A broadly cross-reactive monoclonal antibody against hepatitis E virus capsid antigen.

To generate a hepatitis E virus (HEV) genotype 3 (HEV-3)-specific monoclonal antibody (mAb), the Escherichia coli-expressed carboxy-terminal part of its capsid protein was used to immunise BALB/c mice. The immunisation resulted in the induction of HEV-specific antibodies of high titre. The mAb G117-AA4 of IgG1 isotype was obtained showing a strong reactivity with the homologous E. coli, but also yeast-expressed capsid protein of HEV-3. The mAb strongly cross-reacted with ratHEV capsid protein derivatives produced in both expression systems and weaker with an E. coli-expressed batHEV capsid protein fragment. In addition, the mAb reacted with capsid protein derivatives of genotypes HEV-2 and HEV-4 and common vole hepatitis E virus (cvHEV), produced by the cell-free synthesis in Chinese hamster ovary (CHO) and Spodoptera frugiperda (Sf21) cell lysates. Western blot and line blot reactivity of the mAb with capsid protein derivatives of HEV-1 to HEV-4, cvHEV, ratHEV and batHEV suggested a linear epitope. Use of truncated derivatives of ratHEV capsid protein in ELISA, Western blot, and a Pepscan analysis allowed to map the epitope within a partially surface-exposed region with the amino acid sequence LYTSV. The mAb was also shown to bind to human patient-derived HEV-3 from infected cell culture and to hare HEV-3 and camel HEV-7 capsid proteins from transfected cells by immunofluorescence assay. The novel mAb may serve as a useful tool for further investigations on the pathogenesis of HEV infections and might be used for diagnostic purposes. KEY POINTS: • The antibody showed cross-reactivity with capsid proteins of different hepeviruses. • The linear epitope of the antibody was mapped in a partially surface-exposed region. • The antibody detected native HEV-3 antigen in infected mammalian cells.

Characterization of the lytic archaeal virus Drs3 infecting Methanobacterium formicicum.

Viruses are ubiquitous in the biosphere and greatly affect the hosts they infect. It is generally accepted that members of every microbial taxon are susceptible to at least one virus, and a plethora of bacterial viruses are known. In contrast, knowledge of the archaeal virosphere is still limited. Here, a novel lytic archaeal virus is described, designated "Drs3", as well as its host, Methanobacterium formicicum strain Khl10. This hydrogenotrophic methanogenic archaeon and its virus were isolated from the anaerobic digester of an experimental biogas plant in Germany. The tailed virus has an icosahedral head with a diameter of approximately 60 nm and a long non-contractile tail of approximately 230 nm. These structural observations suggest that the new isolate belongs to the family Siphoviridae, but it could not be assigned to an existing genus. Lysis of the host Khl10 was observed 40-44 h after infection. Lysis of the type strain Methanobacterium formicicum DSMZ 1535 was not observed in the presence of Drs3, pointing towards resistance in the type strain or a rather narrow host range of this newly isolated archaeal virus. The complete 37-kb linear dsDNA genome of Drs3 contains 39 open reading frames, only 12 of which show similarity to genes with predicted functions.

Generation of an Avian-Mammalian Rotavirus Reassortant by Using a Helper Virus-Dependent Reverse Genetics System.

UNLABELLED: The genetic diversity of rotavirus A (RVA) strains is facilitated in part by genetic reassortment. Although this process of genome segment exchange has been reported frequently among mammalian RVAs, it remained unknown if mammalian RVAs also could package genome segments from avian RVA strains. We generated a simian RVA strain SA11 reassortant containing the VP4 gene of chicken RVA strain 02V0002G3. To achieve this, we transfected BSR5/T7 cells with a T7 polymerase-driven VP4-encoding plasmid, infected the cells with a temperature-sensitive SA11 VP4 mutant, and selected the recombinant virus by increasing the temperature. The reassortant virus could be stably passaged and exhibited cytopathic effects in MA-104 cells, but it replicated less efficiently than both parental viruses. Our results show that avian and mammalian rotaviruses can exchange genome segments, resulting in replication-competent reassortants with new genomic and antigenic features. IMPORTANCE: This study shows that rotaviruses of mammals can package genome segments from rotaviruses of birds. The genetic diversity of rotaviruses could be broadened by this process, which might be important for their antigenic variability. The reverse genetics system applied in the study could be useful for targeted generation and subsequent characterization of distinct rotavirus reassortant strains.

