Phylogenetic analysis of dengue virus types 1 and 3 isolated in Jakarta,Indonesia in 1988

Dengue viruses are mosquito-borne viruses that cause dengue fever and dengue hemorrhagic fever, both of which are globally important diseases. These viruses have evolved in a transmission cycle between human hosts and mosquito vectors in various tropical and subtropical environments. We previously isolated three strains of dengue type 1 virus (DENV1) and 14 strains of dengue type 3 virus (DENV3) during an outbreak of dengue fever and dengue hemorrhagic fever in Jakarta, Indonesia in 1988. Here, we compared the nucleotide sequences of the entire envelope protein-coding region among these strains. The isolates were 97.6–100% identical for DENV1 and 98.8–100% identical for DENV3. All DENV1 isolates were included in two different clades of genotype IV and all DENV3 isolates were included in a single clade of genotype I. For DENV1, three Yap Island strains isolated in 2004 were the only strains closely related to the present isolates; the recently circulated Indonesian strains were in different clades. Molecular clock analyses estimated that ancestors of the genotype IV strains of DENV1 have been indigenous in Indonesia since 1948. We predict that they diverged frequently around 1967 and that their offspring distributed to Southeast Asia, the Western Pacific, and Africa. For DENV3, the clade containing all the present isolates also contained strains isolated from other Indonesian regions and other countries including Malaysia, Singapore, China, and East Timor from 1985–2010. Molecular clock analyses estimated that the common ancestor of the genotype I strains of DENV3 emerged in Indonesia around 1967 and diverged frequently until 1980, and that their offspring distributed mainly in Southeast Asia. The first dengue outbreak in 1968 and subsequent outbreaks in Indonesia might have influenced the divergence and distribution of the DENV1 genotype IV strains and the DENV3 genotype I strains in many countries.     

Molecular characterization of chicken-derived genotype VIId Newcastle disease virus isolates in China during 2005–2012 reveals a new length in hemagglutinin–neuraminidase

Newcastle disease (ND) is one of the most important diseases of poultry, and causes severe economic losses in the global poultry industry. Although all Newcastle disease virus (NDV) isolates belong to a single serotype, significant genetic diversity has been described between different NDV isolates. Here, we report the molecular characterization of 23 virulent genotype VIId NDV isolates of class II circulating in China. Phylogenetic construction and analysis revealed the existence of distinctly genomic and amino acid differences that clearly distinguished these isolates from other typical NDV genotypes and vaccine strains. We also report a new 582-amino-acid hemagglutinin–neuraminidase in genotype VII NDV strains. This is believed to be the first study to investigate systematically the most predominant NDV strains, and provides more information on the genetic nature of genotype VIId NDV of class II circulating in China.     

A vaccine candidate of attenuated genotype VII Newcastle disease virus generated by reverse genetics

Genotype VII Newcastle disease virus (NDV) has been documented as the predominant epidemic genotype in China and some other Asian countries since 1990s. Recent work has demonstrated that NDV vaccines phylogenetically closer to epidemic viruses provide better protection than conventional vaccines in terms of reducing virus shedding and transmission. Since there is currently no available vaccine which possesses a close antigenic relationship to the prevalent virulent NDV, a new vaccine to protect against the infection of this genotype NDV is in urgent need. Here, we describe the generation of a pathogenicity-attenuated genotype VII NDV (NDV/ZJ1HN) from a velogenic NDV by mutating the velogenic amino acid motif at the F protein cleavage site using reverse genetics techniques. The attenuated-pathogenicity of NDV/ZJ1HN was confirmed by examination of mean death time (MDT) in embryonated eggs and intracerebral pathogenicity index (ICPI) in day-old chickens. Subsequently, 2 weeks old birds were immunized with live and inactivated NDV/ZJ1HN-based vaccines and challenged 3 or 4 weeks post-immunization with a lethal dose of a virulent genotype VII NDV strain. Results showed that NDV/ZJ1HN effectively protected the vaccinated birds from morbidity and mortality against genotype VII virus challenge and significantly reduced virus shedding from the vaccinated birds when compared with La Sota vaccinated animals, suggesting that NDV/ZJ1HN is a promising vaccine candidate for the control of current ND epidemic in China.     

