What a Difference a Gene Makes: Identification of Virulence Factors of Cowpox Virus.

Cowpox virus (CPXV) is a zoonotic orthopoxvirus (OPV) that causes spillover infections from its animal hosts to humans. In 2009, several human CPXV cases occurred through transmission from pet rats. An isolate from a diseased rat, RatPox09, exhibited significantly increased virulence in Wistar rats and caused high mortality compared to that caused by the mildly virulent laboratory strain Brighton Red (BR). The RatPox09 genome encodes four genes which are absent in the BR genome. We hypothesized that their gene products could be major factors influencing the high virulence of RatPox09. To address this hypothesis, we employed several BR-RatPox09 chimeric viruses. Using Red-mediated mutagenesis, we generated BR-based knock-in mutants with single or multiple insertions of the respective RatPox09 genes. High-throughput sequencing was used to verify the genomic integrity of all recombinant viruses, and transcriptomic analyses confirmed that the expression profiles of the genes that were adjacent to the modified ones were unaltered. While the in vitro growth kinetics were comparable to those of BR and RatPox09, we discovered that a knock-in BR mutant containing the four RatPox09-specific genes was as virulent as the RatPox09 isolate, causing death in over 75% of infected Wistar rats. Unexpectedly, the insertion of gCPXV0030 (g7tGP) alone into the BR genome resulted in significantly higher clinical scores and lower survival rates matching the rate for rats infected with RatPox09. The insertion of gCPXV0284, encoding the BTB (broad-complex, tramtrack, and bric-à-brac) domain protein D7L, also increased the virulence of BR, while the other two open reading frames failed to rescue virulence independently. In summary, our results confirmed our hypothesis that a relatively small set of four genes can contribute significantly to CPXV virulence in the natural rat animal model.IMPORTANCE With the cessation of vaccination against smallpox and its assumed cross-protectivity against other OPV infections, waning immunity could open up new niches for related poxviruses. Therefore, the identification of virulence mechanisms in CPXV is of general interest. Here, we aimed to identify virulence markers in an experimental rodent CPXV infection model using bacterial artificial chromosome (BAC)-based virus recombineering. We focused our work on the recent zoonotic CPXV isolate RatPox09, which is highly pathogenic in Wistar rats, unlike the avirulent BR reference strain. In several animal studies, we were able to identify a novel set of CPXV virulence genes. Two of the identified virulence genes, encoding a putative BTB/POZ protein (CPXVD7L) and a B22R-family protein (CPXV7tGP), respectively, have not yet been described to be involved in CPXV virulence. Our results also show that single genes can significantly affect virulence, thus facilitating adaptation to other hosts.

Generation of a complete single-gene knockout bacterial artificial chromosome library of cowpox virus and identification of its essential genes.

Cowpox virus (CPXV) belongs to the genus Orthopoxvirus in the Poxviridae family. It infects a broad range of vertebrates and can cause zoonotic infections. CPXV has the largest genome among the orthopoxviruses and is therefore considered to have the most complete set of genes of all members of the genus. Since CPXV has also become a model for studying poxvirus genetics and pathogenesis, we created and characterized a complete set of single gene knockout bacterial artificial chromosome (BAC) clones of the CPXV strain Brighton Red. These mutants allow a systematic assessment of the contribution of single CPXV genes to the outcome of virus infection and replication, as well as to the virus host range. A full-length BAC clone of CPXV strain Brighton Red (pBRF) harboring the gene expressing the enhanced green fluorescent protein under the control of a viral late promoter was modified by introducing the mrfp1 gene encoding the monomeric red fluorescent protein driven by a synthetic early vaccinia virus promoter. Based on the modified BAC (pBRFseR), a library of targeted knockout mutants for each single viral open reading frame (ORF) was generated. Reconstitution of infectious virus was successful for 109 of the 183 mutant BAC clones, indicating that the deleted genes are not essential for virus replication. In contrast, 74 ORFs were identified as essential because no virus progeny was obtained upon transfection of the mutant BAC clones and in the presence of a helper virus. More than 70% of all late CPXV genes belonged to this latter group of essential genes.