Campylobacter group II phage CP21 is the prototype of a new subgroup revealing a distinct modular genome organization and host specificity.

BACKGROUND: The application of phages is a promising tool to reduce the number of Campylobacter along the food chain. Besides the efficacy against a broad range of strains, phages have to be safe in terms of their genomes. Thus far, no genes with pathogenic potential (e.g., genes encoding virulence factors) have been detected in Campylobacter phages. However, preliminary studies suggested that the genomes of group II phages may be diverse and prone to genomic rearrangements. RESULTS: We determined and analysed the genomic sequence (182,761 bp) of group II phage CP21 that is closely related to the already characterized group II phages CP220 and CPt10. The genomes of these phages are comprised of four modules separated by very similar repeat regions, some of which harbouring open reading frames (ORFs). Though, the arrangement of the modules and the location of some ORFs on the genomes are different in CP21 and in CP220/CPt10. In this work, a PCR system was established to study the modular genome organization of other group II phages demonstrating that they belong to different subgroups of the CP220-like virus genus, the prototypes of which are CP21 and CP220. The subgroups revealed different restriction patterns and, interestingly enough, also distinct host specificities, tail fiber proteins and tRNA genes. We additionally analysed the genome of group II phage vB_CcoM-IBB_35 (IBB_35) for which to date only five individual contigs could be determined. We show that the contigs represent modules linked by long repeat regions enclosing some yet not identified ORFs (e.g., for a head completion protein). The data suggest that IBB_35 is a member of the CP220 subgroup. CONCLUSION: Campylobacter group II phages are diverse regarding their genome organization. Since all hitherto characterized group II phages contain numerous genes for transposases and homing endonucleases as well as similar repeat regions, it cannot be excluded that these phages are genetically unstable. To answer this question, further experiments and sequencing of more group II phages should be performed.

Genetic characterization of German Mycobacterium avium strains isolated from different hosts and specimens by multilocus sequence typing.

Infections caused by Mycobacterium avium and its subspecies are reported as emerging disease in many countries worldwide. In our study we applied the multilocus sequence typing technology to 98 German M. avium strains originating from different hosts and specimens to examine the degree of the genetic diversity. By MLST, 80% of strains were identified as subspecies 'M. avium hominissuis', and 20% as subspecies M. avium avium/M. avium silvaticum. Distinctly different MLST profiles were identified for both subspecies. Based on the analysis of 4 and 5 loci, 87 and 106 SNPs and 1 codon deletion could be detected, respectively, resulting in 40 different strain profiles. Twelve out of these have recently been described for strains coming from different countries, yet in our study, additional new strain profiles (n=28) were found. The high degree of diversity within 'M. avium subsp. hominissuis' as well as the relatedness of human, porcine and environmental strains could be confirmed by IS1245 RFLP fingerprinting. The detection of ISMav6 and hsp65 code 15 in one adult patient strain being positive for IS901, but displaying 'M. avium subsp. hominissuis' MLST profile revealed that PCR for detection of IS901 is not a definitive proof of M. avium subsp. avium/M. avium subsp. silvaticum.

Hepatitis E virus in pork liver sausage, France.

We investigated viability of hepatitis E virus (HEV) identified in contaminated pork liver sausages obtained from France. HEV replication was demonstrated in 1 of 4 samples by using a 3-dimensional cell culture system. The risk for human infection with HEV by consumption of these sausages should be considered to be high.

Identification of an avian group A rotavirus containing a novel VP4 gene with a close relationship to those of mammalian rotaviruses.

Group A rotaviruses (RVAs) are an important cause of diarrhoeal illness in humans, as well as in mammalian and avian animal species. Previous sequence analyses indicated that avian RVAs are related only distantly to mammalian RVAs. Here, the complete genomes of RVA strain 03V0002E10 from turkey (Meleagris gallopavo) and RVA strain 10V0112H5 from pheasant (Phasianus colchicus) were analysed using a combination of 454 deep sequencing and Sanger sequencing technologies. An adenine-rich insertion similar to that found in the chicken RVA strain 02V0002G3, but considerably shorter, was found in the 3' NCR of the NSP1 gene of the pheasant strain. Most genome segments of both strains were related closely to those of avian RVAs. The novel genotype N10 was assigned to the NSP2 gene of the pheasant RVA, which is related most closely to genotype N6 found in avian RVAs. However, this virus contains a VP4 gene of the novel genotype P[37], which is related most closely to RVAs from pigs, dogs and humans. This strain either may represent an avian/mammalian rotavirus reassortant, or it carries an unusual avian rotavirus VP4 gene, thereby broadening the potential genetic and antigenic variability among RVAs.

