LAMP real-time turbidity detection for fowl adenovirus.

BACKGROUND: Fowl adenovirus (FAdV) is an infectious agent that can cause jaundice, severe anaemia, dyspnoea and reproductive disorders in fowls. Since 2015, FAdV disease has been rapidly spreading among broiler chickens in China, where it has caused significant economic losses. In this study, a loop-mediated isothermal amplification (LAMP) real-time turbidity method with strong specificity to FAdV was established. RESULTS: The established assay was specific to FAdV-4, and the lower limit of detection was 75 copies/µL of extracted DNA. The positive detection rate for the suspected tissue samples was 33.3% (14/42), which was consistent with that of the real-time PCR. CONCLUSION: The proposed LAMP method can quickly and accurately detect prevalent FAdV via real-time turbidity assay, thereby providing a diagnostic platform for the prevention and control of the FAdV disease.

Improvement of the Immunogenicity of Porcine Circovirus Type 2 DNA Vaccine by Recombinant ORF2 Gene and CpG Motifs.

Nowadays, adjuvant is still important for boosting immunity and improving resistance in animals. In order to boost the immunity of porcine circovirus type 2 (PCV2) DNA vaccine, CpG motifs were inserted. In this study, the dose-effect was studied, and the immunity of PCV2 DNA vaccines by recombinant open reading frame 2 (ORF2) gene and CpG motifs was evaluated. Three-week-old Changbai piglets were inoculated intramuscularly with 200 µg, 400 µg, and 800 µg DNA vaccines containing 14 and 18 CpG motifs, respectively. Average gain and rectum temperature were recorded everyday during the experiments. Blood was collected from the piglets after vaccination to detect the changes of specific antibodies, interleukin-2, and immune cells every week. Tissues were collected for histopathology and polymerase chain reaction. The results indicated that compared to those of the control piglets, all concentrations of two DNA vaccines could induce PCV2-specific antibodies. A cellular immunity test showed that PCV2-specific lymphocytes proliferated the number of TH, TC, and CD3+ positive T-cells raised in the blood of DNA vaccine immune groups. There was no distinct pathological damage and viremia occurring in pigs that were inoculated with DNA vaccines, but there was some minor pathological damage in the control group. The results demonstrated that CpG motifs as an adjuvant could boost the humoral and cellular immunity of pigs to PCV2, especially in terms of cellular immunity. Comparing two DNA vaccines that were constructed, the one containing 18 CpG motifs was more effective. This is the first report that CpG motifs as an adjuvant insert to the PCV2 DNA vaccine could boost immunity.

Isolation and genetic characterization of avian influenza virus H4N6 from ducks in China.

An avian influenza virus (AIV) strain belonging to the H4 subtype and provisionally designated as A/duck/China/J1/2012(H4N6) was isolated from diseased ducks with respiratory disease at a commercial poultry farm in Shandong, China, in 2012. The genomic coding sequences of all eight segments of this J1 isolate were determined and used for subsequent analysis. Phylogenetic analysis of all eight segments showed that this duck H4N6 virus was of Eurasian lineage and not American lineage. The results show that the virus probably emerged because of a reassortment event involving other avian H4N6 and H6N1 viruses. Interestingly, this H4N6 virus had all the conserved features common to low-pathogenic AIVs, including the HA cleavage sequence, receptor-binding sequences for the 2,3-linked sialic acid receptor in avian species, and the PB2 627E motif. These results suggest that the duck H4N6 isolate could not cross the species barrier to infect and replicate in mammals, including humans. In addition, screening of the duck serum samples showed that only 0.57 % (2/352) of the individuals had weak but measurable hemagglutination inhibition (HI) antibody titers. The low antibody prevalence data were also supported by the failure to detect H4N6 virus (0/56) in clinical nasal swabs of the ducks. These data indicate an alternate reservoir for the H4N6 virus.

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.

[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.

[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.