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Newcastle disease (ND) has been enzootic in China for several decades since the first recognition of the disease in 1946 in China. Continuous surveillance revealed that the sub-genotype VIId Newcastle disease virus (NDV) has been predominantly responsible for most of ND outbreak in China in recent years. But in the present study, three virulent NDVs isolated from poultry in southern China were classified as sub-genotype VIIh, which is highly related to the viruses circulating in some Southeast Asia countries. Continuous isolation of genotype VIIh NDV strains in the region suggests its panzootic potential. This is the first report of the sub-genotype VIIh NDVs in domestic poultry in China. The complete genome length of the three isolates was 15,192 nucleotides, and the motif at the cleavage site of F protein was 112RRRRR/F117 or 112RRRKR/F117, which was typical of virulent NDV. Phylogenetic analysis based on the F gene revealed that the three viruses had close relationship with the sub-genotype VIIh virus isolated from wild bird in 2011 in China. These viruses might have formed a stable lineage in poultry during 2012–2016 and have the potential to cause enzootic in China. Our study revealed the genetic and phylogenetic characteristics of the three sub-genotype VIIh isolates, which could help us to better understand the epidemiological context of these viruses.

The risk-based active surveillance for Newcastle disease virus (NDV) was carried out in China from 2011 to 2020. A total of 110,018 swabs were collected from 28 provinces. 2,389 class I NDVs were isolated and identified by RT-PCR and sequencing. The average annual positivity rate of class I NDVs from 2011 to 2020 was 2.17%. In the last 10 years, the positivity rate was highest in 2011 (4.76%), and has since decreased. Most viruses were isolated from chickens, while others were collected from ducks, geese and pigeons, as well as from the environment. The positivity rates for class I NDVs in poultry ranged from 0.55% to 2.40%. The viruses were isolated from 373 sampling sites in 24 provinces, mainly in East, Central, South and Southwest China. The positivity rates of NDVs in wholesale markets (51.58%) and retail markets (42.83%) were much higher than those in poultry farms (7.14%) and slaughterhouses (3.85%). Phylogenetic analyses showed that most isolates belonged to sub-genotype 1.1.2, while only 22 viruses belonged to sub-genotype 1.2, indicating the viruses in sub-genotype 1.1.2 were the predominant strains in China. The F and HN genes of six strains in the two sub-genotypes were sequenced and analyzed. The cleavage sites of F protein in the six viruses were 112ERQER/L117, 112ERQGR/L117 or 112GRQERL117, which were 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. This study revealed the distribution, genetic and phylogenetic characteristics of class I NDVs in China, and could help us to better understand the epidemiological context of class I NDVs in China.

Recent epidemiological surveys have shown that class I Newcastle disease virus (NDV) is widely distributed in China. However, little is currently known about its transmission. Therefore, in this study, we compared the transmission of class I and class II NDV. Specific-pathogen-free chickens were divided into a class I NDV inoculation group and an aerosol-exposed infection group and kept in 2 separate isolators (A and B, respectively) that were connected with an airtight plastic pipe. After inoculation, air samples were collected regularly with an All-Glass Impinger-30 (Liaoyang, China), and the airborne virus contents were analyzed using the plaque count method. In addition, oral and cloacal swabs were collected regularly to detect virus shedding using quantitative reverse transcription PCR. Similar trials were conducted simultaneously with class II NDV in isolators C and D. We consistently detected class I NDV aerosols in both isolators A and B up to 40 D post-inoculation (dpi). The aerosol concentration reached a maximum of 13.81 × 103 plague-forming units per cubic meter of air at 18 dpi and was significantly higher than that of class II NDV at 21 and 24 dpi. We also detected class I virus shedding from 2 to 40 dpi in the inoculated chickens and from 7 to 40 D post-aerosol-exposed infection in the aerosol-exposed chickens. This phenomenon may explain why class I NDV has been the primary epidemic strain of NDV in recent years.

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.