The complete genome sequence of bacteriophage CP21 reveals modular shuffling in Campylobacter group II phages.

Campylobacter group II phages described so far share a high degree of sequence similarity. We report the 182,833-bp genomic sequence of the closely related group II phage CP21 and show that it has a completely different genomic organization. As in other group II phages, the CP21 genome is composed of large modules separated by long DNA repeat regions which obviously trigger recombination and modular shuffling.

Spread of a distinct Stx2-encoding phage prototype among Escherichia coli O104:H4 strains from outbreaks in Germany, Norway, and Georgia.

Shiga toxin 2 (Stx2)-producing Escherichia coli (STEC) O104:H4 caused one of the world's largest outbreaks of hemorrhagic colitis and hemolytic uremic syndrome in Germany in 2011. These strains have evolved from enteroaggregative E. coli (EAEC) by the acquisition of the Stx2 genes and have been designated enteroaggregative hemorrhagic E. coli. Nucleotide sequencing has shown that the Stx2 gene is carried by prophages integrated into the chromosome of STEC O104:H4. We studied the properties of Stx2-encoding bacteriophages which are responsible for the emergence of this new type of E. coli pathogen. For this, we analyzed Stx bacteriophages from STEC O104:H4 strains from Germany (in 2001 and 2011), Norway (2006), and the Republic of Georgia (2009). Viable Stx2-encoding bacteriophages could be isolated from all STEC strains except for the Norwegian strain. The Stx2 phages formed lysogens on E. coli K-12 by integration into the wrbA locus, resulting in Stx2 production. The nucleotide sequence of the Stx2 phage P13374 of a German STEC O104:H4 outbreak was determined. From the bioinformatic analyses of the prophage sequence of 60,894 bp, 79 open reading frames were inferred. Interestingly, the Stx2 phages from the German 2001 and 2011 outbreak strains were found to be identical and closely related to the Stx2 phages from the Georgian 2009 isolates. Major proteins of the virion particles were analyzed by mass spectrometry. Stx2 production in STEC O104:H4 strains was inducible by mitomycin C and was compared to Stx2 production of E. coli K-12 lysogens.

Shiga toxin-producing Escherichia coli strains from cattle as a source of the Stx2a bacteriophages present in enteroaggregative Escherichia coli O104:H4 strains.

Enteroaggregative, Shiga toxin-producing E. coli (EAEC-STEC) O104:H4 strains are emerging pathogens causing life threatening diseases in humans. EAEC-STEC O104:H4 strains isolated between 2001 and 2011 were found to harbor a distinct type of Shiga toxin 2a- (Stx2a) encoding prophage. This phage type shows only <65% genetic similarity to so far described viable Stx phages due to differences in the modules for DNA replication, metabolism, regulation and host specificity. Stx production in EAEC is rarely observed and the source of the Stx2a phage in the EAEC-STEC O104:H4 strains is not known. We identified two DNA segments derived from orf15 and the cI gene of the O104:H4 Stx2a phage P13374 that are characteristic for Stx2a prophages present in EAEC-STEC O104:H4 strains. By PCR, these sequences were detected in 14 (5.8%) of 241 Stx2-positive STEC from animals and food. Infectious Stx2a phages could be isolated from four bovine STEC strains. These were found highly similar to P13374 for orf15, cI and stx2a sequences, the chromosomal integration site (wrbA), for phage DNA restriction profiles, virion morphology and superinfection immunity. Stx2a phages of the four bovine STEC strains formed lysogens on the E. coli K-12 strain C600. Phage P13374 from an EAEC-STEC O104:H4 outbreak strain and one of the bovine STEC phages (P13803) lysogenized the Stx-negative EAEC O104:H4 strain CB14647 by integrating in the wrbA gene of CB14647 and converted it into a Stx2a producer. Our findings provide experimental evidence that EAEC-STEC O104:H4 strains have evolved by uptake of Stx2a phages from the bovine reservoir.

Genotypes and virulence characteristics of Shiga toxin-producing Escherichia coli O104 strains from different origins and sources.

