Rapid Generation of a Recombinant Genotype VIII Newcastle Disease Virus (NDV) Using Full-Length Synthetic cDNA.

Rescue of (-)ssRNA viruses involves the sequential assembly and cloning of the full-length cDNA, which is often a challenging and time-consuming process. The objective of this study was to develop a novel method to rapidly clone the full-length cDNA of a very virulent NDV by only one assembly step. A completely synthetic 15 kb cDNA of a Malaysian genotype VIII NDV known as strain AF2240-I with additional flanking BsmBI sites was synthesised. However, to completely follow the rule-of-six, the additional G residues that are traditionally added after the T7 promoter transcription initiation site were not synthesised. The synthetic fragment was then cloned into low-copy number transcription vector pOLTV5-phiX between the T7 promoter and HDV Rz sequences through digestion with BbsI. The construct was co-transfected with helper plasmids into BSRT7/5 cells. A recombinant NDV called rAF was successfully rescued using transfection supernatant harvested as early as 16 h post-transfection. Virus from each passage showed an intracerebral pathogenicity index (ICPI) and a mean death time (MDT) similar to the parent strain AF2240-I. Moreover, rAF possessed an introduced mutation which was maintained for several passages. The entire rescue using the one-step assembly procedure was completed within a few weeks, which is extremely fast compared to previously used methods.

Genome-wide gene expression analysis of anguillid herpesvirus 1.

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.

Anguillid herpesvirus 1 transcriptome.

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.

Recovery of avirulent, thermostable Newcastle disease virus strain NDV4-C from cloned cDNA and stable expression of an inserted foreign gene.

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.

Intranasal administration of a live-attenuated recombinant newcastle disease virus expressing the SARS-CoV-2 Spike protein induces high neutralizing antibody levels and protects from experimental challenge infection in hamsters.

The emergence of SARS-CoV-2 in December 2019 resulted in the COVID-19 pandemic. Recurring disease outbreaks repeatedly overloaded the public health sector and severely affected the global economy. We developed a candidate COVID-19 vaccine based on a recombinant Newcastle disease virus (NDV) vaccine vector, encoding a pre-fusion stabilized full-length Spike protein obtained from the original SARS-CoV-2 Wuhan isolate. Vaccination of hamsters by intra-muscular injection or intra-nasal instillation induced high neutralizing antibody responses. Intranasal challenge infection with SARS-CoV-2 strain Lelystad demonstrated that both vaccination routes provided partial protection in the upper respiratory tract, and almost complete protection in the lower respiratory tract, as measured by suppressed viral loads and absence of histological lung lesions. Activity wheel measurements demonstrated that animals vaccinated by intranasal inoculation rapidly recovered to normal activity. NDV constructs encoding the spike of SARS-CoV-2 may be attractive candidates for development of intra-nasal COVID-19 booster vaccines.

Virulence of Newcastle disease virus: what is known so far?

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.

Identification and localization of the structural proteins of anguillid herpesvirus 1.

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.

Complete genome sequence and taxonomic position of anguillid herpesvirus 1.

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.

Exploring the Prospects of Engineered Newcastle Disease Virus in Modern Vaccinology.

Many traditional vaccines have proven to be incapable of controlling newly emerging infectious diseases. They have also achieved limited success in the fight against a variety of human cancers. Thus, innovative vaccine strategies are highly needed to overcome the global burden of these diseases. Advances in molecular biology and reverse genetics have completely restructured the concept of vaccinology, leading to the emergence of state-of-the-art technologies for vaccine design, development and delivery. Among these modern vaccine technologies are the recombinant viral vectored vaccines, which are known for their incredible specificity in antigen delivery as well as the induction of robust immune responses in the vaccinated hosts. Although a number of viruses have been used as vaccine vectors, genetically engineered Newcastle disease virus (NDV) possesses some useful attributes that make it a preferable candidate for vectoring vaccine antigens. Here, we review the molecular biology of NDV and discuss the reverse genetics approaches used to engineer the virus into an efficient vaccine vector. We then discuss the prospects of the engineered virus as an efficient vehicle of vaccines against cancer and several infectious diseases of man and animals.

Development of an Effective and Stable Genotype-Matched Live Attenuated Newcastle Disease Virus Vaccine Based on a Novel Naturally Recombinant Malaysian Isolate Using Reverse Genetics.

