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Recombinant Newcastle disease virus (rNDV) strains engineered to express foreign genes from an additional transcription unit (ATU) are considered as candidate live-attenuated vector vaccines for human and veterinary use. Early during the COVID-19 pandemic we and others generated COVID-19 vaccine candidates based on rNDV expressing a partial or complete SARS-CoV-2 spike (S) protein. In our studies, a number of the rNDV constructs did not show high S expression levels in cell culture or seroconversion in immunized hamsters. Sanger sequencing showed the presence of frequent A-to-G transitions characteristic of adenosine deaminase acting on RNA (ADAR). Subsequent whole genome rNDV sequencing revealed that this biased hypermutation was exclusively localized in the ATU expressing the spike gene, and was related to deamination of adenosines in the negative strand viral genome RNA. The biased hypermutation was found both after virus rescue in chicken cell line DF-1 followed by passaging in embryonated chicken eggs, and after direct virus rescue and subsequent passaging in Vero E6 cells. Levels of biased hypermutation were higher in constructs containing codon-optimized as compared to native S gene sequences, suggesting potential association with increased GC content. These data show that deep sequencing of candidate recombinant vector vaccine constructs in different phases of development is of crucial importance in the development of NDV-based vaccines.

Newcastle disease (ND) is a severe threat to the poultry industry and is caused by virulent strains of Newcastle disease virus (NDV). Many countries maintain a vaccination policy, but NDV is rapidly evolving as shown by the discovery of several new genotypes in the last decades. We tested the efficacy of the currently used classical commercial ND vaccine based on the genotype II strain VG/GA, applied under standard field conditions, against outbreak strains. Field vaccinated broilers were challenged with four different viruses belonging to genotype II, V or VII. A large proportion of field vaccinated broilers showed suboptimal immunity and the protection level against early and recent NDV isolates was dramatically low. Furthermore, there were no significant differences in protection afforded by a genotype II vaccine against a genotype II virus challenge compared to a challenge with viruses belonging to the other genotypes. This study suggests that the susceptibility of vaccinated poultry to NDV infection is not the result of vaccine mismatch, but rather of poor vaccination practices., (Copyright © 2014 Elsevier B.V. All rights reserved.)

A reverse genetics system for thermostable Newcastle disease virus (NDV) is not currently available. In this study, we developed a reverse genetics system for the avirulent and thermostable NDV4-C strain. Successful recovery of NDV4-C was achieved by using either T7 RNA polymerase or cellular RNA polymerase II to drive transcription of the full-length virus antigenome from cloned cDNA. The recovered viruses rNDV4-C (T7) and rNDV4-C (CMV) showed similar growth properties, thermostability, and virulence as the parental strain NDV4-C. The potential of rNDV4-C (T7) to serve as a viral vector was assessed by generating a recombinant virus, rNDV4-eGFP, which expressed enhanced green fluorescent protein. The rNDV4-eGFP could stably carry and express eGFP for at least fifteen passages. The reverse genetics system for NDV4-C will make it possible to analyze the genetic elements that determine thermostability and the oncolytic properties of NDV.

Laboratory response networks (LRNs) have been established for security reasons in several countries including the Netherlands, France, and Sweden. LRNs function in these countries as a preparedness measure for a coordinated diagnostic response capability in case of a bioterrorism incident or other biocrimes. Generally, these LRNs are organized on a national level. The EU project AniBioThreat has identified the need for an integrated European LRN to strengthen preparedness against animal bioterrorism. One task of the AniBioThreat project is to suggest a plan to implement laboratory biorisk management CWA 15793:2011 (CWA 15793), a management system built on the principle of continual improvement through the Plan-Do-Check-Act (PDCA) cycle. The implementation of CWA 15793 can facilitate trust and credibility in a future European LRN and is an assurance that the work done at the laboratories is performed in a structured way with continuous improvements. As a first step, a gap analysis was performed to establish the current compliance status of biosafety and laboratory biosecurity management with CWA 15793 in 5 AniBioThreat partner institutes in France (ANSES), the Netherlands (CVI and RIVM), and Sweden (SMI and SVA). All 5 partners are national and/or international laboratory reference institutes in the field of public or animal health and possess high-containment laboratories and animal facilities. The gap analysis showed that the participating institutes already have robust biorisk management programs in place, but several gaps were identified that need to be addressed. Despite differences between the participating institutes in their compliance status, these variations are not significant. Biorisk management exercises also have been identified as a useful tool to control compliance status and thereby implementation of CWA 15793. An exercise concerning an insider threat and loss of a biological agent was performed at SVA in the AniBioThreat project to evaluate implementation of the contingency plans and as an activity in the implementation process of CWA 15793. The outcome of the exercise was perceived as very useful, and improvements to enhance biorisk preparedness were identified. Gap analyses and exercises are important, useful activities to facilitate implementation of CWA 15793. The PDCA cycle will enforce a structured way to work, with continual improvements concerning biorisk management activities. Based on the activities in the AniBioThreat project, the following requirements are suggested to promote implementation: support from the top management of the organizations, knowledge about CWA 15793, a compliance audit checklist and gap analysis, training and exercises, networking in LRNs and other networks, and interinstitutional audits. Implementation of CWA 15793 at each institute would strengthen the European animal bioterrorism response capabilities by establishing a well-prepared LRN.

Background: Whereas temporal gene expression in mammalian herpesviruses has been studied extensively, little is known about gene expression in fish herpesviruses. Here we report a genome-wide transcription analysis of a fish herpesvirus, anguillid herpesvirus 1, in cell culture, studied during the first 6 hours of infection using reverse transcription quantitative PCR., Results: Four immediate-early genes - open reading frames 1, 6A, 127 and 131 - were identified on the basis of expression in the presence of a protein synthesis inhibitor and unique expression profiles during infection in the absence of inhibitor. All of these genes are located within or near the terminal direct repeats. The remaining 122 open reading frames were clustered into groups on the basis of transcription profiles during infection. Expression of these genes was also studied in the presence of a viral DNA polymerase inhibitor, enabling classification into early, early-late and late genes. In general, clustering by expression profile and classification by inhibitor studies corresponded well. Most early genes encode enzymes and proteins involved in DNA replication, most late genes encode structural proteins, and early-late genes encode non-structural as well as structural proteins., Conclusions: Overall, anguillid herpesvirus 1 gene expression was shown to be regulated in a temporal fashion, comparable to that of mammalian herpesviruses.