Temporal,geographic, and host distribution of avian paramyxovirus 1 (Newcastle disease virus)

Newcastle disease is caused by virulent forms of avian paramyxovirus of serotype 1 (APMV-1) and has global economic importance. The disease reached panzootic proportions within two decades after first being identified in 1926 in the United Kingdom and Indonesia and still remains endemic in many countries across the world. Here we review information on the host, temporal, and geographic distribution of APMV-1 genetic diversity based on the evolutionary systematics of the complete coding region of the fusion gene. Strains of APMV-1 are phylogenetically separated into two classes (class I and class II) and further classified into genotypes based on genetic differences. Class I viruses are genetically less diverse, generally present in wild waterfowl, and are of low virulence. Class II viruses are genetically and phenotypically more diverse, frequently isolated from poultry with occasional spillovers into wild birds, and exhibit a wider range of virulence. Waterfowl, cormorants, and pigeons are natural reservoirs of all APMV-1 pathotypes, except viscerotropic velogenic viruses for which natural reservoirs have not been identified. Genotypes I and II within class II include isolates of high and low virulence, the latter often being used as vaccines. Viruses of genotypes III and IX that emerged decades ago are now isolated rarely, but may be found in domestic and wild birds in China. Containing only virulent viruses and responsible for the majority of recent outbreaks in poultry and wild birds, viruses from genotypes V, VI, and VII, are highly mobile and have been isolated on different continents. Conversely, virulent viruses of genotypes XI (Madagascar), XIII (mainly Southwest Asia), XVI (North America) and XIV, XVII and XVIII (Africa) appear to have a more limited geographic distribution and have been isolated predominantly from poultry.     

A genotype VII Newcastle disease virus-like particles confer full protection with reduced virus load and decreased virus shedding

Newcastle disease (ND) is one of the most severe avian infectious disease inflicting a great loss on poultry industry worldwide. The control of ND relies on proper vaccination strategies. The vaccine strains of Newcastle disease virus (NDV) mainly belong to genotype I, II or III, which cannot fully prohibit virus shedding against the prevalent genotype VII virulent strain attack. To develop a safe, genotype matched vaccine candidate, we employed a bac-to-bac expression system and constructed a genotype VII NDV strain based virus-like particles (NDV VLPs). It was constructed with NDV M protein as the skeleton, and protective antigen F and HN proteins displayed on the surface. The NDV VLPs exhibited a similar appearance to the live NDV particles, but with denser F and HN proteins displayed on the surface. The immunization assay indicated that NDV VLPs stimulated a longer protection period, less tissue virus loading and shorter virus shedding period than the commercialized LaSota-formulated vaccine when challenged with genotype VII NDV strain. These results proposed the potential role of NDV VLPs as an alternative to current live genotype unmatched vaccine for the control and eliminate NDV in the avian flocks.     

Molecular and antigenic characteristics of Newcastle disease virus isolates from domestic ducks in China

Newcastle disease (ND) is one of the most devastating diseases to the poultry industry. The causative agents of ND are virulent strains of Newcastle disease virus (NDV), which are members of the genus Avulavirus within the family Paramyxoviridae. Waterfowl, such as ducks and geese, are generally considered potential reservoirs of NDV and may show few or no clinical signs when infected with viruses that are obviously virulent in chickens. However, ND outbreaks in domestic waterfowl have been frequently reported in many countries in the past decade. In this study, 18 NDV strains isolated from domestic ducks in southern and eastern China, between 2005 and 2013, were genetically and phylogenetically characterized. The complete genomes of these strains were sequenced, and they exhibited genome sizes of 15,186 nucleotides (nt), 15,192 nt, and 15,198 nt, which follow the “rule of six” that is required for the replication of NDV strains. Based on the cleavage site of the F protein and pathogenicity tests in chickens, 17 of our NDV isolates were categorized as lentogenic viruses, and one was characterized as a velogenic virus. Phylogenetic analysis based on the partial sequences of the F gene and the complete genome sequences showed that there are at least four genotypes of NDV circulating in domestic ducks; GD1, AH224, and AH209 belong to genotypes VIId, Ib, and II of class II NDVs, respectively, and the remaining 15 isolates belong to genotype 1b of class I NDVs. Cross-reactive hemagglutination inhibition tests demonstrated that the antigenic relatedness between NDV strains may be associated with their genotypes, rather than their hosts. These results suggest that though those NDV isolates were from duck, they still don’t form a phylogenetic group because they came from the same species; however, they may play an important role in promoting the evolution of NDVs.     