Genotype VII Newcastle disease viruses are associated with huge economic losses in the global poultry industry. Despite the intensive applications of vaccines, disease outbreaks caused by those viruses continue to occur frequently even among the vaccinated poultry farms. An important factor in the suboptimal protective efficacy of the current vaccines is the genetic mismatch between the prevalent strains and the vaccine strains. Therefore, in the present study, an effective and stable genotype-matched live attenuated Newcastle disease virus (NDV) vaccine was developed using reverse genetics, based on a recently isolated virulent naturally recombinant NDV IBS025/13 Malaysian strain. First of all, the sequence encoding the fusion protein (F) cleavage site of the virus was modified in silico from virulent polybasic (RRQKRF) to avirulent monobasic (GRQGRL) motif. The entire modified sequence was then chemically synthesized and inserted into pOLTV5 transcription vector for virus rescue. A recombinant virus termed mIBS025 was successfully recovered and shown to be highly attenuated based on OIE recommended pathogenicity assessment indices. Furthermore, the virus was shown to remain stably attenuated and retain the avirulent monobasic F cleavage site after 15 consecutive passages in specific-pathogen-free embryonated eggs and 12 passages in one-day-old chicks. More so, the recombinant virus induced a significantly higher hemagglutination inhibition antibody titre than LaSota although both vaccines fully protected chicken against genotype VII NDV induced mortality and morbidity. Finally, mIBS025 was shown to significantly reduce both the duration and quantity of cloacal and oropharyngeal shedding of the challenged genotype VII virus compared to the LaSota vaccine. These findings collectively indicate that mIBS025 provides a better protective efficacy than LaSota and therefore can be used as a promising vaccine candidate against genotype VII NDV strains.

Diagnostic and Vaccination Approaches for Newcastle Disease Virus in Poultry: The Current and Emerging Perspectives.

Newcastle disease (ND) is one of the most devastating diseases that considerably cripple the global poultry industry. Because of its enormous socioeconomic importance and potential to rapidly spread to naïve birds in the vicinity, ND is included among the list of avian diseases that must be notified to the OIE immediately upon recognition. Currently, virus isolation followed by its serological or molecular identification is regarded as the gold standard method of ND diagnosis. However, this method is generally slow and requires specialised laboratory with biosafety containment facilities, making it of little relevance under epidemic situations where rapid diagnosis is seriously needed. Thus, molecular based diagnostics have evolved to overcome some of these difficulties, but the extensive genetic diversity of the virus ensures that isolates with mutations at the primer/probe binding sites escape detection using these assays. This diagnostic dilemma leads to the emergence of cutting-edge technologies such as next-generation sequencing (NGS) which have so far proven to be promising in terms of rapid, sensitive, and accurate recognition of virulent Newcastle disease virus (NDV) isolates even in mixed infections. As regards disease control strategies, conventional ND vaccines have stood the test of time by demonstrating track record of protective efficacy in the last 60 years. However, these vaccines are unable to block the replication and shedding of most of the currently circulating phylogenetically divergent virulent NDV isolates. Hence, rationally designed vaccines targeting the prevailing genotypes, the so-called genotype-matched vaccines, are highly needed to overcome these vaccination related challenges. Among the recently evolving technologies for the development of genotype-matched vaccines, reverse genetics-based live attenuated vaccines obviously appeared to be the most promising candidates. In this review, a comprehensive description of the current and emerging trends in the detection, identification, and control of ND in poultry are provided. The strengths and weaknesses of each of those techniques are also emphasised.

Genotype Diversity of Newcastle Disease Virus in Nigeria: Disease Control Challenges and Future Outlook.

Newcastle disease (ND) is one of the most important avian diseases with considerable threat to the productivity of poultry all over the world. The disease is associated with severe respiratory, gastrointestinal, and neurological lesions in chicken leading to high mortality and several other production related losses. The aetiology of the disease is an avian paramyxovirus type-1 or Newcastle disease virus (NDV), whose isolates are serologically grouped into a single serotype but genetically classified into a total of 19 genotypes, owing to the continuous emergence and evolution of the virus. In Nigeria, molecular characterization of NDV is generally very scanty and majorly focuses on the amplification of the partial F gene for genotype assignment. However, with the introduction of the most objective NDV genotyping criteria which utilize complete fusion protein coding sequences in phylogenetic taxonomy, the enormous genetic diversity of the virus in Nigeria became very conspicuous. In this review, we examine the current ecological distribution of various NDV genotypes in Nigeria based on the available complete fusion protein nucleotide sequences (1662 bp) in the NCBI database. We then discuss the challenges of ND control as a result of the wide genetic distance between the currently circulating NDV isolates and the commonest vaccines used to combat the disease in the country. Finally, we suggest future directions in the war against the economically devastating ND in Nigeria.

The effect on pathogenesis of Newcastle disease virus LaSota strain from a mutation of the fusion cleavage site to a virulent sequence.

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.

Harmonization of European laboratory response networks by implementing CWA 15793: use of a gap analysis and an "insider" exercise as tools.

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.

Newcastle disease virus outbreaks: vaccine mismatch or inadequate application?

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.

Two genetically closely related pigeon paramyxovirus type 1 (PPMV-1) variants with identical velogenic fusion protein cleavage sites but with strongly contrasting 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.

The nsp1alpha and nsp1 papain-like autoproteinases are essential for porcine reproductive and respiratory syndrome virus RNA synthesis.

