin-swabs of parapsoriasis patients (6/13, 46.2%), compared to those of healthy adults (1/51, 1.96%). Eight patients (8/12, 66.7%) had CuV-DNA in biopsied skin, and four developed CTCL.Peer reviewe

Human Protoparvovirus DNA and IgG in Children and Adults with and without Respiratory or Gastrointestinal Infections

Three human protoparvoviruses, bufavirus (BuV), tusavirus (TuV) and cutavirus (CuV), have recently been discovered in diarrheal stool. BuV has been associated with diarrhea and CuV with cutaneous T-cell lymphoma, but there are hardly any data for TuV or CuV in stool or respiratory samples. Hence, using qPCR and IgG enzyme immunoassays, we analyzed 1072 stool, 316 respiratory and 445 serum or plasma samples from 1098 patients with and without gastroenteritis (GE) or respiratory-tract infections (RTI) from Finland, Latvia and Malawi. The overall CuV-DNA prevalences in stool samples ranged between 0–6.1% among our six patient cohorts. In Finland, CuV DNA was significantly more prevalent in GE patients above rather than below 60 years of age (5.1% vs 0.2%). CuV DNA was more prevalent in stools among Latvian and Malawian children compared with Finnish children. In 10/11 CuV DNA-positive adults and 4/6 CuV DNA-positive children with GE, no known causal pathogens were detected. Interestingly, for the first time, CuV DNA was observed in two nasopharyngeal aspirates from children with RTI and the rare TuV in diarrheal stools of two adults. Our results provide new insights on the occurrence of human protoparvoviruses in GE and RTI in different countries