Molecular and phylogenetic characterization based on the complete genome of a virulent pathotype of Newcastle disease virus isolated in the 1970s in Brazil

Newcastle disease (ND) is caused by the avian paramyxovirus type 1 (APMV-1) or Newcastle disease virus (NDV) that comprises a diverse group of viruses with a single-stranded, negative-sense RNA genome. ND is one of the most important diseases of chickens, because it severely affects poultry production worldwide. In the 1970s, outbreaks of virulent ND were recorded in Brazil, and the strain APMV-1/Chicken/Brazil/SJM/75 (SJM) of NDV was isolated. This strain was characterized as highly pathogenic for chickens but not pathogenic for other bird species. Here we present the complete genome of NDV strain SJM and investigate the phylogenetic relationships of this virus with other NDV strains in terms of genome and proteins composition, as well as characterizing its evolution process. The NDV strain SJM is categorized as a velogenic virus and the complete genome is 15,192 nucleotides in length, consisting of six genes in the order 3′-NP-P-M-F-HN-L-5′. The presence of the major pathogenic determinant of NDV strains (¹¹²R-R-Q-K-R↓F¹¹⁷) was identified in the Fusion protein of the NDV strain SJM. In addition, phylogenetic analysis classified the NDV strain SJM as a member of class II, genotype V, and indicates that this virus help us in the understanding of the evolutionary process of strains belonging to this genotype. This study contributes to the growing interest involving the characterization of NDV isolates to improve our current understanding about the epidemiology, surveillance and evolution of the pathogenic strains.     

中国15个民族HBV基因型及基因亚型分布特征分析

目的 了解我国不同民族人群感染HBV的基因型及基因亚型分布特征。方法 利用多阶段分层整群随机抽样结合系统抽样的方法从2020年全国乙型肝炎血清流行病学调查HBsAg阳性样本库中抽取样本,利用巢式PCR扩增阳性样本HBV S区基因,构建系统发育树分析判定HBV基因型和亚型,结合社会人口学资料进行综合分析。结果 成功扩增15个民族的1 539份样本,检出B、C、D、I型和C/D重组型5种基因型。B型占比较高的民族包括汉（74.52%,623/836）、壮（49.28%,34/69）、彝（53.19%,25/47）、苗（94.12%,32/34）和布依族（81.48%,22/27）;C型占比较高的民族为瑶族（70.91%,39/55）;D型占比较高的民族为维吾尔族（83.78%,31/37）;C/D重组型占比较高的民族为藏族（92.35%,326/353）;检出I型11例中,8例来自于壮族。除藏族外,各民族的B型中B2亚型均>80.00%;在C型中,C2亚型占比较高的民族包括汉、藏、彝、维吾尔、蒙古、满、回和苗族8个民族,C5亚型占比较高的民族包括壮（55.56%,15/27）和瑶族（84.62%,33/39）;在D型中,彝族均为D3亚型,维吾尔和哈萨克族均为D1亚型。C/D1和C/D2亚型在藏族占比分别为43.06%（152/353）和49.29%（174/353）。I型均为I1亚型。结论 我国15个民族中发现HBV 5种基因型和15种基因亚型,不同民族的HBV基因型和基因亚型分布差异明显。     