The two N-terminal cleavage products, nsp1alpha and nsp1beta, of the replicase polyproteins of porcine reproductive and respiratory syndrome virus (PRRSV) each contain a papain-like autoproteinase domain, which have been named PCPalpha and PCPbeta, respectively. To assess their role in the PRRSV life cycle, substitutions and deletions of the presumed catalytic cysteine and histidine residues of PCPalpha and PCPbeta were introduced into a PRRSV infectious cDNA clone. Mutations that inactivated PCPalpha activity completely blocked subgenomic mRNA synthesis, but did not affect genome replication. In contrast, mutants in which PCPbeta activity was blocked proved to be non-viable and no sign of viral RNA synthesis could be detected, indicating that the correct processing of the nsp1beta/nsp2 cleavage site is essential for PRRSV genome replication. In conclusion, the data presented here show that a productive PRRSV life cycle depends on the correct processing of both the nsp1alpha/nsp1beta and nsp1beta/nsp2 junctions.

Virulence of Newcastle disease virus is determined by the cleavage site of the fusion protein and by both the stem region and globular head of the haemagglutinin-neuraminidase protein.

Virulence of Newcastle disease virus (NDV) is mainly determined by the amino acid sequence surrounding the fusion (F) protein cleavage site, since host proteases that cleave the F protein of virulent strains are present in more tissues than those that cleave the F protein of non-virulent strains. Nevertheless, comparison of NDV strains that carry exactly the same F protein cleavage site shows that significant differences in virulence still exist. For instance, virulent field strain Herts/33 with the F cleavage site 112RRQRRF117 had an intracerebral pathogenicity index of 1.88 compared with 1.28 for strain NDFLtag, which has the same cleavage site. This implies that additional factors contribute to virulence. After generating an infectious clone of Herts/33 (FL-Herts), we were able to map the location of additional virulence factors by exchanging sequences between FL-Herts and NDFLtag. The results showed that, in addition to the F protein cleavage site, the haemagglutinin-neuraminidase (HN) protein also contributed to virulence. The effect of the HN protein on virulence was most prominent after intravenous inoculation. Interestingly, both the stem region and the globular head of the HN protein seem to be involved in determining virulence.

Recombinant Newcastle disease virus as a viral vector: effect of genomic location of foreign gene on gene expression and virus replication.

Newcastle disease virus (NDV) was examined for its suitability as a vector for the expression and delivery of foreign genes for vaccination and gene therapy. A reporter gene encoding human secreted alkaline phosphatase (SEAP) was inserted as an additional transcription unit at four different positions in the NDV genome, between the NP and P, M and F, and HN and L genes and behind the L gene. Eight infectious recombinant NDV (rNDV) viruses, four in the non-virulent strain NDFL and four in the virulent derivative NDFLtag, were generated by reverse genetics. SEAP expression levels, replication kinetics and virus yield were examined. Replication kinetics of the rNDV viruses in primary chicken embryo fibroblasts showed that the insertion of an additional gene resulted in a delay in the onset of replication. This effect was most prominent when the gene was inserted between the NP and P genes. With the exception of the strain that carried the SEAP gene behind the L gene, all recombinant strains expressed high levels of SEAP, both in cell culture and in embryonated chicken eggs. In embryonated eggs, the rNDV viruses showed a 2.6- to 5.6-fold (NDFL) or 2.1- to 8.1-fold (NDFLtag) reduction in yield compared with the parent strains. These results show that foreign genes can be inserted at different positions in the NDV genome without severely affecting replication efficiency or virus yield.

Effect of fusion protein cleavage site mutations on virulence of Newcastle disease virus: non-virulent cleavage site mutants revert to virulence after one passage in chicken brain.

Virulence of Newcastle disease virus (NDV) is mainly determined by the amino acid sequence of the fusion (F0) protein cleavage site. Full-length NDV cDNA clone pNDFL was used to generate infectious NDV with defined mutations in the F0 cleavage site (RRQRR downward arrow L, GRQGR downward arrow F, RRQGR downward arrow F, RGQRR downward arrow F and RKQKR downward arrow F). All the mutants were viable and the mutations were maintained after virus propagation in embryonated eggs. The mutants showed single-cell infections on chicken embryo fibroblasts, which suggested that they were non-virulent. However, virulence tests in 1-day-old chickens resulted in an intracerebral pathogenicity index (ICPI) between 0 and 1.3. Moreover, virulent virus was isolated from chickens that had died in the virulence tests. Subsequent sequence analysis showed that the mutants RRQRR downward arrow L, RRQGR downward arrow F, RGQRR downward arrow F and RKQKR downward arrow F gave rise to the appearance of revertants containing the virulent cleavage site RRQ(K/R)R downward arrow F and an ICPI of 1.4 or higher. This indicated that reversion to virulence was caused by alteration of the amino acid sequence of the F0 cleavage site from a non-virulent to a virulent type. Furthermore, the ICPI of the revertants was higher than that of cDNA-derived strain NDFLtag, which has the same cleavage site, RRQRR downward arrow F (ICPI=1.3). NDFLtag(Pass), which was isolated from dead chickens after intracerebral inoculation of NDFLtag, also showed an increase in the ICPI from 1.3 to 1.5. This study proves that reversion to virulence occurs within non-virulent NDV populations and that the virulence may increase after one passage in chicken brain.