Sixty-two Escherichia coli strains carrying the wzxO104-gene from different sources, origins and time periods were analyzed for their serotypes, virulence genes and compared for genomic similarity by pulsed-field gel-electrophoresis (PFGE). The O104 antigen was present in 55 strains and the structurally and genetically related capsular antigen K9 in five strains. The presence of 49 genes associated with enteropathogenic E. coli (EPEC), enteroaggregative E. coli (EAEC) and enterohemorrhagic E. coli (EHEC) was investigated. Fifty-four strains of serotypes O104:H2 (n=1), O104:H4 (n=37), O104:H7 (n=5) and O104:H21 (n=11) produced Shiga-toxins (Stx). Among STEC O104, a close association between serotype, virulence gene profile and genomic similarity was found. EAEC virulence genes were only present in STEC O104:H4 strains. EHEC-O157 plasmid-encoded genes were only found in STEC O104:H2, O104:H7 and O104:H21 strains. None of the 62 O104 or K9 strains carried an eae-gene involved in the attaching and effacing phenotype. The 38 O104:H4 strains formed a single PFGE-cluster (>83.7% similarity). Thirty-one of these strains were from the European O104:H4 outbreak in 2011. The outbreak strains and older O104:H4 strains from Germany (2001), Georgia and France (2009) clustered together at>86.2% similarity. O104:H4 strains isolated between 2001 and 2009 differed for some plasmid-encoded virulence genes compared to the outbreak strains from 2011. STEC O104:H21 and STEC O104:H7 strains isolated in the U.S. and in Europe showed characteristic differences in their Stx-types, virulence gene and PFGE profiles indicating that these have evolved separately. E. coli K9 strains were not associated with virulence and were heterogeneous for their serotypes and PFGE profiles.

The simultaneous occurrence of human norovirus and hepatitis E virus in a Norway rat (Rattus norvegicus).

Wild rats can be reservoirs and vectors for several human pathogens. An initial RT-PCR screening of the intestinal contents of Norway rats trapped in the sewer system of Copenhagen, Denmark, for caliciviruses revealed the presence of a human norovirus in one of 11 rodents. Subsequent phylogenetic analysis of the ~4.0-kb 3'-terminus of the norovirus genome resulted in the identification of a recombinant GI.b/GI.6 strain. The simultaneous detection of hepatitis E virus-like particles in the feces of this rat by transmission electron microscopy was confirmed by RT-PCR and sequence determination, resulting in the identification of a novel rat hepatitis E virus.

Isolation and characterization of phages with lytic activity against methicillin-resistant Staphylococcus aureus strains belonging to clonal complex 398.

Some years ago, MRSA clonal complex (CC) 398 emerged, which spread extensively in livestock animals. People in contact with food production animals are at high risk of colonization. A reduction of MRSA CC398 in livestock might be achieved by application of virulent phages. However, there have not yet been any reports published on phages lysing MRSA CC398 strains. In this study, three virulent phages (PSa1, PSa2 and PSa3) with lytic activity against MRSA CC398 strains were isolated from German pig farms. Morphologically, the phages are members of the family Podoviridae, and they exhibited an identical host range. They lysed 52 (60 %) out of 86 tested MRSA CC398 strains representing 18 different spa types. While the PSa1 and PSa3 genomes have a similar size of approximately 17.5 kb, the PSa2 genome is somewhat larger (ca. 18.5 kb). Southern hybridization revealed strong DNA homologies between the phages, which was confirmed by sequence analysis of cloned restriction fragments and PCR products. Moreover, the whole PSa3 genomic sequence (17,602 bp) showed a close relationship to 44AHJD-like phages, which are not known to contain virulence-associated genes. To assess whether these phages might be candidates for applications, in vitro experiments were carried out in which the number of MRSA CC398 cells could be reduced by up to four log10 units. The phages were stable at a wide range of temperatures and pH values.

Serological cross-reactions between four polyomaviruses of birds using virus-like particles expressed in yeast.