Baby hamster kidney (BHK21) cells are used to produce vaccines against various viral veterinary diseases, including rabies and foot-and-mouth-disease. Although particular influenza virus strains replicate efficiently in BHK21 cells the general use of these cells for influenza vaccine production is prohibited by the poor replication of most strains, including model strain A/PR/8/34 [H1N1] (PR8). We now show that in contrast to PR8, the related strain A/WSN/33 [H1N1] (WSN) replicates efficiently in BHK21 cells. This difference is determined by the haemagglutinin (HA) protein since reciprocal reassortant viruses with swapped HAs behave similarly with respect to growth on BHK21 cells as the parental virus from which their HA gene is derived. The ability or inability of six other influenza virus strains to grow on BHK21 cells appears to be similarly dependent on the nature of the HA gene since reassortant PR8 viruses containing the HA of these strains grow to similar titres as the parental virus from which the HA gene was derived. However, the growth to low titres of a seventh influenza strain was not due to the nature of the HA gene since a reassortant PR8 virus containing this HA grew efficiently on BHK21 cells. Taken together, these results suggest that the HA gene often primarily determines influenza replication efficiency on BHK21 cells but that in some strains other genes are also involved. High virus titres could be obtained with reassortant PR8 strains that contained a chimeric HA consisting of the HA1 domain of PR8 and the HA2 domain of WSN. HA1 contains most antigenic sites and is therefore important for vaccine efficacy. This method of producing the HA1 domain as fusion to a heterologous HA2 domain could possibly also be used for the production of HA1 domains of other viruses to enable the use of BHK21 cells as a generic platform for veterinary influenza vaccine production., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Newcastle disease (ND) is one of the most important diseases of poultry, and may cause devastating losses in the poultry industry worldwide. Its causative agent is Newcastle disease virus (NDV), also known as avian paramyxovirus type 1. Many countries maintain a stringent vaccination policy against ND, but there are indications that ND outbreaks can still occur despite intensive vaccination. It has been argued that this may be due to antigenic divergence between the vaccine strains and circulating field strains. Here we present the complete genome sequence of a highly virulent genotype VII virus (NL/93) obtained from vaccinated poultry during an outbreak of ND in the Netherlands in 1992-1993. Using this strain, we investigated whether the identified genetic evolution of NDV is accompanied by antigenic evolution. In this study we show that a live vaccine that is antigenically adapted to match the genotype VII NL/93 outbreak strain does not provide increased protection compared to a classic genotype II live vaccine. When challenged with the NL/93 strain, chickens vaccinated with a classic vaccine were completely protected against clinical disease and mortality and virus shedding was significantly reduced, even with a supposedly suboptimal vaccine dose. These results suggest that it is not antigenic variation but rather poor flock immunity due to inadequate vaccination practices that may be responsible for outbreaks and spreading of virulent NDV field strains., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Influenza viruses unable to express NS1 protein (delNS1) replicate poorly and induce large amounts of interferon (IFN). They are therefore considered candidate viruses for live-attenuated influenza vaccines. Their attenuated replication is generally assumed to result from the inability to counter the antiviral host response, as delNS1 viruses replicate efficiently in Vero cells, which lack IFN expression. In this study, delNS1 virus was parallel passaged on IFN-competent MDCK cells, which resulted in two strains that were able to replicate to high virus titers in MDCK cells due to adaptive mutations especially in the M-gene segment but also in the NP and NS gene segments. Most notable were clustered U-to-C mutations in the M segment of both strains and clustered A-to-G mutations in the NS segment of one strain, which presumably resulted from host cell-mediated RNA editing. The M segment mutations in both strains changed the ratio of M1 to M2 expression, probably by affecting splicing efficiency. In one virus, 2 amino acid substitutions in M1 additionally enhanced virus replication, possibly through changes in the M1 distribution between the nucleus and the cytoplasm. Both adapted viruses induced levels of IFN equal to that of the original delNS1 virus. These results show that the increased replication of the adapted viruses is not primarily due to altered IFN induction but rather is related to changes in M1 expression or localization. The mutations identified in this paper may be used to enhance delNS1 virus replication for vaccine production.

We used deep sequencing of poly(A) RNA to characterize the transcriptome of an economically important eel virus, anguillid herpesvirus 1 (AngHV1), at a stage during the lytic life cycle when infectious virus was being produced. In contrast to the transcription of mammalian herpesviruses, the overall level of antisense transcription from the 248,526-bp genome was low, amounting to only 1.5% of transcription in predicted protein-coding regions, and no abundant, nonoverlapping, noncoding RNAs were identified. RNA splicing was found to be more common than had been anticipated previously. Counting the 10,634-bp terminal direct repeat once, 100 splice junctions were identified, of which 58 were considered likely to be involved in the expression of functional proteins because they represent splicing between protein-coding exons or between 5' untranslated regions and protein-coding exons. Each of the 30 most highly represented of these 58 splice junctions was confirmed by RT-PCR. We also used deep sequencing to identify numerous putative 5' and 3' ends of AngHV1 transcripts, confirming some and adding others by rapid amplification of cDNA ends (RACE). The findings prompted a revision of the AngHV1 genome map to include a total of 129 protein-coding genes, 5 of which are duplicated in the terminal direct repeat. Not counting duplicates, 11 genes contain integral, spliced protein-coding exons, and 9 contain 5' untranslated exons or, because of alternative splicing, 5' untranslated and 5' translated exons. The results of this study sharpen our understanding of AngHV1 genomics and provide the first detailed view of a fish herpesvirus transcriptome.

In the last decade many studies have been performed on the virulence of Newcastle disease virus (NDV). This is mainly due to the development of reverse genetics systems which made it possible to genetically modify NDV and to investigate the contribution of individual genes and genome regions to its virulence. However, the available information is scattered and a comprehensive overview of the factors and conditions determining NDV virulence is lacking. This review summarises, compares and discusses the available literature and shows that virulence of NDV is a complex trait determined by multiple genetic factors.

Many of the known fish herpesviruses have important aquaculture species as their natural host, and may cause serious disease and mortality. Anguillid herpesvirus 1 (AngHV-1) causes a hemorrhagic disease in European eel, Anguilla anguilla. Despite their importance, fundamental molecular knowledge on fish herpesviruses is still limited. In this study we describe the identification and localization of the structural proteins of AngHV-1. Purified virions were fractionated into a capsid-tegument and an envelope fraction, and premature capsids were isolated from infected cells. Proteins were extracted by different methods and identified by mass spectrometry. A total of 40 structural proteins were identified, of which 7 could be assigned to the capsid, 11 to the envelope, and 22 to the tegument. The identification and localization of these proteins allowed functional predictions. Our findings include the identification of the putative capsid triplex protein 1, the predominant tegument protein, and the major antigenic envelope proteins. Eighteen of the 40 AngHV-1 structural proteins had sequence homologues in related Cyprinid herpesvirus 3 (CyHV-3). Conservation of fish herpesvirus structural genes seemed to be high for the capsid proteins, limited for the tegument proteins, and low for the envelope proteins. The identification and localization of the structural proteins of AngHV-1 in this study adds to the fundamental knowledge of members of the Alloherpesviridae family, especially of the Cyprinivirus genus.