Identification of 10 cowpox virus proteins that are necessary for induction of hemorrhagic lesions (red pocks) on chorioallantoic membranes.

UNLABELLED: Cowpox viruses (CPXV) cause hemorrhagic lesions ("red pocks") on infected chorioallantoic membranes (CAM) of embryonated chicken eggs, while most other members of the genus Orthopoxvirus produce nonhemorrhagic lesions ("white pocks"). Cytokine response modifier A (CrmA) of CPXV strain Brighton Red (BR) is necessary but not sufficient for the induction of red pocks. To identify additional viral proteins involved in the induction of hemorrhagic lesions, a library of single-gene CPXV knockout mutants was screened. We identified 10 proteins that are required for the formation of hemorrhagic lesions, which are encoded by CPXV060, CPXV064, CPXV068, CPXV069, CPXV074, CPXV136, CPXV168, CPXV169, CPXV172, and CPXV199. The genes are the homologues of F12L, F15L, E2L, E3L, E8R, A4L, A33R, A34R, A36R, and B5R of vaccinia virus (VACV). Mutants with deletions in CPXV060, CPXV168, CPXV169, CPXV172, or CPXV199 induced white pocks with a comet-like shape on the CAM. The homologues of these five genes in VACV encode proteins that are involved in the production of extracellular enveloped viruses (EEV) and the repulsion of superinfecting virions by actin tails. The homologue of CPXV068 in VACV is also involved in EEV production but is not related to actin tail induction. The other genes encode immunomodulatory proteins (CPXV069 and crmA) and viral core proteins (CPXV074 and CPXV136), and the function of the product of CPXV064 is unknown. IMPORTANCE: It has been known for a long time that cowpox virus induces hemorrhagic lesions on chicken CAM, while most of the other orthopoxviruses produce nonhemorrhagic lesions. Although cowpox virus CrmA has been proved to be responsible for the hemorrhagic phenotype, other proteins causing this phenotype remain unknown. Recently, we generated a complete single-gene knockout bacterial artificial chromosome (BAC) library of cowpox virus Brighton strain. Out of 183 knockout BAC clones, 109 knockout viruses were reconstituted. The knockout library makes possible high-throughput screening for studying poxvirus replication and pathogenesis. In this study, we screened all 109 single-gene knockout viruses and identified 10 proteins necessary for inducing hemorrhagic lesions. The identification of these genes gives a new perspective for studying the hemorrhagic phenotype and may give a better understanding of poxvirus virulence.

Prevalence of equine gammaherpesviruses on breeding farms in Turkey and development of a TaqMan MGB real-time PCR to detect equine herpesvirus 5 (EHV-5).

Equine herpesvirus type 2 (EHV-2) and EHV-5 are members of the subfamily Gammaherpesvirinae. The viruses are detected in horses with upper respiratory tract disease and are associated with low performance in racehorses. The aim of the current study was to use nested PCR to investigate the epidemiology of EHV-2 and EHV-5 in Arabian horse populations from breeding farms located in three different cities (Eskisehir, Malatya, and Bursa) in Turkey, using a real-time quantitative PCR (qPCR) with a TaqMan® minor-groove-binder (MGB) probe to detect EHV-5. Screening of blood and ocular and nasal swab samples by nested PCR showed the prevalence of EHV-2 and EHV-5 to be 59 % and 62 %, respectively, with a coinfection rate of 45 %. Thirty-seven isolates from blood samples were identified as EHV-2 using nested PCR. To develop the EHV-5 qPCR, a pair of primers and an MGB probe were designed based on a highly conserved genomic region encoding glycoprotein B (gB). The detection limit of the qPCR was 10 molecules per reaction, and it specifically detected EHV-5 and no other herpesviruses infecting horses (EHV-1, EHV-2, or EHV-4). When applied to field samples, the assay proved to be more sensitive than a well-established nested PCR. Therefore, the qPCR developed in this study provides a rapid, reliable, and sensitive diagnostic assay for the detection of EHV-5, and it complements other diagnostic procedures for equine respiratory disease.