The complete genomic sequence of Newcastle disease virus (NDV) strain NDV08-004, isolated from domestic ducks in China, was determined in this study. The genome is 15198 nucleotides (nt) in length, follows the "rule of six" and contains a 55-nt leader sequence at the 3' end and a 114-nt trailer sequence at the 5' end. Compared with the full genome sequences of Class II NDV strains, the NDV08-004 isolate has a 12-nt insertion (TGGGAGACGGGG) in the phosphoprotein gene between nucleotides 2381 and 2382 of the genome (numbered according to the genomic sequence of the La Sota strain, which consists of 15186 nt). Strain NDV08-004 has the motif (112)E-Q-Q-E-R-L(117) at the cleavage site of the fusion protein, which is typical of lentogenic NDV strains, and this is in agreement with the results of pathogenic tests based on the mean death time (MDT) and the intracerebral pathogenicity index (ICPI). Phylogenetic analysis based on the full genome revealed that all the NDV strains studied could be divided into two distinct clades, namely class I and class II, and the NDV08-004 isolate characterized in this study was grouped in class I. Further phylogenetic analysis based on a 374-bp fragment of the F gene in class I strains of NDV demonstrated that NDV08-004 belongs to genotype 3, and should be therefore similar to strains obtained from live bird markets in Hong Kong in recent years.

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.

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.

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 ICPI1.2). 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.

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.

To evaluate the resistance against the adamantine of multi-genotype (H1N1, H3N2 and H9N2) swine influenza viruses isolated from China in recent years by pyrosequencing.Mutation in one of five key amino acid residues (positions 26, 27, 30, 31 and 34) within the M2 protein of influenza A viruses, leading to resistance against the adamantine class of anti-influenza drugs. The residues L26, V27, A30, S31, and G34 in the M2 protein were targeted for pyrosequencing,and 10 swine influenza viruses obtained from China during 2004 to 2008 were used to perform the amantadine resistance analysis.All 5 H1N1 swine influenza viruses were adamantine resistance, three mutations were founded in these isolates, namely V27T, V27I and S31N. Other five isolates, including four H3N2 and one H9N2 swine influenza virus, were proved to be sensitive to amantadine.Pyrosequencing technology based on the M2 gene can be used to determine the amantadine resistance for multi-genotype swine influenza viruses.

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.

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 (112)RRQKRF(117) motif at the cleavage site of F protein as the virulent strain.

Eighty-three strains of Newcastle disease virus (NDV) were obtained from outbreaks in chickens, pigeons, geese, and ducks in China in 2005 and characterized genotypically. The main functional region of the F gene (535 nucleotides) was amplified and sequenced. A phylogenetic tree based on nucleotides 47-435 of the F gene was created using sequences from 83 isolates and representative NDV sequences obtained from GenBank. Phylogenetic analysis showed that all newly characterized strains belonged to six genetic groups: I, II, III, VIb, VIIc, and VIId. All the isolates belonging to groups I and II (14 total) were lentogenic according to the amino acid sequences of the fusion protein cleavage site, and either V4 or LaSota-type, depending on the vaccines that were used. Most isolates (64 total) were classified in group VIId, a predominant genotype responsible for most Newcastle disease outbreaks since the end of the last century. One strain, NDV05-055, was in group VIIc, three pigeon strains were in group VIb, and one isolate, NDV05-041, was in group III, and characterized as a velogenic strain. This study revealed that genotype VIId was the major NDV strain responsible for the 2005 ND epizoonosis that occurred in China.

A codon optimized DNA sequence coding for foot-and-mouth disease virus (FMDV) capsid protein complex epitopes of VP1 amino acid residues 21-40, 135-160, and 200-213 was genetically fused to the C-terminal end of a glutathione-S-transferase (GST) gene in pGEX-6P-1 vector with the synonymous codons preferred by Escherichia coli . The gene was synthesized using PCR and subsequently expressed in E. coli producing an intracellular, soluble fusion protein that retained antigenicity associated with FMDV antibodies by western blot analysis. The chimera was purified from bacterial lysates by affinity chromatography and could be used in ELISA tests for antibodies against FMDV.