Characterization of two pigeon paramyxovirus type 1 isolates in China.

For over three decades, there has been a continuing panzootic caused by a virulent variant avian paramyxovirus type 1 strain, the so-called pigeon paramyxovirus type 1. It is found primarily in racing pigeons, but it has also spread to wild birds and poultry. In this study, two pigeon paramyxovirus type 1 strains, SD12 and BJ13, obtained from diseased pigeons in China, were characterized. Phylogenetic analysis based on complete sequences allowed characterization of both strains as genotype VI, class II. Further phylogenetic analysis of a 374-nucleotide section of the fusion gene showed that SD12 fell into lineage VIbii-d and BJ13 into VIbii-f. The deduced amino acid sequence of the cleavage site of the fusion protein confirmed that both isolates contained the virulent motif (112)K/RRQKR↓F(117) at the cleavage site. Nevertheless, the values of intracerebral pathogenicity indices showed the SD12 isolate to be a velogenic strain and BJ13 isolate to be a mesogenic strain. The SD12 isolate was further investigated via clinical observation, RNA detection, histopathology and viral serology in experimentally infected 3-week-old chickens. It showed a mild pathological phenotype in chickens, with viral replication restricted to a few tissues. The molecular mechanism for the SD12 isolate to have a virulent motif but low levels of virulence for chickens requires further study.

Generation by reverse genetics of an effective attenuated Newcastle disease virus vaccine based on a prevalent highly virulent Chinese strain.

OBJECTIVES: To investigate whether the differences between the circulating Newcastle disease virus (NDV) isolates and the used vaccine might account for the current ND outbreaks in vaccinated poultry flocks. RESULTS: A reverse genetics system using prevalent genotype VIId isolate SG10 was constructed and a mutant virus, named aSG10, was developed by changing the virulent F protein cleavage site motif "(112)RRQKR↓F(117)" into an avirulent motif "(112)GRQGR↓L(117)". The attenuated pathogenicity of aSG10 was confirmed from the mean death time and intracerebral pathogenicity index. aSG10 and LaSota both protected vaccinated birds from death after challenge with highly virulent genotype VII NDV, strain SG10. However, aSG10 significantly reduced the challenge virus shedding from the vaccinated birds compared to LaSota vaccine. We also generated a recombinant virus, aSG10-enhanced green fluorescent protein (EGFP), which expresses EGFP. aSG10-EGFP stably expressed EGFP for at least 10 passages. CONCLUSIONS: The mutant, aSG10, can be safely used as a vaccine vector and is a potential vaccine candidate in increasing the protective efficacy for the control of current ND epidemic in China.

Pathotypical characterization and molecular epidemiology of Newcastle disease virus isolates from different hosts in China from 1996 to 2005.

Thirty Newcastle disease virus (NDV) strains isolated from outbreaks in China during 1996 to 2005 were characterized pathotypically and genotypically. All strains except one were velogenic. An analysis of the variable region (nucleotides 47 to 420) of the F gene indicated that 6 isolates belonged to genotype II, 3 to genotype III, 1 (isolated from a pigeon) to genotype VI, and 20 to genotype VII. Isolates belonging to genotype VII were further divided into five subtypes, VIIa, VIIb, VIIc, VIId, and VIIe, and subtype VIId was made up of VIId1 to VIId5. These results showed that genotype VII isolates might have been the most prevalent in China during the past two decades. Genotype VII isolates shared high homology, but the homology was less than that between genotype VII viruses and the vaccine virus LaSota. Among these NDV isolates, 25 isolates had the velogenic motif (112)R/K-R-Q-K/R-R-F(117) that is consistent with results of the biological tests. However, four of five LaSota-type isolates that contained the lentogenic motif (112)G-R-Q-G-R-L(117) were velogenic, except SY/03, in the view of the biological test. The majority of genotype VII isolates had lost one or two N-glycosylation sites. Finally, a cross-protection experiment in which specific-pathogen-free chickens vaccinated with LaSota were challenged by six NDV isolates showed that more than three isolates were antigenic variants that could be responsible for recent outbreaks of Newcastle disease.

