Hualei Liu

Genotyping of Newcastle Disease Viruses Isolated from 2002 to 2004 in China

Abstract:  The main function region of the fusion (F) protein gene of 124 strains of Newcastle disease virus isolated from 2002 to 2004 in China was amplified and sequenced for further phylogenetic and residue substitutive analysis. Most of the isolates were classified into genotype VIIc, VIId, VIf, and VIb, while others into genotype IX, III, or II. The genotype IX, a unique genotype which includes strain F48, the first Chinese virulent NDV strain isolated in 1948, were still found inducing sporadic infections in certain areas. Subgenotype VIIc, VIId, and VIIe viruses, which were distributed in clusters in the phylogenetic tree distinct from members of subgenotypes VIIa and VIIb, were responsible for most outbreaks in China and circulated predominantly in China in recent years. Strain NDV03‐026, an isolate of the genotype II which was normally lentogenic, was found carrying 112RRQKRF117 motif at the cleavage site of F protein as the virulent strain.

Characterization of Newcastle Disease Virus Isolated from Waterfowl in China

Abstract Ten representative isolates of Newcastle disease virus (NDV) obtained from outbreaks in waterfowl (geese and ducks) in China since 1997 were characterized both pathotypically and genotypically. The mean death time and intracerebral pathogenicity index were used to evaluate the virulence of the isolates. Pathogenicity tests showed that all 10 isolates were velogenic strains. The main functional region of the F gene made up of 535 nucleotides was amplified by reverse transcription-polymerase chain reaction and sequenced. The deduced amino acid sequence of the fusion protein cleavage site in all 10 isolates was 112RRQKRF117, which is a typical sequence of velogenic strains and is in agreement with the results of in vivo pathogenicity tests. For genotyping, a phylogenetic tree based on nucleotides 47–435 of the F gene was constructed. Phylogenetic analysis showed that most of the isolates were of the genotype VII virus. Only one strain, WG, was found to be of the genotype IX virus. This strain was closest to F48E9, which was isolated in China in 1946 and has been used as a standard challenge strain in vaccine evaluation in China. So, genotype IX virus still causes sporadic infections in geese in China. Further phylogenetic analyses on the genotype VII strains found that all these strains can be subdivided into 5 subgenotypes, and most of the isolates (8 strains) were classified as VIId, a predominant genotype responsible for most Newcastle disease (ND) outbreaks since the end of the past century in China. Only 1 strain, NDV03-053, was shown to be of genotype VIIc virus. Results indicate that the strains of genotype VIId NDV have been the major pathogen, responsible for most epizootic ND outbreaks in waterfowl in China since 1997.

Molecular characterization and phylogenetic analysis of new Newcastle disease virus isolates from the mainland of China.

Seventy-nine velogenic Newcastle disease virus (NDV) isolates were obtained from infected chicken flocks during the outbreaks of Newcastle disease (ND) in various regions of the mainland of China in 2006. The F gene fragment (535bp, from nt 47 to 581 of the F gene) which codes the main functional region of the F protein was obtained by RT-PCR and sequenced. All sequences obtained in this study have been submitted to GenBank. All the isolates have the motif (112)R-R-Q/R-K/R-R-F(117) at the cleavage site of the fusion protein, which is typical of velogenic NDV isolates. For genotyping, a phylogenetic tree based on nucleotides 47-435 of the F gene was constructed, and the 79 isolates could be divided into two genotypes, namely VIId and III. Most of the isolates proved to be of genotype VIId; only two isolates were of genotype III. Genotype VIId NDV has been the predominant pathogen responsible for most Newcastle disease outbreaks in China. The proportion of isolates of genotype VIId NDV shows an increasing trend, according to studies on the molecular epidemiology of NDV in China from 2002 to 2006.

Characterization of Pigeon-Origin Newcastle Disease Virus Isolated in China

Abstract Fourteen pigeon-origin Newcastle disease virus (NDV) isolates were obtained from sick pigeons in China between 1996 and 2005. The mean death time (MDT) of embryonated eggs and the intracerebral pathogenicity indices (ICPI) were tested to determine the virulence of the field isolates. The result indicated that most isolates were proved to be mesogenic (MDT 60∼90 hr and ICPI > 1.2). The main function regions of F protein gene of the isolates were amplified and sequenced for phylogenetic and residue substitutive analysis. The fusion protein cleavage site sequences of most isolates had multiple basic amino acids R/KRQKRF at positions 112–116 and a phenyl alanine at position 117, characteristic of velogenic isolates. In the phylogenetic tree, the majority of the isolates were clustered into a single genetic lineage, termed genotype VIb, and were typical pigeon paramyxovirus type 1, whereas a small number of recent isolates (three strains) were grouped into genotype VIId, a predominant genotype responsible for most Newcastle disease outbreaks in chickens and geese since the end of last century. One isolate, PK9901, was proved to be a lentogenic strain, of genotype II NDV, to which the vaccine strain La Sota belongs.