Updated unified phylogenetic classification system and revised nomenclature for Newcastle disease virus

Several Avian paramyxoviruses 1 (synonymous with Newcastle disease virus or NDV, used hereafter) classification systems have been proposed for strain identification and differentiation. These systems pioneered classification efforts; however, they were based on different approaches and lacked objective criteria for the differentiation of isolates. These differences have created discrepancies among systems, rendering discussions and comparisons across studies difficult. Although a system that used objective classification criteria was proposed by Diel and co-workers in 2012, the ample worldwide circulation and constant evolution of NDV, and utilization of only some of the criteria, led to identical naming and/or incorrect assigning of new sub/genotypes. To address these issues, an international consortium of experts was convened to undertake in-depth analyses of NDV genetic diversity. This consortium generated curated, up-to-date, complete fusion gene class I and class II datasets of all known NDV for public use, performed comprehensive phylogenetic neighbor-Joining, maximum-likelihood, Bayesian and nucleotide distance analyses, and compared these inference methods. An updated NDV classification and nomenclature system that incorporates phylogenetic topology, genetic distances, branch support, and epidemiological independence was developed. This new consensus system maintains two NDV classes and existing genotypes, identifies three new class II genotypes, and reduces the number of sub-genotypes. In order to track the ancestry of viruses, a dichotomous naming system for designating sub-genotypes was introduced. In addition, a pilot dataset and sub-trees rooting guidelines for rapid preliminary genotype identification of new isolates are provided. Guidelines for sequence dataset curation and phylogenetic inference, and a detailed comparison between the updated and previous systems are included. To increase the speed of phylogenetic inference and ensure consistency between laboratories, detailed guidelines for the use of a supercomputer are also provided. The proposed unified classification system will facilitate future studies of NDV evolution and epidemiology, and comparison of results obtained across the world.     

2005年山东省流动人口成人麻疹暴发的病毒基因特征分析

[目的]确定麻疹病毒株的来源、传播途径及变异情况,探讨流动人口和成人麻疹发病的特点。[方法]2005年在山东省邹平县麻疹暴发疫区采集病人血清和咽拭子标本,进行血清学、病原学和分子流行病学特征检测。[结果]从6例病人咽拭子标本分离4个病毒流行株．均为H1基因型．3株属于H1a．1株属于H1b。4株之间核苷酸、氨基酸同源性为95．80％～100．00％、97．30％～100．00％,分成2个亚支、2个亚型,与H2基因型参考株中国疫苗株S191之间存在较大的遗传差异（8．60％）．与山东省2005年另外7株病毒株的基因型别分布相似。3株H1a病毒株又分为2个小分支．1株与2005年另外2株H1b病毒株（枣庄市、泰安市）的亲缘关系最近,与2000年的3株H1b病毒株（淄博市）亲缘关系较远．不属于同一祖先传播链病毒株。[结论]2005年邹平县成人麻疹暴发是由H1a、H1b两个亚型病毒株引起．以本地流行优势基因亚型H1a为主,H1b亚型为外来输入并在山东省少数市地传播。     

Genetic variation in V gene of class II Newcastle disease virus

The genetic variation and molecular evolution of the V gene of the class II Newcastle disease virus (NDV) isolates with genotypes I–XVIII were determined using bioinformatics. Results indicated that low homology existed in different genotype viruses, whereas high homology often for the same genotypes, exception may be existed within genotypes I, V, VI, and XII. Sequence analysis showed that the genetic variation of V protein was consistent with virus genotype, and specific signatures on the V protein for nine genotypes were identified. Phylogenetic analysis demonstrated that the phylogenetic trees were highly consistent between the V and F genes, with slight discrepancies in the sub-genotypes. Evolutionary rate analyses based on V and F genes revealed the evolution rates varied in genotypes. These data indicate that the genetic variation of V protein is genotype-related and will help in elucidating the molecular evolution of NDV.     