Molecular characterization of three new virulent Newcastle disease virus variants isolated in China.

Three cases of Newcastle disease virus (NDV) found in nature had the lentogenic motif (112)G-R-Q-G-R-L(117) in their fusion protein cleavage sites. However, both intracerebral pathogenicity and intravenous pathogenicity indexes showed that these NDV isolates were virulent. In comparison with the LaSota live virus vaccine, these viruses had significant genetic variations in the hemagglutinin-neuraminidase gene.

[Study on immune efficacy of Newcastle disease chitosan microsphere vaccine].

Newcastle disease is an acute and highly contagious disease caused by Newcastle disease virus (NDV), one of which does great harms to the poultry industry. The most basic measure of controlling New Castle disease is to alid vaccine, now we usually use La Sota live vaccine and inactivated NDV vaccine, but these two vaccines both have more or less limitation. It can produce higher mucosal immunity titers by taking vaccine orally, meanwhile it can induce humoral and cell-mediated immune response and mucosal immunity strongly. Therefore, it becomes the focus of the research, which prepare new pattern vaccines taking orally. NDV chitosan microsphere vaccine was prepared using chitosan as capsule wall material, NDV as core material, glutaraldehyde as cross-linking material, and its even particle diameter was 5.83um, and its surface was smooth and glossy, no obviously pore space, yellow brown pykno-ball, and its safety and potency were evaluated. The SPF chickens were immunized with NDV chitosan microsphere vaccine, La Sota live vaccine and inactivated NDV vaccine respectively. To evaluate vaccine's immune efficacy, using MTT to measure lymphocytes proliferation in vitro, using HI to measure serum special IgG and using ELISA tests to detect mucosal sIgA titers. The results show that NDV chitosan microsphere vaccine was safe, could induce humoral and cell-mediated immune response and mucosal immunity strongly. The results of the potency tests conformed that the vaccine could produce good protective effect.

[Newcastle disease virus heredity mutation and correlation of HN and F gene].

Prevailed Newcastle disease virus isolates were collected during 1999-2005 in China. These isolates were purified by CEF plaque assay and replicated in SPF embryos. The fusion protein (F) gene and hemagglutinin-neuraminidase (HN) gene of these isolated viruses were cloned and sequenced. Some of the F gene and HN gene sequences from GenBank were also used in this study. The homologies of nucleotide and amino acids were compared and correlations were analyzed by SPSS8.0 software among different length sequences of the F gene or HN gene. The nucleotide homologies and correlation among the F gene and HN gene were also analyzed. The results indicated there are good correlation among different length sequences of the F gene or HN gene and the F genome or HN genome (r > or = 0.973). There was also good correlations among different length amino acids of NDV F protein or HN protein (0.911< or = r < or = 0.968). But, there was only a less correlation between the whole F gene and HN gene (r = 0.312). The heredity mutation of HN genes had the character of geographical areas. The sequences of HN gene in Chinese isolates had an identity of more than 97%. But there was only 79.2% - 80.7% in HN nucleotide homology among the Chinese isolates and La Sota (vaccine).

[Molecular characterization of a genotype II virulent Newcastle disease virus isolate from chicken].