Polyomaviruses are aetiological agents of fatal acute diseases in various bird species. Genomic analysis revealed that avian polyomavirus (APyV), crow polyomavirus (CPyV), finch polyomavirus (FPyV) and goose hemorrhagic polyomavirus (GHPyV) are closely related to each other, but nevertheless form separate viral species; however, their serological relationship was previously unknown. As only APyV can be grown efficiently in tissue culture, virus-like particles (VLPs) were generated by expression of the genomic regions encoding the major structural protein VP1 of these viruses in yeast; these were used to elicit type-specific antibodies in rabbits and as antigens in serological reactions. For increased VLP assembly, a nuclear-localization signal was introduced into APyV-VP1. VLPs derived from the VP1 of the monkey polyomavirus simian virus 40 served as control. APyV-, GHPyV- and CPyV-VLPs showed haemagglutinating activity with chicken and human erythrocytes. CPyV- and GHPyV-specific sera showed slight cross-reactions in immunoblotting, haemagglutination-inhibition assay and indirect ELISA. The FPyV-specific serum inhibited the haemagglutination activity of APyV-VLPs slightly and showed a weak cross-neutralizing activity against APyV in cell-culture tests. Generally, these data indicate that the four polyomaviruses of birds are serologically distinct. However, in accordance with genetic data, a relationship between CPyV and GHPyV as well as between APyV and FPyV is evident, and grouping into two different serogroups may be suggested. The haemagglutinating activity of APyV, CPyV and GHPyV may indicate similar receptor-binding mechanisms for these viruses. Our data could be useful for the development of vaccines against the polyomavirus-induced diseases in birds and for interpretation of diagnostic test results.

Campylobacter jejuni group III phage CP81 contains many T4-like genes without belonging to the T4-type phage group: implications for the evolution of T4 phages.

CP81 is a virulent Campylobacter group III phage whose linear genome comprises 132,454 bp. At the nucleotide level, CP81 differs from other phages. However, a number of its structural and replication/recombination proteins revealed a relationship to the group II Campylobacter phages CP220/CPt10 and to T4-type phages. Unlike the T4-related phages, the CP81 genome does not contain conserved replication and virion modules. Instead, the respective genes are scattered throughout the phage genome. Moreover, most genes for metabolic enzymes of CP220/CPt10 are lacking in CP81. On the other hand, the CP81 genome contains nine similar genes for homing endonucleases which may be involved in the attrition of the conserved gene order for the virion core genes of T4-type phages. The phage apparently possesses an unusual modification of C or G bases. Efficient cleavage of its DNA was only achieved with restriction enzymes recognizing pure A/T sites. Uncommonly, phenol extraction leads to a significant loss of CP81 DNA from the aqueous layer, a property not yet described for other phages belonging to the T4 superfamily.

Infection of calves with bovine norovirus GIII.1 strain Jena virus: an experimental model to study the pathogenesis of norovirus infection.

The experimental infection of newborn calves with bovine norovirus was used as a homologous large animal model to study the pathogenesis of norovirus infection and to determine target cells for viral replication. Six newborn calves were inoculated orally with Jena virus (JV), a bovine norovirus GIII.1 strain, and six calves served as mock-inoculated controls. Following infection, calves were euthanized before the onset of diarrhea (12 h postinoculation [hpi]), shortly after the onset of diarrhea (18 to 21 hpi), and postconvalescence (4 days pi [dpi]). Calves inoculated with JV developed severe watery diarrhea at 14 to 16 hpi, and this symptom lasted for 53.5 to 67.0 h. Intestinal lesions were characterized by severe villus atrophy together with loss and attenuation of villus epithelium. Viral capsid antigen (JV antigen) was detected by immunohistochemistry in the cytoplasm of epithelial cells on villi. In addition, granular material positive for JV antigen was detected in the lamina propria of villi. Lesions first appeared at 12 hpi and were most extensive at 18 to 19 hpi, extending from midjejunum to ileum. The intestinal mucosa had completely recovered at 4 dpi. There was no indication of systemic infection as described for norovirus infection in mice. JV was found in intestinal contents by reverse transcription-PCR (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) as early as 12 hpi. Fecal shedding of the virus started at 13 hpi and stopped at 23 hpi or at necropsy (4 dpi), respectively. Throughout the trial, none of the control calves tested positive for JV by ELISA or RT-PCR.

Discovery and genetic characterization of novel caliciviruses in German and Dutch poultry.