A virus-encoded telomerase RNA promotes malignant T cell lymphomagenesis.

Telomerase is a ribonucleoprotein complex consisting of two essential core components: a reverse transcriptase and an RNA subunit (telomerase RNA [TR]). Dysregulation of telomerase has been associated with cell immortalization and oncogenesis. Marek's disease herpesvirus (MDV) induces a malignant T cell lymphoma in chickens and harbors in its genome two identical copies of a viral TR (vTR) with 88% sequence identity to chicken TR. MDV mutants lacking both copies of vTR were significantly impaired in their ability to induce T cell lymphomas, although lytic replication in vivo was unaffected. Tumor incidences were reduced by >60% in chickens infected with vTR- viruses compared with animals inoculated with MDV harboring at least one intact copy of vTR. Lymphomas in animals infected with the vTR- viruses were also significantly smaller in size and less disseminated. Constitutive expression of vTR in the chicken fibroblast cell line DF-1 resulted in a phenotype consistent with transformation as indicated by morphological alteration, enhanced anchorage-independent cell growth, cell growth beyond saturation density, and increased expression levels of integrin alpha v. We concluded that vTR plays a critical role in MDV-induced T cell lymphomagenesis. Furthermore, our results provide the first description of tumor-promoting effects of TR in a natural virus-host infection model.

Viral bacterial artificial chromosomes: generation, mutagenesis, and removal of mini-F sequences.

Maintenance and manipulation of large DNA and RNA virus genomes had presented an obstacle for virological research. BAC vectors provided a solution to both problems as they can harbor large DNA sequences and can efficiently be modified using well-established mutagenesis techniques in Escherichia coli. Numerous DNA virus genomes of herpesvirus and pox virus were cloned into mini-F vectors. In addition, several reverse genetic systems for RNA viruses such as members of Coronaviridae and Flaviviridae could be established based on BAC constructs. Transfection into susceptible eukaryotic cells of virus DNA cloned as a BAC allows reconstitution of recombinant viruses. In this paper, we provide an overview on the strategies that can be used for the generation of virus BAC vectors and also on systems that are currently available for various virus species. Furthermore, we address common mutagenesis techniques that allow modification of BACs from single-nucleotide substitutions to deletion of viral genes or insertion of foreign sequences. Finally, we review the reconstitution of viruses from BAC vectors and the removal of the bacterial sequences from the virus genome during this process.

Simian varicella virus open reading frame 63/70 expression is required for efficient virus replication in culture.

Simian varicella virus (SVV) open reading frame (ORF) 63, duplicated in the virus genome as ORF 70, is homologous to varicella zoster virus ORF 63/70. Transfection of bacterial artificial chromosome clones containing the wild-type SVV genome and mutants with stop codons in ORF 70, in both ORFs 63 and 70 and the repaired virus DNA sequences into Vero cells produced a cytopathic effect (CPE). The onset of CPE was much slower with the double-mutant transfectants (10 days vs. 3 days) and plaques were smaller. While SVV ORF 63 is not required for replication in culture, its expression leads to robust virus replication.

Recovery of infectious virus from full-length cowpox virus (CPXV) DNA cloned as a bacterial artificial chromosome (BAC).

Transmission from pet rats and cats to humans as well as severe infection in felids and other animal species have recently drawn increasing attention to cowpox virus (CPXV). We report the cloning of the entire genome of cowpox virus strain Brighton Red (BR) as a bacterial artificial chromosome (BAC) in Escherichia coli and the recovery of infectious virus from cloned DNA. Generation of a full-length CPXV DNA clone was achieved by first introducing a mini-F vector, which allows maintenance of large circular DNA in E. coli, into the thymidine kinase locus of CPXV by homologous recombination. Circular replication intermediates were then electroporated into E. coli DH10B cells. Upon successful establishment of the infectious BR clone, we modified the full-length clone such that recombination-mediated excision of bacterial sequences can occur upon transfection in eukaryotic cells. This self-excision of the bacterial replicon is made possible by a sequence duplication within mini-F sequences and allows recovery of recombinant virus progeny without remaining marker or vector sequences. The in vitro growth properties of viruses derived from both BAC clones were determined and found to be virtually indistinguishable from those of parental, wild-type BR. Finally, the complete genomic sequence of the infectious clone was determined and the cloned viral genome was shown to be identical to that of the parental virus. In summary, the generated infectious clone will greatly facilitate studies on individual genes and pathogenesis of CPXV. Moreover, the vector potential of CPXV can now be more systematically explored using this newly generated tool.