2002-2007年福建省麻疹野病毒的分离与基因特性分析

[目的]收集福建省流行的麻疹野毒株,分析麻疹野病毒的基因特征。[方法]应用B95a或Vero/slam细胞,从疑似病例的咽拭子或尿液标本分离麻疹病毒,用RT-PCR方法扩增分离株病毒的N基因3'端450个核苷酸片段,分析其基因型别及遗传特征。[结果]2002年、2006-2007年共从5个设区市分离了14株麻疹野病毒,均为H1a基因亚型。14株病毒核苷酸同源性为97.8%~100%,在遗传树图中分属于4个独立分支,具有不同的来源。福建省分离株与H1基因型中国代表株、H2基因型及疫苗株S191相比,与S191差异性最大(核苷酸同源性91.0%~92.0%,氨基酸同源性88.1%~90.1%)。MV06-32、MV07-30、MV07-39和MV07-46这4株间N基因3'端450个核苷酸间同源性为100%,显示不同地区和年份来源的病毒株具有高度同源性。[结论]福建省目前监测到的麻疹野病毒均为H1基因型、H1a基因亚型,未发现其它型别;不同地区或同一地区内存在多传播链同时循环,也存在同一野病毒株可在不同地区、不同年份间持续循环传播。     

Genetic diversity of avian paramyxovirus type 1: Proposal for a unified nomenclature and classification system of Newcastle disease virus genotypes

The avian paramyxovirus type 1 (APMV-1), or Newcastle disease virus (NDV), comprise a diverse group of viruses with a single-stranded, negative-sense RNA genome. Historically, two systems have been simultaneously used to classify NDV isolates into lineages or genotypes, generating confusion in the nomenclature and discrepancies in the assignment of genetic groups. In the present study we assessed the genetic diversity of the avian paramyxovirus type-1 (APMV-1) and propose a unified nomenclature and a classification system based on objective criteria to separate NDV into genotypes. Complete F gene sequences of class I (n = 110) and class II (n = 602) viruses were used for the phylogenetic reconstruction and to identify distinct taxonomic groups. The mean interpopulational evolutionary distance was estimated (10%) and set as the cutoff value to assign new genotypes. Results of our study revealed that class I viruses comprise a single genotype, while class II contains 15 genetic groups including 10 previously established (I–IX, and XI) and five new genotypes (X, XII, XIII, XIV and XV). Sub-genotypes were identified among class I and class II genotypes. Adoption of a unified nomenclature and of objective criteria to classify NDV isolates will facilitate studies on NDV epidemiology, evolution, disease control and diagnostics.     

Can genotype mismatch really affect the level of protection conferred by Newcastle disease vaccines against heterologous virulent strains?

Newcastle disease (ND), caused by virulent class II avian paramyxovirus 1 (Newcastle disease virus, NDV), occurs sporadically in poultry despite their having been immunized with commercial vaccines. These vaccines were all derived from NDV strains isolated around 70 years ago. Since then, class II NDV strains have evolved into 18 genotypes. Whether the vaccination failure results from genotype mismatches between the currently used vaccine strains and field-circulating velogenic strains or from an impaired immune response in the vaccination remains unclear. To test the first hypothesis, we performed a heterologous genotype II vaccine/genotype XI challenge in one-day old specific pathogen free (SPF) chicks and reproduced viral shedding. We then produced two attenuated strains of genotype II and XI by reverse genetics and used them to immunize two-week old SPF chickens that were subsequently challenged with velogenic strains of genotypes II, VII and XI. We found that both vaccines could induce antibodies with hemagglutination inhibition titers higher than 6.5 log₂. Vaccination also completely prevented disease, viral shedding in swabs, and blocked viral replication in tissues from different genotypes in contrast to unvaccinated chickens that died shortly after challenge. Taken together, our results support the hypothesis that, in immunocompetent poultry, genotype mismatch is not the main reason for vaccination failure.     