Newcastle disease virus (NDV) field strain SQZ04 was isolated from a broiler flock with typical symptoms and lesions, and cloned by plaque-purification three times. NDV SQZ04 was determined as a virulent strain with MDT of 50.5h and 51.2h, ICPI of 2.0 and 1.92, IVPI of 2.8 and 2.68 respectively before and after plaque-purification. Analysis of F gene indicated that SQZO4 was determined as a virulent gene type II , and its protein amino acid sequence has homologies of 99.3% , 98.7% and 96.9% with published gene type II vaccine strains LaSota, B1, virulent strain Taxas48,much higher than homologies of 88.3% - 88.6% or 91.3% - 92.1% with published gene types VI and IX. This is the first virulent field strain of gene type UI reported in China. Further more, the amino acid sequence 111 GGRQGRL117 in the F protein cleavage site in SQZ04 strain is identical to lentogenic strains of NDV, such as vaccine strains LaSota, B1. This is the first report that virulent NDV could have lentogenic amino acid sequence in the cleavage site of F protein, where HN genes was compared SQZ04 has a higher homologies of 95.3%- 97.3% with known velogenic strains, but lower homologies of 87.8% - 89.5% with published lentogenic strains.

[Laboratory and clinical study of levofloxacin against Helicobacter pylori].

OBJECTIVE: To assess antibacterial activity of levofloxacin to Helicobacter pylori (Hp) strains In Vitro and In Vivo. METHODS: The Minimum inhibitory concentration (MIC) for 52 clinical isolates was detected by agar dilution method and was compared with those of amoxicillin and clarithromycin. To examine the effects of pH variation on the susceptibility of Hp to levofloxacin, Mueller-Hinton agar with 7% defibrinated sheep blood was adjusted to a pH range of 4.0, 5.0, 7.0 by adding hydrochloric acid. 85 Hp-positive Patients with chronic active gastritis or active peptic ulcer disease were consecutively recruited in a prospective, open-label study. The enrolled patients were randomised to receive a seven-day course of omeprazole 20 mg bid plus amoxicillin 1000mg bid and levofloxacin 200mg bid. Their Hp status was assessed by (13)C-urea breath test and/or endoscopy 4 - 6 weeks after the end of treatment. RESULTS: The resistant rates of strains to levofloxacin, amoxicillin and clarithromycin were 1.9%, 11.5% and 25%, respectively. A dual-resistance to amoxicillin and clarithromycin was demonstrated in five Hp strains (9.6%), which were all susceptible to levofloxacin. The prevalence of strains with resistance to levofloxacin was lower than that of strains with resistance to clarithromycin (P < 0.01), and was no statistically different with amoxicillin (P > 0.05). The activity of levofloxacin was diminished under acidic environment (P < 0.01). 84 enrolled patients completed the study. 76 patients (PP and ITT analysis, 91.7%; 90.6%) become Hp-negative. Slight side-effects occurred in 5 patients (5.9%). CONCLUSION: In the present study, we report a high rate of resistance to amoxicillin and clarithromycin in this region. Omeprazole/levofloxacin-based triple therapy, including amoxicillin, is attractive because they combine a high eradication efficacy with an excellent tolerability and safety profile.

[Genetic characterization and correlation among fragments of HN gene of the field Newcastle disease viruses].

Twenty-four isolates of Newcastle disease virus (NDV) prevailing during 1997 -- 2005 in China were collected. These isolates were purified by CEF plaque assay and replicated in SPF chicken embryos. The hemagglutinin-neuraminidase (HN) genes of these viruses were cloned and sequenced. The HN gene sequences of thirty-six NDV reference strains in GenBank were also used in this study. The amino acid homologing of these viruses were compared and analyzed. The correlations among different fragments of HN gene were also analyzed. The results indicated that the homology of Chinese field NDV strains was 94.4%-99.4%, but 86.9%-89% compared with LaSota and Clone30, 87.9%-89.9% to F48E9, and 87.2%-96.2% to foreign NDV strains. There had the nearest distances among Chinese NDV isolates as compared with that of the LaSota, Clone30 and F48E9 by the phylogenetic tree. However, the distances of seven foreign NDV isolates were very close to Chinese NDV isolates as compared with these of the other foreign NDV isolates. We also found that all the Chinese field isolates were devoid of glycosylation site in position 538 -- 540. There were good correlations between different length amino acid fragments and the genomes of HN, especially the 5'-terminus first 80aa.