Cloning the simian varicella virus genome in E. coli as an infectious bacterial artificial chromosome.

Simian varicella virus (SVV) is closely related to human varicella-zoster virus and causes varicella and zoster-like disease in nonhuman primates. In this study, a mini-F replicon was inserted into a SVV cosmid, and infectious SVV was generated by co-transfection of Vero cells with overlapping SVV cosmids. The entire SVV genome, cloned as a bacterial artificial chromosome (BAC), was stably propagated upon serial passage in E. coli. Transfection of pSVV-BAC DNA into Vero cells yielded infectious SVV (rSVV-BAC). The mini-F vector sequences flanked by loxP sites were removed by co-infection of Vero cells with rSVV-BAC and adenovirus expressing Cre-recombinase. Recombinant SVV generated using the SVV-BAC genetic system has similar molecular and in vitro replication properties as wild-type SVV. To demonstrate the utility of this approach, a SVV ORF 10 deletion mutant was created using two-step Red-mediated recombination. The results indicate that SVV ORF 10, which encodes a homolog of the HSV-1 virion VP-16 transactivator protein, is not essential for in vitro replication but is required for optimal replication in cell culture.

Marek's disease virus: from miasma to model.

Marek's disease virus (MDV) is an oncogenic herpesvirus that causes various clinical syndromes in its natural host, the chicken. MDV has long been of interest as a model organism, particularly with respect to the pathogenesis and immune control of virus-induced lymphoma in an easily accessible small-animal system. Recent advances in MDV genetics and the determination of the chicken genome sequence, aided by functional genomics, have begun to dramatically increase our understanding not only of lytic MDV replication, but also of the factors and mechanisms leading to latency and tumour formation. This new information is helping to elucidate cellular signalling pathways that have undergone convergent evolution and are perturbed by different viruses, and emphasizes the value of MDV as a comparative biomedical model. Furthermore, the door is now open for rational and efficient engineering of new vaccines against one of the most important and widespread infectious diseases in chickens.

First transmission of human immunodeficiency virus Type 1 by a cellular blood product after mandatory nucleic acid screening in Germany.

BACKGROUND: In February 2007, a 63-year-old man underwent surgery. Retrospective testing with nucleic acid testing (NAT) showed that the patient was human immunodeficiency virus Type 1 (HIV-1) positive 10 days after transfusion. The transfusion-transmitted infection had been identified by a donor-related lookback started in April 2007 after anti-HIV seroconversion. METHODS: Sequence analysis was performed in the gag-pol region as well as in the V3 loop env region. Archived plasma from the transmitting donation was investigated for the individual-donation NAT with the Roche COBAS AmpliPrep/COBAS TaqMan HIV-1 test (Roche CAP/CTM HIV-1 test) and for HIV antigen/antibody combination testing (Abbott Architect). Additional testing was done on the donor's follow-up sample and on the recipient's sample. RESULTS: The Roche CAP/CTM HIV-1 test failed to detect viral RNA by minipool NAT in the index donation (April 2007) as well as in the donation that caused the infection (January 2007). Phylogenetic analysis showed a very high genetic similarity among viral sequences from both donor and recipient, proving the HIV-1 transmission by sequence data. CONCLUSION: This case represents the first documented HIV-1 transmission by transfusion of red blood cells after mandatory introduction of HIV-1 NAT for blood screening in Germany. Low viral load and mismatches in the primer/probe region might explain the detection failure of the NAT screening assay. A certain risk remains that new virus variants contain mutations at positions critical for amplification or detection of viral genomes. An option to reduce the risk of a detection failure by NAT is the simultaneous use of several conserved regions as amplification targets.