Influenza A viruses lacking the gene encoding the non-structural NS1 protein (delNS1) have potential use as live attenuated vaccines. However, due to the lack of NS1, virus replication in cell culture is considerably reduced, prohibiting commercial vaccine production. We therefore established two stable MDCK cell lines that show inducible expression of the allele B NS1 protein. Upon induction, both cell lines expressed NS1 to about 1000-fold lower levels than influenza virus-infected cells. Nevertheless, expression of NS1 increased delNS1 virus titres to levels comparable to those obtained with an isogenic virus strain containing an intact NS1 gene. Recombinant NS1 expression increased the infectious virus titres 244 to 544-fold and inhibited virus induced apoptosis. However, NS1 expression resulted in only slightly, statistically not significant, reduced levels of interferon-β production. Thus, the low amount of recombinant NS1 is sufficient to restore delNS1 virus replication in MDCK cells, but it remains unclear whether this occurs in an interferon dependent manner. In contrast to previous findings, recombinant NS1 expression did not induce apoptosis, nor did it affect cell growth. These cell lines thus show potential to improve the yield of delNS1 virus for vaccine production., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The pathogenesis of pigeon paramyxovirus type 1 (PPMV-1) isolate AV324/96 and of its recombinant derivative, rgAV324, was studied in pigeons. For comparison, the virulent chicken virus FL-Herts, which is a recombinant derivative of strain Herts/33, was also included. After inoculation by the combined intraocular, intranasal and intratracheal route, clinical signs, virus shedding and serological responses were examined. Clinical signs were observed only in the FL-Herts-infected group. All virus-inoculated pigeons had positive tracheal swabs until 5 days post infection. However, only the AV324/96-infected and rgAV324-infected birds, and not the FL-Herts-infected birds, shed virus in the cloaca. The AV324/96-infected pigeons showed higher mean antibody titres than the rgAV324-infected birds, whereas the antibody titres of the FL-Herts-infected group were rather low. The results show that the pigeon strain AV324 is not virulent for pigeons, but underlines the potential risk of poultry becoming infected by PPMV-1 shed by non-symptomatic pigeons.

Some Newcastle disease virus (NDV) variants isolated from pigeons (pigeon paramyxovirus type 1; PPMV-1) do not show their full virulence potential for domestic chickens but may become virulent upon spread in these animals. In this study we examined the molecular changes responsible for this gain of virulence by passaging a low-pathogenic PPMV-1 isolate in chickens. Complete genome sequencing of virus obtained after 1, 3 and 5 passages showed the increase in virulence was not accompanied by changes in the fusion protein--a well known virulence determinant of NDV--but by mutations in the L and P replication proteins. The effect of these mutations on virulence was confirmed by means of reverse genetics using an infectious cDNA clone. Acquisition of three amino acid mutations, two in the L protein and one in the P protein, significantly increased virulence as determined by intracerebral pathogenicity index tests in day-old chickens. The mutations enhanced virus replication in vitro and in vivo and increased the plaque size in infected cell culture monolayers. Furthermore, they increased the activity of the viral replication complex as determined by an in vitro minigenome replication assay. Our data demonstrate that PPMV-1 replication in chickens results in mutations in the polymerase complex rather than the viral fusion protein, and that the virulence level of pigeon paramyxoviruses is directly related to the activity of the viral replication complex.

Virulent strains of Newcastle disease virus ([NDV] also known as avian paramyxovirus type 1) can be discriminated from low-virulence strains by the presence of multiple basic amino acid residues at the proteolytic cleavage site of the fusion (F) protein. However, some NDV variants isolated from pigeons (pigeon paramyxovirus type 1 [PPMV-1]) have low levels of virulence, despite the fact that their F protein cleavage sites contain a multibasic amino acid sequence and have the same functionality as that of virulent strains. To determine the molecular basis of this discrepancy, we examined the role of the internal proteins in NDV virulence. Using reverse genetics, the genes encoding the nucleoprotein (NP), phosphoprotein (P), matrix protein (M), and large polymerase protein (L) were exchanged between the nonvirulent PPMV-1 strain AV324 and the highly virulent NDV strain Herts. Recombinant viruses were evaluated for their pathogenicities and replication levels in day-old chickens, and viral genome replication and plaque sizes were examined in cell culture monolayers. We also tested the contributions of the individual NP, P, and L proteins to the activity of the viral replication complex in an in vitro replication assay. The results showed that the replication proteins of Herts are more active than those of AV324 and that the activity of the viral replication complex is directly related to virulence. Although the M protein affected viral replication in vitro, it had only a minor effect on virulence.

Two pathogenetically different pigeon paramyxovirus type 1 (PPMV-1) virus clones were recently derived by passage of a single isolate with an intracerebral pathogenicity index (ICPI) of 0.32. The virus clones had an ICPI of 0.025 and 1.3, respectively (Fuller et al., 2007). Remarkably both viruses contained a cleavage site motif in the precursor fusion (F) protein that is usually associated with virulent viruses. In the current study, both viral genomes were completely sequenced and only four amino acid differences were observed. Of these, two were considered irrelevant on theoretical grounds and two amino acid changes were unique for virus 0.025. The latter were introduced into an infectious clone of a virulent Newcastle disease virus strain, individually and combined, and the effects of the mutations on pathogenicity were examined. The results indicate that only the S453P substitution in the F protein had a modest effect on pathogenicity. We were not able to identify the molecular basis for the pathogenicity difference between both viruses. However, our observations emphasize the need to determine both the virulence (ICPI) and the sequence of the cleavage site of the F protein to avoid dismissing of potential virulent PPMV-1 isolates., ((c) 2009 Elsevier B.V. All rights reserved.)

Eel herpesvirus or anguillid herpesvirus 1 (AngHV1) frequently causes disease in freshwater eels. The complete genome sequence of AngHV1 and its taxonomic position within the family Alloherpesviridae were determined. Shotgun sequencing revealed a 249 kbp genome including an 11 kbp terminal direct repeat that contains 7 of the 136 predicted protein-coding open reading frames. Twelve of these genes are conserved among other members of the family Alloherpesviridae and another 28 genes have clear homologues in cyprinid herpesvirus 3. Phylogenetic analyses based on amino acid sequences of five conserved genes, including the ATPase subunit of the terminase, confirm the position of AngHV1 within the family Alloherpesviridae, where it is most closely related to the cyprinid herpesviruses. Our analyses support a recent proposal to subdivide the family Alloherpesviridae into two sister clades, one containing AngHV1 and the cyprinid herpesviruses and the other containing Ictalurid herpesvirus 1 and the ranid herpesviruses.