Evaluating viral interference between Influenza virus and Newcastle disease virus using real-time reverse transcription–polymerase chain reaction in chicken eggs

BackgroundSimultaneous and sequential allantoic cavity inoculations of Specific-pathogen-free (SPF) chicken eggs with Influenza virus (AIV) and Newcastle disease virus (NDV) demonstrated that the interaction of AIV and NDV during co-infection was variable. Our research revisited the replication interference potential of AIV and NDV using real-time reverse transcription–polymerase chain reaction (real-time RT-PCR) for AIV and NDV to specifically detect the viral genomes in mixed infections.ResultsData from this survey showed that when different doses of NDV (Lasota or F48E8) and AIV (F98 or H5N1) were simultaneously inoculated into embryonating chicken eggs (ECE), interference with the growth of NDV occurred, while interference with the growth of AIV did not occur. When equal amount of the two viruses were sequentially employed, the degree of interference was dependent upon the time of superinfection and the virulence of virus.ConclusionAIV have a negative impact on NDV growth if they are inoculated simultaneously or sequentially and that the degree of interference depended upon the quantity and relative virulence of the virus strains used; however, interference with AIV was not observed. Only if NDV were inoculated at an earlier time will NDV able to interfere with the growth of AIV.

Genomic Characterizations of Six Pigeon Paramyxovirus Type 1 Viruses Isolated from Live Bird Markets in China during 2011 to 2013

The genomes of six pigeon paramyxovirus type 1 (PPMV-1) isolated from symptomless pigeons in live poultry markets during the national active surveillance from 2011 to 2013 were sequenced and analyzed in this study. The complete genome lengths of all isolates were 15,192 nucleotides with the gene order of 3’-NP-P-M-F-HN-L-5’. All isolates had the same motif of 112RRQKRF117 at the cleavage site of the fusion protein, which was typical of velogenic Newcastle disease virus (NDV). Several mutations were identified in the functional domains of F and HN proteins, including fusion peptide, heptad repeat region, transmembrane domains and neutralizing epitopes. Phylogenetic analysis based on sequences of complete genomes and six genes revealed that all isolates belonged to genotype VI in class II, but at least 2 sub-genotypes were identified. Most isolates were placed into sub-genotype VIb with the exception of pi/GX/1015/13, which was classified in sub-genotype VIa. The obvious antigenic difference between PPMV-1 isolates and La Sota strain was found based on the R-value calculated by cross hemagglutination inhibition (HI) assay. These results provided the evidence that PPMV-1 could be detected from healthy pigeons, and our study may be useful in designing vaccines used in pigeon, and developing molecular diagnostic tools to monitor and prevent future PPMV-1 outbreaks.

Multiplex RT-PCR for rapid detection and differentiation of class I and class II Newcastle disease viruses

A multiplex RT-PCR was developed for detection and differentiation of class I and class II strains of Newcastle disease virus (NDV). The method was shown to have high specificity and sensitivity. The results obtained from the multiplex RT-PCR for a total of 67 NDV field isolates obtained in 2009 were consistent with those obtained by nucleotide sequencing and phylogenetic analysis. A phylogenetic tree based on the partial sequences of the F gene revealed that the 67 field isolates of NDV could be divided into two classes. Twenty-seven NDV isolates were grouped into class I, and two genotypes were identified. Most of the class I isolates were determined to be of genotype 3, with the exception of isolate NDV09-034, which belonged to genotype 2. Forty class II NDV isolates were divided into three genotypes, namely genotype VII (27 isolates), genotype I (2 isolates) and genotype II (11 isolates). Isolates of genotypes I and II in class II were shown to be related to commercial vaccine strains used commonly in China. All isolates of genotype VII were predicted to be virulent, on the basis of the sequence motif at the cleavage site of the F gene. This genotype has become predominantly responsible for most outbreaks of ND in China in recent years. In conclusion, this multiplex RT-PCR provides a new assay for rapid detection and differentiation of both classes of NDV isolates.

Complete Genome Sequences of New Emerging Newcastle Disease Virus Strains Isolated from China

ABSTRACT Five Newcastle disease virus strains isolated from geese were classified into a new genotype, designated genotype XII. The complete genome sequences of two strains indicated that these viruses were distinct from viruses of genotype VII. More investigations need to be conducted for us to understand the origin of these new strains.

Genomic Characterizations of a Newcastle Disease Virus Isolated from Ducks in Live Bird Markets in China.

One class I Newcastle disease virus (NDV), designated as duck/Guangxi/1261/2015 (GX1261), was isolated from asymptomatic ducks in live bird markets (LBM) from southern China during the national active surveillance for NDVs in 2015. The complete genome length of GX1261 isolate was 15,198 nucleotides with the gene order of 3’-NP-P-M-F-HN-L-5’. The motif at the cleavage site of F protein was 112ERQER/L117, which was typical of low virulence NDV. Several mutations were identified in the functional domains of F and HN proteins, including fusion peptide, heptad repeat region, transmembrane domains and neutralizing epitopes. Phylogenetic analysis based on the complete F gene revealed that the isolate was clustered into sub-genotype 1c in class I, and showed a high level of similarity with the strains isolated from waterfowl in the United States of America. This is the first report of this kind of virus in the mainland of China. These results demonstrated that GX1261-like viruses might exist in asymptomatic waterfowl, and remain undetected or unidentified. Thus, more investigation needs to be done in order to identify the source of the virus. This study revealed the genetic and phylogenetic characteristics of GX1261 isolate and could help us to better understand the epidemiological context of class I NDV in China.

Genome Sequences of an H5N1 Highly Pathogenic Avian Influenza Virus Isolated from Vaccinated Layers in China in 2012

ABSTRACT An H5N1 virus was isolated from vaccinated layers during an outbreak of highly pathogenic avian influenza (HPAI) in Ningxia, China, in 2012. Phylogenetic analysis revealed that the virus is a novel variant in clade 7.2, and the outbreak likely resulted from mutations in the viral hemagglutinin (HA) gene.