A self-excisable infectious bacterial artificial chromosome clone of varicella-zoster virus allows analysis of the essential tegument protein encoded by ORF9.

In order to facilitate the generation of mutant viruses of varicella-zoster virus (VZV), the agent causing varicella (chicken pox) and herpes zoster (shingles), we generated a full-length infectious bacterial artificial chromosome (BAC) clone of the P-Oka strain. First, mini-F sequences were inserted into a preexisting VZV cosmid, and the SuperCos replicon was removed. Subsequently, mini-F-containing recombinant virus was generated from overlapping cosmid clones, and full-length VZV DNA recovered from the recombinant virus was established in Escherichia coli as an infectious BAC. An inverted duplication of VZV genomic sequences within the mini-F replicon resulted in markerless excision of vector sequences upon virus reconstitution in eukaryotic cells. Using the novel tool, the role in VZV replication of the major tegument protein encoded by ORF9 was investigated. A markerless point mutation introduced in the start codon by two-step en passant Red mutagenesis abrogated ORF9 expression and resulted in a dramatic growth defect that was not observed in a revertant virus. The essential nature of ORF9 for VZV replication was ultimately confirmed by restoration of the growth of the ORF9-deficient mutant virus using trans-complementation via baculovirus-mediated gene transfer.

Live-attenuated recombinant equine herpesvirus type 1 (EHV-1) induces a neutralizing antibody response against West Nile virus (WNV).

The immunogenicity in horses of a recombinant equine herpesvirus type 1 (EHV-1) vaccine expressing West Nile virus (WNV) prM and E proteins was studied. To construct the recombinant EHV-1, two-step en passant mutagenesis was employed for manipulation of a bacterial artificial chromosome (BAC) of vaccine strain RacH. Recombinant EHV-1 stably expressed the WNV prM and E proteins as demonstrated by indirect immunofluorescence and Western blotting. In addition, growth properties in vitro of the EHV-1/WNV recombinant were found to not be significantly different from those of the parental virus. To determine if vaccination of horses induces an antibody response, 10 horses were allocated in two groups. Group A consisted of six horses that were vaccinated three times with the recombinant EHV-1/WNV virus in 28- to 31-day intervals. Group B consisted of four horses that were sham-vaccinated using the same regimen. Serum was collected on days 0, 31, 45 and 66. Plaque reduction neutralization test and IgG(T)- and IgGb-specific WNV E antibody-capture ELISAs were used. After a single vaccination (day 31), at least four of the six horses from group A had detectable levels of serum neutralizing antibodies against WNV, and three horses retained SN titers until the end of the study. None of the horses in the control group B sero-converted. On days 31 and 45, five of the six horses in group A had a marked increase of WNV-specific IgG(T), and at least four exhibited modestly elevated WNV-specific IgGb titers. From the results, we concluded that the EHV-1 vectored virus is able to express the WNV structural proteins and that vaccination of horses results in the induction of WNV E-protein-specific IgG(T), IgGb, and neutralizing antibodies.

Two-step red-mediated recombination for versatile high-efficiency markerless DNA manipulation in Escherichia coli.

Red recombination using PCR-amplified selectable markers is a well-established technique for mutagenesis of large DNA molecules in Escherichia coli. The system has limited efficacy and versatility, however, for markerless modifications including point mutations, deletions, and particularly insertions of longer sequences. Here we describe a procedure that combines Red recombination and cleavage with the homing endonuclease I-SceI to allow highly efficient, PCR-based DNA engineering without retention of unwanted foreign sequences. We applied the method to modification of bacterial artificial chromosome (BAC) constructs harboring an infectious herpesvirus clone to demonstrate the potential of the mutagenesis technique, which was used for the insertion of long sequences such as coding regions or promoters, introduction of point mutations, scarless deletions, and insertion of short sequences such as an epitope tag. The system proved to be highly reliable and efficient and can be adapted for a variety of different modifications of BAC clones, which are fundamental tools for applications as diverse as the generation of transgenic animals and the construction of gene therapy or vaccine vectors.

Phocine herpesvirus 1 (PhHV-1) in harbor seals from Svalbard, Norway.