In the past decade, the use of Newcastle disease virus (NDV) as a vaccine vector for the prevention of economically important livestock diseases as well as for human diseases has been extensively explored. In this study, we have constructed a recombinant NDV vaccine virus, named NDFL-Gn, that produces the Rift Valley fever virus (RVFV) Gn glycoprotein. Calves were immunized via either the intranasal route or the intramuscular route. Delivery via the intranasal route elicited no detectable antibody responses, whereas delivery via the intramuscular route elicited antibodies against both NDV and the Gn protein. The RVFV-neutralizing activity of the antisera from intramuscularly vaccinated calves was demonstrated, suggesting that NDV is a promising vaccine vector for the prevention of RVF in calves., (Copyright 2010 Elsevier Ltd. All rights reserved.)

The principal molecular determinant of virulence of Newcastle disease virus (NDV) is the amino acid sequence at the fusion cleavage activation site. To extend the understanding of the role of the fusion cleavage activation site in NDV virulence, the pathogenesis in chickens of a lentogenic LaSota isolate and two infectious clones, NDFL and NDFLtag, were compared. NDFL is an infectious clone of a lentogenic NDV strain (LaSota E13-1), and NDFLtag is the infectious clone with the fusion cleavage site sequence mutated to the virulent motif. NDFL and NDFLtag were described by Peeters et al. The viruses were inoculated intraconjunctivally into groups of 4-wk-old white leghorn chickens and compared in a pathogenesis study for determination of disease causation (clinical signs of disease, gross lesions, histology, virus isolation, and serology) and viral distribution (presence of viral nucleoprotein and mRNA was detected by immunohistochemistry and in situ hybridization, respectively). The modification of the fusion cleavage activation site to the virulent motif in the infectious clone only slightly increased disease severity and viral distribution in the pathogenesis assessment, even though dramatically increased pathogenicity of NDFLtag was confirmed by standard pathogenicity index tests. The result, that the mutated fusion cleavage site of NDV-NDFLtag had only a small influence on pathogenesis in chickens compared to either E13-1 or NDFL, suggests that the pathogenic effects of NDV are not dependent on the fusion cleavage site alone.

Infectious bursal disease virus (IBDV), a member of the family Birnaviridae, is a non-enveloped, double-stranded RNA virus. Viral protein 1 (VP1), the putative RNA-dependent RNA polymerase, occurs in virions both as a free polypeptide and as a genome-linked protein, called VPg. To gain more insight in its function, we initiated a yeast two-hybrid screen. With this approach we identified the carboxy-terminal domain of eukaryotic translation initiation factor 4AII (eIF4AII) as an interactor for VP1. The association between these molecules was confirmed by co-immunoprecipitation analyses. eIF4A plays an essential role in the initiation of translation of both capped and uncapped mRNAs. Its association with IBDV VP1 suggests an involvement of this viral protein in IBDV mRNA translation. An interaction between VP1 and full-length eIF4AII was, however, not observed. In view of the known two-domain structure of eIF4AII it is conceivable that the interaction of VP1 with full-length eIF4AII requires collaborating proteins that open up its structure and expose the VP1-binding site in the carboxy-terminal domain. The biological relevance of the potential VP1-eIF4AII interaction is discussed.

The tissue tropism of non-virulent and virulent Newcastle disease virus (NDV) was investigated using 8-day-old and 14-day-old embryonating chicken eggs (ECE), inoculated with an infectious clone of the non-virulent La Sota strain (NDFL-GFP) or its virulent derivative (NDFLtag-GFP). Both strains expressed the gene encoding jellyfish green fluorescence protein (GFP) as a marker. The GFP was readily expressed in chicken embryo cells infected with the NDV strains indicating virus replication. Whereas both strains replicated in the chorioallantoic membrane (CAM) and infected the skin of 8-day-old ECE, only the virulent strain (NDFLtag-GFP) spread to internal organs (pleura/peritoneum). In 14-day-old ECE, the initial target organs appeared to be the CAM and the lungs for both strains. At 48 h after inoculation, the virulent strain (NDFLtag-GFP) had also spread to the spleen and heart and was detected in a wide-range of embryonic cell types. The kinetics of virus replication and spread in the CAM closely resembled each other in both the 8-day-old and 14-day-old ECE. Infection of 8-day-old and 14-day-old ECE forms a convenient model to investigate tissue tropism of NDV, as well as the kinetics of viral infection. The advantage of using GFP is that samples can be easily screened by direct fluorescence microscopy without any pre-treatment.

Infectious bursal disease virus (IBDV), a nonenveloped double-stranded RNA virus of chicken, encodes five proteins. Of these, the RNA-dependent RNA polymerase (VP1) is specified by the smaller genome segment, while the large segment directs synthesis of a nonstructural protein (VP5) and a structural protein precursor from which the capsid proteins pVP2 and VP3 as well as the viral protease VP4 are derived. Using the recently redefined processing sites of the precursor, we have reevaluated the homotypic interactions of the viral proteins using the yeast two-hybrid system. Except for VP1, which interacted weakly, all proteins appeared to self-associate strongly. Using a deletion mutagenesis approach, we subsequently mapped the interacting domains in these polypeptides, where possible confirming the observations made in the two-hybrid system by performing coimmunoprecipitation analyses of tagged protein constructs coexpressed in avian culture cells. The results revealed that pVP2 possesses multiple interaction domains, consistent with available structural information about this external capsid protein. VP3-VP3 interactions were mapped to the amino-terminal part of the polypeptide. Interestingly, this domain is distinct from two other interaction domains occurring in this internal capsid protein: while binding to VP1 has been mapped to the carboxy-terminal end of the protein, interaction with the genomic dsRNA segments has been suggested to occur just upstream thereof. No interaction sites could be assigned to the VP4 protein; any deletion applied abolished its self-association. Finally, one interaction domain was detected in the central, most hydrophobic region of VP5, supporting the idea that this virulence determinant may function as a membrane pore-forming protein in infected cells.