Continuous dengue type 1 virus genotype shifts followed by co-circulation,clade shifts and subsequent disappearance in Surabaya,Indonesia, 2008–2013

Four serotypes of dengue virus (DENV-1 to DENV-4) and their genotypes are distributed in tropical and subtropical regions. Indonesia has been recently suggested as the origin of some dengue virus genotypes. In Surabaya, the second biggest city of Indonesia, we previously reported a shift of the predominantly circulating serotype from DENV-2 to DENV-1 in November 2008, followed by a genotype shift of DENV-1 from genotype IV (GIV) to genotype I (GI) in September 2009, based on nucleotide sequences in the envelope protein coding region. Since then, GI strains had predominantly circulated until December 2010. In this report, we investigated further DENV-1 transitions in Surabaya during 2011–2013 in order to comprehend dengue dynamics during 2008–2013 in more detail. From January 2011 through December 2011, only GIV strains were isolated, indicating that a genotype shift again took place from GI to GIV. In January 2012, GI and GIV strains started co-circulating, which continued until June 2013. To further investigate this phenomenon, analysis was performed at a clade level. GI and GIV strains isolated in Surabaya formed four and three distinct clades, respectively. Concomitant with co-circulation, new clade strains appeared in both genotypes. In contrast, some previously circulating clades were not isolated during co-circulation, indicating clade shifts. Among our Surabaya isolates, nucleotide and amino acid differences in the E region were, respectively, 1.0–2.3% and 0.2–1.0% for GI isolates and 2.0–6.3% and 0.0–1.8% for GIV isolates. Several characteristic amino acid substitutions in the envelope ectodomain were observed in some clades. After July 2013, DENV-1 strains were not isolated and were replaced with DENV-2. This study showed that continuous shifts of more than one genotype resulted in their co-circulation and subsequent disappearance and suggested the relevance of clade replacement to genotype co-circulation and disappearance in Surabaya.     

四川省2003～2005年麻疹野病毒分离株基因特征分析

目的了解四川省2003~2005年流行的麻疹野病毒分离株基因特征,为控制、消除麻疹提供科学依据。方法用B95a、Vero/Slam细胞从疑似麻疹爆发和散发患者的标本中分离麻疹病毒,通过逆转录-聚合酶链反应(RT-PCR)从分离到的22株麻疹病毒中扩增出核蛋白(nucleoprotein,N)基因羧基末端676个核苷酸片段,再对扩增产物进行核苷酸序列测定和分析,并以C末端456个核苷酸片段构建基因亲缘性关系树,进行核苷酸、氨基酸同源性分析。结果四川省2003~2005年从6个市(自治州,下同)分离的22株麻疹野病毒全部为H1基因型,除2株为H1b基因亚型外,其余均为H1a基因亚型。22株麻疹野病毒的核苷酸同源性为96.2%~100.0%,氨基酸同源性为95.3%~100.0%。四川省11株麻疹病毒代表株与中国S191疫苗株的核苷酸同源性为90.0%~92.5%,氨基酸同源性为86.7%~91.6%。结论四川省2003~2005年流行的麻疹野病毒以H1a为绝对优势基因亚型,H1b为弱势基因亚型,未发现H1c基因亚型。其中以H1a基因亚型为主的病毒株引起的多个传播链造成四川省各市的麻疹流行。     

Antigenic variation of LaSota and genotype VII Newcastle disease virus (NDV) and their efficacy against challenge with velogenic NDV