Phocine herpesvirus 1 (PhHV-1) infections in seals are associated with disease and sometimes high mortality, primarily in young animals. PhHV-1 has been detected in seals from European waters as well as in waters on both coasts of North America. Serological surveys of various pinniped species have indicated a wide geographical distribution of PhHV-1. A quantitative and sensitive real-time PCR assay targeting the gene encoding glycoprotein B of PhHV-1 was developed for detection of PhHV-1 in ocular and nasal swab samples from wild harbor seals (Phoca vitulina) from Svalbard (Norway). PhHV-1 DNA was detected in samples from 6 (8%) seals collected in 2009 and 2 (3%) in 2010; all had herpesvirus DNA in the ocular swab sample, whereas only one of these animals also had herpesvirus DNA in the nasal swab sample. Four PCR positive animals were approximately 1 year of age and four were pups of the year. Serum samples obtained in 1998 (n=59), 1999 (n=74), 2000 (n=81), 2009 (n=69) and 2010 (n=83) were tested for anti-PhHV-1 antibodies in an indirect ELISA. The PhHV-1 seroprevalence in the population remained high throughout this period, varying from 77 to 100% between years. No eye disease was observed in this harbor seal population, but the ELISA and PCR findings reported here suggest that PhHV-1 is endemic in this globally northernmost harbor seal colony, and that the virus is shed on the mucosa of the eye and nose.

Generation and characterization of a Cowpox virus mutant lacking host range factor CP77.

Cowpox virus (CPXV) host range factor CP77 was identified to be required for virus replication in Chinese hamster ovary (CHO) cells, but the underlying molecular mechanism by which CP77 modulates host range has remained unclear. Therefore, a CPXVΔCP77 deletion mutant was constructed by applying bacterial artificial chromosome (BAC) technology. Integrity of BAC-derived viral DNA was confirmed by whole genome sequencing. In vitro growth characteristics of CPXV wild type (WT), BAC-derived vCPXV WT and vCPXVΔCP77 were virtually indistinguishable in HEK293T cells, whereas in CHO-K1 cells replication of virus lacking CP77 was unambiguously attenuated. This block of viral replication was confirmed by lack of late viral protein expression. The replication defect of various Orthopoxviruses lacking CP77 in CHO cells could be restored by recombinant expression of CP77. Thus, for the first time, the described CP77-dependent host range effect in CHO cells was shown in the background of CPXV as well as Camelpox virus. To further characterize the mutant virus, cells of several different species were comparably infected with vCPXV WT and vCPXVΔCP77, respectively. Interestingly, except for CHO-K1 cells, vCPXV WT and vCPXVΔCP77 showed no significant difference in terms of morphology of cytopathic effects, expression of a late transcribed virus-encoded green fluorescent protein and virus reproduction, even in other hamster-derived cells. Additionally, in ovo inoculation with either virus revealed the same red-pock phenotype on chicken egg chorioallantoic membranes. Since the data presented indicate a CP77-dependent host range effect only for CHO cells, we conclude that the protein might mediate additional functions not identified yet. The vCPXVΔCP77 deletion mutant generated can now be applied as a useful tool to investigate the function of the putative host range protein CP77.

Cowpox virus serpin CrmA is necessary but not sufficient for the red pock phenotype on chicken chorioallantoic membranes.