Infectious bursal disease virus (IBDV) is a double-stranded RNA (dsRNA) virus of the Birnaviridae family. Its two genome segments are encapsidated together with multiple copies of the viral RNA-dependent RNA polymerase, VP1, in a single-shell capsid that is composed of VP2 and VP3. In this study we identified the domains responsible for the interaction between VP3 and VP1. Using the yeast two-hybrid system we found that VP1 binds to VP3 through an internal domain, while VP3 interacts with VP1 solely by its carboxy-terminal 10 amino acids. These results were confirmed by using a reverse-genetics system that allowed us to analyze the interaction of carboxy-terminally truncated VP3 molecules with VP1 in infected cells. Coimmunoprecipitations with VP1- and VP3-specific antibodies revealed that the interaction is extremely sensitive to truncation of VP3. The mere deletion of the C-terminal residue reduced coprecipitation almost completely and also fully abolished production of infectious virions. Surprisingly, these experiments additionally revealed that VP3 also binds to RNA. RNase treatments and reverse transcription-PCR analyses of the immunoprecipitates demonstrated that VP3 interacts with dsRNA of both viral genome segments. This interaction is not mediated by the carboxy-terminal domain of VP3 since C-terminal truncations of 1, 5, or 10 residues did not prevent formation of the VP3-dsRNA complexes. VP3 seems to be the key organizer of birnavirus structure, as it maintains critical interactions with all components of the viral particle: itself, VP2, VP1, and the two genomic dsRNAs.

Infectious bursal disease virus (IBDV) is the major viral pathogen in the poultry industry. Live attenuated serotype 1 vaccine strains are commonly used to protect susceptible chickens during their first 6 weeks of life. Wild-type serotype 1 IBDV strains are highly pathogenic only in chickens, whereas serotype 2 strains are apathogenic in chickens and other birds. Here we describe the replacement of the genomic double-stranded RNA (dsRNA) encoding the N- or C-terminal part of VP3 of serotype 1 very virulent IBDV (vvIBDV) (isolate D6948) with the corresponding part of serotype 2 (isolate TY89) genomic dsRNA. The modified virus containing the C-terminal part of serotype 2 VP3 significantly reduced the virulence in specific-pathogen-free chickens, without affecting the distinct bursa tropism of serotype 1 IBDV strains. Furthermore, by using serotype-specific antibodies we were able to distinguish bursas infected with wild-type vvIBDV from bursas infected with the modified vvIBDV. We are currently evaluating the potential of this recombinant strain as an attenuated live vaccine that induces a unique serological response (i.e., an IBDV marker vaccine).

Infectious Bursal Disease Virus (IBDV) is the causative agent of one of the most important and wide-spread infectious diseases among commercial chicken flocks. IBDV causes a depletion of B-lymphoid cells in the bursa of Fabricius, inducing immunosuppression, morbidity, or even acute mortality. Because currently used live IBDV vaccines are derivatives from field isolates no serologic discrimination between field isolates and live vaccines can be made. The recently developed reverse genetics techniques for IBDV allows one to generate genetically modified IBDVs which might have altered biological and antigenic properties. Here, we describe the rescue of mosaic serotype I IBDVs, of which the polyprotein encoding region was partly replaced by the corresponding region of a serotype II strain. A mosaic virus, containing the C-terminal part of serotype II VP3 showed only a slightly delayed release of progeny virus compared to unmodified serotype I virus, while maximum viral titers at 25 h post infection were equal. Since serotype specific epitope(s) are present in the C-terminal part of VP3, we were able to discriminate this rescued virus from serotype I and II IBDV strains. These findings make the use of a chimeric VP3 a promising approach to develop an IBDV marker vaccine.

Specific alterations in the genetic material of RNA viruses rely on a technique known as reverse genetics. Transfection of cells with the altered generic material is a critical step of this procedure. In this report we have compared RNA and cDNA transfection methods for the efficiency of transient protein expression and rescue of (recombinant) infectious bursal disease virus (IBDV). Quantitative expression analysis of the secreted alkaline phosphatase reporter protein, and qualitative expression levels of an IBDV protein showed both that cDNA transfection results in a much higher level of protein expression than RNA transfection. Because the rescue of a crippled variant of IBDV was achieved consistently using the cDNA transfection method, but failed when we used the RNA transfection method, we favor the cDNA transfection method for the rescue of (recombinant) IBDV from cloned cDNA.

Chickens (Gallus gallus domesticus) can suffer from AA amyloidosis featuring the joints as major targets of amyloid accumulation. Analysis of post-mortem recordings from commercial chickens revealed that amyloid arthropathy frequently occurred in brown layer chickens, but never in white layers. The suspected higher susceptibility of brown layers was confirmed experimentally by inducing amyloidosis with an arthropathic and amyloidogenic strain of E. faecalis. Sequence analysis of cDNA coding for SAA, the amyloid precursor protein, revealed that the SAA proteins are identical in both breeds, although some nucleotide differences existed in the untranslated regions of the mRNAs. The chicken SAA gene was found to be a single copy gene which comprises 4 exons. The first of these exons apparently occupies a conserved position and is not translated. Investigation of the affected joints using in situ hybridization demonstrated local SAA gene expression. It is concluded that the likelihood of an E. faecalis induced arthritis to progress to amyloidosis is breed-dependent, but is not a consequence of a more amyloidogenic SAA.

Using a recently developed reverse genetics system, we have generated a recombinant Newcastle disease virus (NDV) vaccine in which the gene encoding the hemagglutinin-neuraminidase (HN) has been replaced by a hybrid HN gene consisting of the cytoplasmic domain, transmembrane region, and stalk region of HN of NDV, and the immunogenic globular domain of HN of avian paramyxovirus type 4 (APMV4). The objective was to generate a chimeric live vaccine that induces a protective immune response against NDV by eliciting neutralizing antibodies against the fusion (F) protein, but which can be differentiated from wild-type NDV on the basis of different antibodies elicited by their HN proteins. Pathogenicity tests in day-old chickens showed that the recombinant was non-virulent (intracerebral pathogenicity index [ICPI]=0.00). A vaccination-challenge experiment in 4-week-old specific pathogen free chickens demonstrated that the recombinant was completely safe and was able to protect chickens from challenge with a lethal dose of virulent NDV. By using a secreted form of HN produced in Pichia pastoris, a test was developed that allowed serological differentiation between animals vaccinated with the recombinant vaccine and animals infected with NDV. These results demonstrate that genetically modified marker vaccines can be generated from small RNA viruses that lack non-essential genes.