Continued monitoring and evaluation of vaccine efficacy against prevalent or newly isolated strains has great importance in advising Newcastle disease (ND) immunization strategy. In this study, we systematically analysed the antigenic variation between genotype VII NDV aSG10 and the commercial vaccine strain LaSota, and assessed their efficacy against challenge with velogenic NDV by serological analysis and animal testing. We show that these two viruses are antigenically distinguishable; anti-NDV aSG10 hyper-immune sera demonstrated higher haemagglutination inhibition (HI) titres (11.13 ± 0.30log₂) against the aSG10 virus, compared with titres against LaSota (9.53 ± 0.50log₂). Conversely, the hyper-immune sera from LaSota showed higher HI titres against LaSota virus (9.73 ± 0.36log₂), but 2-fold lower HI titre against aSG10 (8.87 ± 0.38log₂). Each serum neutralised heterologous virus, but neutralisation titres were always 3- to 6-fold higher against its homologous strain than heterologous virus. The cross-reactivity R value between aSG10 and LaSota was 0.23, indicating that they are loosely related with major antigenic differences within a single serotype. The results of animal tests revealed that the aSG10 vaccine had a significantly higher protection rate than the LaSota vaccine against genotype VII NDV, regardless of intramuscular (IM) or eye drop/intranasal (ED/IN) route of SG10 challenge. Compared with IM administration, chicken flocks needed higher HI antibody levels to obtain sufficient protection when challenged by the natural ED/IN route. These results are highly informative for better control of ND in the poultry industry.     

Virulence of variola viruses for suckling mice

We report work done in 1971 to determine the quantitative virulence for suckling mice of 26 variola virus isolates from different countries and from cases of differing severity. Strains of recognized variola major and variola minor viruses differed up to 100-fold (expressed as the harmonic mean dose of inoculum which killed mice 2-4 d old, inoculated intracranially, in 5 d). Isolates from Indonesia and from East and West Africa gave intermediate values. Unlike tests on chick embryos, this test distinguished between African and Indonesian isolates.     

Antigenic differences among Newcastle disease virus strains of different genotypes used in vaccine formulation affect viral shedding after a virulent challenge

Strains of Newcastle disease virus (NDV) can be separated into genotypes based on genome differences even though they are antigenically considered to be of a single serotype. It is widely recognized that an efficacious Newcastle disease (ND) vaccine made with any NDV does induce protection against morbidity and mortality from a virulent NDV challenge. However, those ND vaccines do not protect vaccinates from infection and viral shed from such a challenge. Vaccines prepared from ND viruses corresponding to five different genotypes were compared to determine if the phylogenetic distance between vaccine and challenge strain influences the protection induced and the amount of challenge virus shed. Six groups of 4-week-old specific pathogen-free Leghorn chickens were given oil-adjuvanted vaccines prepared from one of five different inactivated ND viruses including strains B1, Ulster, CA02, Pigeon84, Alaska 196, or an allantoic fluid control. Three weeks post-vaccination, serum was analyzed for antibody content using a hemagglutination inhibition assay against each of the vaccine antigens and a commercial NDV ELISA. After challenge with virulent CA02, the birds were examined daily for morbidity and mortality and were monitored at selected intervals for virus shedding. All vaccines except for the control induced greater than 90% protection to clinical disease and mortality. The vaccine homologous with the challenge virus reduced oral shedding significantly more than the heterologous vaccines. NDV vaccines formulated to be phylogenetically closer to potential outbreak viruses may provide better ND control by reducing virus transmission from infected birds.     

The occurrence of five major Newcastle disease virus genotypes (II,IV, V,VI and VIIb) in Bulgaria between 1959 and 1996

Partial sequence and restriction enzyme cleavage site analyses of the fusion protein gene were used to genotype 47 Newcastle disease virus strains isolated between 1959 and 1996 in Bulgaria. Viruses belonged to five major genotypes that appeared to be associated with epizootics characterized by temporal and/or geographical restrictions. Genotype IV viruses (responsible for the European branch of the first panzootic) dominated the scene up to the early 1980s, interspersed with sporadic outbreaks caused by genotype II (US strains causing pneumoencephalitis) viruses. Genotype V viruses (transmitted by psittacines from South America) were first shown in 1973 and persisted until the late 1980s. Genotype VI (earliest members from the Middle-East 1968/70 outbreaks) was represented by scattered isolations between 1974 and 1996. A genotype VIIb (recent Middle East epizootic) virus was isolated as early as in 1984. Newcastle disease epizootics in Bulgaria were highlighted by multiple infection with more than one genotype at any one time.