It was previously reported that cowpox virus (CPXV) strain Brighton Red (BR) causes red pocks upon inoculation of chorioallantoic membranes (CAMs) of embryonated chicken eggs. Red pocks are characterized by hemorrhage and reduced numbers of inflammatory cells while white pocks induced by other members of the genus Orthopoxvirus lack hemorrhage and have higher numbers of infiltrating heterophils. Analyses of CPXV BR white pock variants identified the cytokine response modifier A (CrmA) as the factor responsible for the differences in pock phenotype through induction of hemorrhage and inhibition of chemotaxis. In the present study CPXV crmA deletion mutants were generated based on a full-length bacterial artificial chromosome clone of CPXV BR (pBR). Deletion of the first crmA start codon was sufficient to abolish protein expression, whereas modification of a potential second start codon had no impact on CrmA production as shown by Western blot analysis. Immunohistochemistry of CAMs inoculated with crmA-positive BR viruses showed accumulation of viral antigen in endothelial cells, which was consistent with the red pock phenotype. On the other hand, crmA-negative mutants were characterized by the induction of white pocks and the absence of CPXV antigen in endothelia. The introduction of the complete CPXV BR crmA gene into the homologous genome region of the attenuated vaccinia virus strain MVA (modified vaccinia virus Ankara), however, resulted in CrmA production but not the red pock phenotype. We therefore conclude that (i) CPXV CrmA is associated with increased accumulation of virus in endothelial cells and (ii) the poxvirus-encoded serpin is necessary but not sufficient for the red pock phenotype and the anti-chemotactic capabilities on CAMs.

Herpesviruses--a zoonotic threat?

Herpesviruses are highly host specific and share a long synchronous evolution with their hosts. Only in rare cases, species barriers fall and allow animal to human or human to animal transmission. Among the zoonotic herpesviruses, Cercopithecine herpesvirus 1 is the most significant and can be transmitted from macaques to human. Conversely, Human herpesvirus 1 is capable of causing severe disease in primates. Besides those two examples, there are several herpesviruses with a certainly limited or only suspected ability to cross species barriers. Those include Saimiriine herpesvirus 2, Phocid herpesvirus 2, Equid herpesvirus 1, Epstein-Barr Virus, Marek's disease virus, and Pseudorabies virus. Concerning xenotransplantations, porcine gammaherpesviruses must be considered as a zoonotic threat.

En passant mutagenesis: a two step markerless red recombination system.

Bacterial artificial chromosomes are used to maintain and modify large sequences of different origins in Escherichia coli. In addition to RecA-based shuttle mutagenesis, Red recombination is commonly used for sequence modification. Since foreign sequences, such as antibiotic resistance genes as well as frt- or loxP-sites are often unwanted in mutant BAC clones, we developed a Red-based technique that allows for the scarless generation of point mutations, deletions, and insertion of smaller and larger sequences. The method employs a sequence duplication that is inserted into the target sequence in the first recombination step and the excision of the selection marker by in vivo I-SceI cleavage and the second Red recombination. To allow for convenient and highly efficient mutagenesis without the use of additional plasmids, the E. coli strain GS1783 with a chromosomal encoded inducible Red- and I-SceI-expression was created.

Red-mediated transposition and final release of the mini-F vector of a cloned infectious herpesvirus genome.

Bacterial artificial chromosomes (BACs) are well-established cloning vehicles for functional genomics and for constructing targeting vectors and infectious viral DNA clones. Red-recombination-based mutagenesis techniques have enabled the manipulation of BACs in Escherichia coli without any remaining operational sequences. Here, we describe that the F-factor-derived vector sequences can be inserted into a novel position and seamlessly removed from the present location of the BAC-cloned DNA via synchronous Red-recombination in E. coli in an en passant mutagenesis-based procedure. Using this technique, the mini-F elements of a cloned infectious varicella zoster virus (VZV) genome were specifically transposed into novel positions distributed over the viral DNA to generate six different BAC variants. In comparison to the other constructs, a BAC variant with mini-F sequences directly inserted into the junction of the genomic termini resulted in highly efficient viral DNA replication-mediated spontaneous vector excision upon virus reconstitution in transfected VZV-permissive eukaryotic cells. Moreover, the derived vector-free recombinant progeny exhibited virtually indistinguishable genome properties and replication kinetics to the wild-type virus. Thus, a sequence-independent, efficient, and easy-to-apply mini-F vector transposition procedure eliminates the last hurdle to perform virtually any kind of imaginable targeted BAC modifications in E. coli. The herpesviral terminal genomic junction was identified as an optimal mini-F vector integration site for the construction of an infectious BAC, which allows the rapid generation of mutant virus without any unwanted secondary genome alterations. The novel mini-F transposition technique can be a valuable tool to optimize, repair or restructure other established BACs as well and may facilitate the development of gene therapy or vaccine vectors.