Many recent outbreaks of infectious bursal disease in commercial chicken flocks worldwide are due to the spread of very virulent strains of infectious bursal disease virus (vvIBDV). The molecular determinants for the enhanced virulence of vvIBDV compared to classical IBDV are unknown. The lack of a reverse genetics system to rescue vvIBDV from its cloned cDNA hampers the identification and study of these determinants. In this report we describe, for the first time, the rescue of vvIBDV from its cloned cDNA. Two plasmids containing a T7 promoter and either the full-length A- or B-segment cDNA of vvIBDV (D6948) were cotransfected into QM5 cells expressing T7 polymerase. The presence of vvIBDV could be detected after passage of the transfection supernatant in either primary bursa cells (in vitro) or embryonated eggs (in vivo), but not QM5 cells. Rescued vvIBDV (rD6948) appeared to have the same virulence as the parental isolate, D6948. Segment-reassorted IBDV, in which one of the two genomic segments originated from cDNA of classical attenuated IBDV CEF94 and the other from D6948, could also be rescued by using this system. Segment-reassorted virus containing the A segment of the classical attenuated isolate (CEF94) and the B segment of the very virulent isolate (D6948) is not released until 15 h after an in vitro infection. This indicates a slightly retarded replication, as the first release of CEF94 is already found at 10 h after infection. Next to segment reassortants, we generated and analyzed mosaic IBDVs (mIBDVs). In these mIBDVs we replaced the region of CEF94 encoding one of the viral proteins (pVP2, VP3, or VP4) by the corresponding region of D6948. Analysis of these mIBDV isolates showed that tropism for non-B-lymphoid cells was exclusively determined by the viral capsid protein VP2. However, the very virulent phenotype was not solely determined by this protein, since mosaic virus containing VP2 of vvIBDV induced neither morbidity nor mortality in young chickens.

Little is known about the intermolecular interactions between the viral proteins of infectious bursal disease virus (IBDV). By using the yeast two-hybrid system, which allows the detection of protein-protein interactions in vivo, all possible interactions were tested by fusing the viral proteins to the LexA DNA-binding domain and the B42 transactivation domain. A heterologous interaction between VP1 and VP3, and homologous interactions of pVP2, VP3, VP5 and possibly VP1, were found by co-expression of the fusion proteins in Saccharomyces cerevisiae. The presence of the VP1-VP3 complex in IBDV-infected cells was confirmed by co-immunoprecipitation studies. Kinetic analyses showed that the complex of VP1 and VP3 is formed in the cytoplasm and eventually is released into the cell-culture medium, indicating that VP1-VP3 complexes are present in mature virions. In IBDV-infected cells, VP1 was present in two forms of 90 and 95 kDa. Whereas VP3 initially interacted with both the 90 and 95 kDa proteins, later it interacted exclusively with the 95 kDa protein both in infected cells and in the culture supernatant. These results suggest that the VP1-VP3 complex is involved in replication and packaging of the IBDV genome.

We examined replication of Newcastle disease virus (NDV) by using minigenomes consisting of the 3' leader and 5' trailer regions of NDV flanking a reporter gene encoding secreted placental alkaline phosphatase (SEAP). Negative-sense minigenome RNA was generated from transfected plasmid DNA by means of in vivo transcription. Subsequent replication of minigenome RNA was determined either after infection with NDV helpervirus or after contransfection with helperplasmids that expressed the essential viral replication proteins NP, P, and L. In both systems, efficient replication of minigenome RNA was observed only if the genome size was a multiple of six nucleotides. Hence, in these systems, replication of NDV minigenome RNA's is strictly dependent on the rule-of-six. When the supernatant from helpervirus-infected, transfected cells was used to infect fresh monolayers, efficient transfer of SEAP activity by virus-like particles was observed only if the size of the minigenome RNA obeyed the rule-of-six. However, after several serial passages, we also observed efficient transfer of SEAP activity by virus-like particles derived from minigenome RNA's that did not obey the rule-of-six. Evidence was obtained which indicated that successful replication of these minigenomes was not due to a change in genome size.

To determine the complete nucleotide sequence of Infectious Bursal Disease virus (IBDV) isolates, an efficient method was developed to generate full-length cDNA of both the genomic A- and B-segments. Reverse transcription was carried out at the highest possible temperature (50 degrees C) for the reverse transcriptase enzyme, and the single stranded cDNA was subsequently amplified by using an optimized PCR. The double stranded, full-length cDNA was efficiently cloned into a high copy number plasmid. Our results show that the entire cDNA of both the A- and B-segment of a classical attenuated isolate (CEF94), and a very virulent field isolate (D6948), can be cloned. The method will simplify greatly the procedure to generate full-length cDNA and determine the nucleotide sequence of the entire genome of IBDV isolates.

To study the mechanism of replication of infectious bursal disease virus (IBDV), and to determine factors on the IBDV RNA which are involved in viral replication, we used cloned full-length cDNA of both the A- and B-segments to generate infectious IBDV. Infectious IBDV was rescued from plasmids that contained full-length IBDV cDNA behind a T7 promoter, by transfecting these plasmids into cells which were infected with a recombinant Fowlpox virus that expressed T7 RNA polymerase. By using the cDNA transfection system we evaluated the effect of the length of the 3' terminus of the A-segment plus strand of IBDV. Although wild-type IBDV predominantly contains four cytosines at the 3' terminus, no difference in virus yield was found when virus was rescued from cDNAs containing three to six adjacent cytosines. When the 3' terminus was shorter than three cytosines the efficiency to generate infectious IBDV from cDNA was reduced, but IBDV could still be recovered reproducibly. The rescued viruses from cDNAs containing 3'-terminal deletions appeared to have a restored 3'-terminal sequence. The missing nucleotides are probably restored by using complementary bases of a stem-loop structure as template., (Copyright 1999 Academic Press.)

A full-length cDNA clone of Newcastle disease virus (NDV) vaccine strain LaSota was assembled from subgenomic overlapping cDNA fragments and cloned in a transcription plasmid between the T7 RNA polymerase promoter and the autocatalytic hepatitis delta virus ribozyme. Transfection of this plasmid into cells that were infected with a recombinant fowlpoxvirus that expressed T7 RNA polymerase, resulted in the synthesis of antigenomic NDV RNA. This RNA was replicated and transcribed by the viral NP, P, and L proteins, which were expressed from cotransfected plasmids. After inoculation of the transfection supernatant into embryonated specific-pathogen-free eggs, infectious virus derived from the cloned cDNA was recovered. By introducing three nucleotide changes in the cDNA, we generated a genetically tagged derivative of the LaSota strain in which the amino acid sequence of the protease cleavage site (GGRQGR downward arrowL) of the fusion protein F0 was changed to the consensus cleavage site of virulent NDV strains (GRRQRR downward arrowF). Pathogenicity tests in day-old chickens showed that the strain derived from the unmodified cDNA was completely nonvirulent (intracerebral pathogenicity index [ICPI] = 0.00). However, the strain derived from the cDNA in which the protease cleavage site was modified showed a dramatic increase in virulence (ICPI = 1.28 out of a possible maximum of 2.0). Pulse-chase labeling of cells infected with the different strains followed by radioimmunoprecipitation of the F protein showed that the efficiency of cleavage of the F0 protein was greatly enhanced by the amino acid replacements. These results demonstrate that genetically modified NDV can be recovered from cloned cDNA and confirm the supposition that cleavage of the F0 protein is a key determinant in virulence of NDV.