Caliciviruses (CV) were identified in the intestinal contents of five chickens and one turkey from various regions in Germany between 2009 and 2011 by degenerate reverse transcription PCR. The full 7,656-nt-long genomic sequence of the turkey CV L11043 was determined. Partial nucleotide sequences were determined for nine chicken strains. Phylogenetic analysis based on partial deduced amino acid sequences of the protease and RNA polymerase and the complete VP1 capsid sequence identified two distinct clusters of avian CVs, the first of which contained chicken CVs that were closely related to strains found in German chickens in Bavaria and that had been proposed to form a novel CV genus (proposed name: Bavovirus). In contrast, the turkey CV strain L11043 and three chicken CV strains formed a genetically distinct second cluster. Distance analysis suggested that the strains of the second cluster may represent members of two distinct genogroups of another novel CV genus (proposed name: Nacovirus). Based on the newly obtained sequence information, two real-time RT-PCR assays were developed and used to identify bavovirus and nacovirus in pooled intestinal contents from 24 chicken farms in Germany and the Netherlands. Of these, 20 (83 %) were positive for bavovirus, 11 (46 %) were positive for nacovirus, and nine (38 %) were positive for both bavovirus and nacovirus. Attempts were made to propagate chicken and turkey CVs from both the bavovirus and nacovirus clusters in primary chicken cecal cells, embryonal liver cells and fibroblast cells, but these attempts were not successful.

The genome segments of a group D rotavirus possess group A-like conserved termini but encode group-specific proteins.

Rotaviruses are a leading cause of viral acute gastroenteritis in humans and animals. They are grouped according to gene composition and antigenicity of VP6. Whereas group A, B, and C rotaviruses are found in humans and animals, group D rotaviruses have been exclusively detected in birds. Despite their broad distribution among chickens, no nucleotide sequence data exist so far. Here, the first complete genome sequence of a group D rotavirus (strain 05V0049) is presented, which was amplified using sequence-independent amplification strategies and degenerate primers. Open reading frames encoding homologues of rotavirus proteins VP1 to VP4, VP6, VP7, and NSP1 to NSP5 were identified. Amino acid sequence identities between the group D rotavirus and the group A, B, and C rotaviruses varied between 12.3% and 51.7%, 11.0% and 23.1%, and 9.5% and 46.9%, respectively. Segment 10 of the group D rotavirus has an additional open reading frame. Generally, phylogenetic analysis indicated a common evolution of group A, C, and D rotaviruses, separate from that of group B. However, the NSP4 sequence of group C has only very low identities in comparison with cogent sequences of all other groups. The avian group A NSP1 sequences are more closely related to those of group D than those of mammalian group A rotaviruses. Most interestingly, the nucleotide sequences at the termini of the 11 genome segments are identical between group D and group A rotaviruses. Further investigations should clarify whether these conserved structures allow an exchange of genome segments between group A and group D rotaviruses.

Genetic characterization of a novel calicivirus from a chicken.

We describe the identification and genetic characterization of a novel enteric calicivirus, detected by transmission electron microscopy and RT-PCR in two clinically normal chickens and in a chicken with runting and stunting syndrome from different flocks in southern Germany. Positive findings were confirmed by sequencing. The complete nucleotide sequence and genome organization of one strain (Bavaria/04V0021) was determined. The genome of the Bavaria virus is 7,908 nt long and contains two coding open reading frames. Phylogenetic analysis of the deduced partial 2C helicase/NTPase, 3C cysteine protease, RNA-dependent RNA polymerase and complete VP1 capsid protein amino acid sequences showed that the virus is genetically related to but distinct from sapoviruses and lagoviruses. Morphologically, the Bavaria virus particles are 37-42 nm in diameter and exhibit characteristic cup-shaped surface depressions.

Characterization of Blf4, an Archaeal Lytic Virus Targeting a Member of the Methanomicrobiales.

Today, the number of known viruses infecting methanogenic archaea is limited. Here, we report on a novel lytic virus, designated Blf4, and its host strain Methanoculleus bourgensis E02.3, a methanogenic archaeon belonging to the Methanomicrobiales, both isolated from a commercial biogas plant in Germany. The virus consists of an icosahedral head 60 nm in diameter and a long non-contractile tail of 125 nm in length, which is consistent with the new isolate belonging to the Siphoviridae family. Electron microscopy revealed that Blf4 attaches to the vegetative cells of M. bourgensis E02.3 as well as to cellular appendages. Apart from M. bourgensis E02.3, none of the tested Methanoculleus strains were lysed by Blf4, indicating a narrow host range. The complete 37 kb dsDNA genome of Blf4 contains 63 open reading frames (ORFs), all organized in the same transcriptional direction. For most of the ORFs, potential functions were predicted. In addition, the genome of the host M. bourgensis E02.3 was sequenced and assembled, resulting in a 2.6 Mbp draft genome consisting of nine contigs. All genes required for a hydrogenotrophic lifestyle were predicted. A CRISPR/Cas system (type I-U) was identified with six spacers directed against Blf4, indicating that this defense system might not be very efficient in fending off invading Blf4 virus.