This paper reviews new findings on the biological functions of pseudorabies virus (PRV) proteins. It focuses on the role of PRV proteins in the pathogenicity, immunogenicity and transmission of PRV vaccine strains in pigs. Furthermore, it evaluates potential risks that are connected with the use of PRV vector strains. Special emphasis is placed upon the spread of genetically engineered vaccine strains within pigs or between pigs.

To better understand the contribution of T cells to the immunity of pigs to pseudorabies virus (PRV), we examined the lymphoproliferation response to this virus. Depletion studies demonstrated that both CD2+CD8+ and CD2+CD4+ cells contributed to lymphoproliferation, but to varying degrees upon stimulation with live and ultraviolet (UV) light-inactivated PRV. Flow cytometric analysis revealed the emergence of both CD2+CD8+ and CD2+CD4+ lymphoblastoid cells. To examine the contribution of specific viral proteins, we prepared immortalized porcine B cells of haplotype d/d that stably expressed a single PRV protein, and used these cells for in vitro stimulation of lymphocytes from PRV-immune miniature pigs of the same haplotype. Cells expressing PRV gB or gC induced proliferation. An immunization/challenge experiment showed that the lymphoproliferation response was stronger upon immunization with the virulent NIA-3 strain than with the attenuated Bartha strain. Upon challenge inoculation, the NIA-3-immunized pigs were almost completely immune, in contrast to the Bartha-immunized pigs. Such poorly protected pigs showed secondary B- and T-cell immune responses upon challenge. In contrast, the better protected NIA-3-immunized pigs did not show a secondary B-cell response. However, they developed a secondary lymphoproliferation response, which was quicker and stronger than in the Bartha-immunized pigs. This dichotomy between secondary B- and T-cell responses indicates that an effective T-cell memory response is able to quickly eliminate challenge virus in immune pigs, so preventing a secondary B-cell response.

To investigate how altered control of expression of the essential immediate-early (IE) gene of pseudorabies virus influences virus replication and virulence, we replaced the IE promoter with the tissue-specific promoters of the bovine cytokeratin IV gene (CKIV), the bovine cytokeratin VIb gene (CKVIb), or the inducible promoter of Drosophila heat shock gene HSP70. We compared expression of the IE gene of the wild-type virus and recombinant viruses in different cell types and at different temperatures and found that IE expression had become cell type or temperature dependent. When a recombinant virus was titrated on nonpermissive cells or was titrated at nonpermissive temperatures in vitro, the plating efficiency was reduced by more than 99%. Mice were inoculated subcutaneously (s.c.), intraperitoneally (i.p.), or intranasally (i.n.) with a dose equal to 100 times the 50% lethal dose of the wild-type virus. After inoculation with temperature-sensitive recombinant N-HSP, two (s.c.), two (i.p.), and four (i.n.) of five mice died. However, at this dose, recombinant N-CKIV, which contains a promoter specific for stratified epithelial tissue of the tongue mucosa, was not lethal when inoculated s.c. or i.p. but killed four mice when inoculated i.n. Recombinant N-CKVIb, which contains a promoter specific for the suprabasal layers of the epidermis, was not lethal after inoculation by any of the three routes. In explant cultures of nasal mucosa of pigs, replication of N-CKIV and N-CKVIb was not markedly reduced in the epithelium. However, in contrast to results obtained with wild-type virus, infection of the stroma was not observed. We conclude that the replicative ability and virulence of pseudorabies virus can be influenced by altering control of expression of the IE gene.

We studied in vivo recombination between a thymidine kinase (TK) negative, glycoprotein E (gE) negative, attenuated strain and a virulent strain of pseudorabies virus (PRV) in pigs. To simplify the detection of recombination we inserted different but overlapping (375 bp) parts of the E1 gene of classical swine fever virus into the gG locus of both virus strains. Recombination between the E1 sequences of these viruses results in reconstitution of the complete E1 coding sequence and expression of the E1 protein. Since E1 is highly immunogenic, we expected to detect in vivo recombination in co-inoculated pigs by the presence of serum antibodies against E1. However, after co-inoculation of pigs with high doses of both virus strains, we were unable to detect antibodies against E1, suggesting that in vivo recombination did not occur or remained below the detection limit. Analysis of individual progeny viruses showed that 13 out of 995 (1.3%) possessed a recombinant TK-negative gE-positive phenotype. In contrast, no E1-positive viruses were detected among 5000 analyzed. This result showed that in vivo recombination between the two virus strains did occur, but was much more frequent between the TK and gE loci than between the E1 sequences. Similar results were obtained in in vitro recombination experiments in which possible growth differences between the various virus strains were excluded. The different recombination frequencies could not be attributed to the difference in distance of the genetic loci since recombination between mutations at a distance of 266 bp in the TK gene occurred as frequent as recombination between the TK and gE genes which are separated by approximately 60 kilobasepairs. These results indicate that some property of the E1 sequence and/or the location of the E1 sequence within the PRV genome affects the frequency of recombination.

We studied in vivo recombination of pseudorabies virus (PRV) by inoculating mice with non-lethal mutants that carry a small deletion or insertion in the thymidine kinase (TK) gene or the ribonucleotide reductase (RR) gene. After co-inoculation of mice with two different mutants, homologous recombination between the viral genomes resulted in the generation of wild-type PRV that was highly lethal for mice. Thus, recombination could easily be assessed by monitoring survival of inoculated animals. Our results demonstrated that recombination was only detectable when high doses of virus were used. Intragenic recombination was more efficient between mutations in the TK gene than between mutations in the RR gene. Efficient intragenic recombination in the TK gene occurred between mutations which were separated by as few as 266 nucleotides. When two mutants were inoculated with an interval of 2 h, recombination still occurred. No recombination could be detected when the viruses were inoculated at the same time but in separate parts of the body. When inoculated separately, none of the mutants tested could be isolated from the brains of mice. Virus could be recovered from the brain, however, after co-inoculation. Surprisingly, of these viruses 36-39% possessed the parental mutant genotype. This observation indicates that complementation enables these mutants to replicate in the brain and suggests that complementation may contribute to pathogenicity of PRV.

We mutagenized and characterized a 41-kilobase-pair subgenomic cloned fragment from the unique long (UL) region of pseudorabies virus (PRV). Forty mutant clones, each carrying a single inserted oligonucleotide, were used for cotransfection with overlapping cloned viral DNA fragments. More than half of these transfections yielded viable virus mutants. Short viral DNA fragments, flanking the oligonucleotide insertions, were cloned and used as probes on Northern blots with RNA isolated from cells infected with wild-type PRV. In this way we were able to construct a partial transcriptional map of this region of the PRV genome. In addition, we used these probes in cross-hybridization studies with cloned genomic fragments from the prototype alphaherpesvirus herpes simplex virus type 1 (HSV-1). This allowed us to define homology between the corresponding regions of these viruses. Most viable PRV mutants were assayed for virulence in mice. Mutagenesis of the identified homologs of HSV-1 genes UL39 (encoding the large subunit of ribonucleotide reductase), UL40 (small subunit of ribonucleotide reductase), UL42 (DNA polymerase accessory factor), UL23 (thymidine kinase), UL21 (a capsid-associated protein), and UL12 (alkaline nuclease) completely abrogated or strongly reduced virulence.

The effects of the rolling-circle mode of replication and the generation of single-stranded DNA (ss DNA) on plasmid deletion formation between short direct repeats in Bacillus subtilis were studied. Deletion units consisting of direct repeats (9, 18, or 27 bp) that do or do not flank inverted repeats (300 bp) were introduced into various plasmid replicons that generate different amounts of ss DNA (from 0% to 40% of the total plasmid DNA). With ss DNA-generating rolling-circle-type plasmids, deletion frequencies between the direct repeats were 3- to 13-fold higher than in plasmids not generating ss DNA. When the direct repeats flanked inverted repeats the deletion frequencies in ss DNA-generating plasmids were increased by as much as 20- to 140-fold. These results support models for deletion formation based on template-switching errors during complementary strand synthesis of rolling-circle-type plasmids. The structural instability (deletion formation between short direct repeats) of the ss DNA-generating plasmid pTA1060 in B. subtilis was very low in the presence of a functional initiation site for complementary strand synthesis (minus origin). This observation suggests that it will be possible to develop stable host-vector cloning systems for B. subtilis.

In an earlier report, we described the construction of the genetically engineered pseudorabies virus strain 2.4N3A which does not express glycoprotein gI. Although this strain showed a strongly reduced virulence in 10-week-old seronegative pigs, it could still cause severe disease or death in 3-day-old piglets. To attenuate the strain further, we constructed mutants with a deletion in the viral thymidine kinase gene. One mutant strain, designated 783, has a deletion of 19 base pairs and was shown to be highly immunogenic and safe for vaccination of pigs against pseudorabies virus.

To study recombination between short homologous sequences in Escherichia coli we constructed plasmids composed of the pBR322 replicon, M13 replication origin and a recombination unit inserted within and inactivating a gene encoding chloramphenicol resistance. The unit was composed of short direct repeats (9, 18 or 27 bp) which flanked inverted repeats (0, 8 or 308 bp) and a gene encoding kanamycin resistance. Recombination between direct repeats restored a functional chloramphenicol resistance gene, and could be detected by a simple phenotype test. The plasmids replicated in a double-stranded form, using the pBR322 replicon, and generated single-stranded DNA when the M13 replication origin was activated. The frequency of chloramphenicol-resistant cells was low (10(-8)-10(-4] when no single-stranded DNA was synthesized but increased greatly (to 100%) after induction of single-stranded DNA synthesis. Recombination between 9 bp direct repeats entailed no transfer of DNA from parental to recombinant plasmids, whereas recombination between 18 or 27 bp repeats entailed massive transfer. The presence or length of inverted repeats did not alter the pattern of DNA transfer. From these results we propose that direct repeats of 9 bp recombine by a copy choice process, while those greater than or equal to 18 bp can recombine by a breakage-reunion process. Genome rearrangements detected in many organisms often occur by recombination between sequences less than 18 bp, which suggests that they may result from copy choice recombination.

Extracts of Methanosarcina barkeri reduced methanol and CO2 to CH4 in the presence of H2 and converted methanol stoichiometrically into CH4 and CO2 in the absence of H2. In dialyzed cell-free extracts these reactions were stimulated by 2-mercaptoethanesulfonic acid (coenzyme M) and some derivatives (acetyl and formylcoenzyme M and the oxidized form of coenzyme M), which could be converted to coenzyme M by enzyme systems present in the extracts. Methylcoenzyme M could not be used in these systems.

Gene II protein is the only phage-encoded protein required for the double-stranded DNA replication of the distantly related filamentous phages IKe and Ff (M13, fd and f1). Complementation studies have demonstrated that, despite a significant degree of homology between the nucleotide sequences of the gene II's of IKe and Ff and the core's (domains A) of their viral strand replication origins, the biological functions of the gene II proteins are not interchangeable. The specificity of these proteins is not determined by the nucleotide sequence (domain B) which is required for efficient initiation of viral strand replication of Ff. In fact, our data indicate that a sequence with a similar function as domain B in Ff does not form part of the viral strand replication origin of IKe.

Using precise excision as a model system, we have quantified the effect of direct repeats, inverted repeats and the size of the spacer between the repeats in the process of deletion formation in Bacillus subtilis. Both in the presence and absence of inverted repeats, the frequency of precise excision was strongly dependent on the direct repeat length. By increasing the direct repeat length from 9 bp to 18 and 27 bp, the precise excision frequency was raised by 3 and 4 orders of magnitude, respectively. In addition, irrespective of the direct repeat length, the presence of flanking inverted repeats enhanced the excision frequency by 3 orders of magnitude. Varying the inverted repeat length and the spacer size over a wide range did not significantly affect the excision frequencies. These results fit well into a model for deletion formation by slipped mispairing during replication of single-stranded plasmid DNA.

A versatile vector plasmid, pKUN9, has been constructed which, simply by infecting cells harboring this plasmid with either bacteriophage IKe or Ff (M13, fd, and fl), permits the selective packaging of both of its DNA strands into, single-stranded (ss) DNA-containing, phage-like particles. The plasmid, which is a derivative of plasmid pUC9 [Vieira and Messing, Gene 19 (1982) 269-276], contains in opposite orientations the replication origins and contiguous packaging signals of the distantly related filamentous phages IKe and Ff. As a result of the selective packaging, both strands of a DNA fragment cloned in pKUN9 can be obtained in a single-stranded form and can be sequenced by the dideoxy method using commercially available (+) and (-) sequencing primers. In addition, plasmid pKUN9 possesses all unique properties incorporated in the M13mp phages and the pUC plasmids.