A set of RT-PCR assays for detection of all known avian paramyxoviruses and application in surveillance of avian paramyxoviruses in China.

BACKGROUND: Avian paramyxoviruses (APMVs), also termed avian avulaviruses, are of a vast diversity and great significance in poultry. Detection of all known APMVs is challenging, and distribution of APMVs have not been well investigated. METHODS: A set of reverse transcription polymerase chain reaction (RT-PCR) assays for detection of all known APMVs were established using degenerate primers targeting the viral polymerase L gene. The assays were preliminarily evaluated using in-vitro transcribed double-stranded RNA controls and 24 known viruses, and then they were employed to detect 4,346 avian samples collected from 11 provinces. RESULTS: The assays could detect 20-200 copies of the double-stranded RNA controls, and detected correctly the 24 known viruses. Of the 4,346 avian samples detected using the assays, 72 samples were found positive. Of the 72 positives, 70 were confirmed through sequencing, indicating the assays were specific for APMVs. The 4,346 samples were also detected using a reported RT-PCR assay, and the results showed this RT-PCR assay was less sensitive than the assays reported here. Of the 70 confirmed positives, 40 were class I Newcastle disease virus (NDV or APMV-1) and 27 were class II NDV from poultry including chickens, ducks, geese, and pigeons, and three were APMV-2 from parrots. The surveillance identified APMV-2 in parrots for the first time, and revealed that prevalence of NDVs in live poultry markets was higher than that in poultry farms. The surveillance also suggested that class I NDVs in chickens could be as prevalent as in ducks, and class II NDVs in ducks could be more prevalent than in chickens, and class II NDVs could be more prevalent than class I NDVs in ducks. Altogether, we developed a set of specific and sensitive RT-PCR assays for detection of all known APMVs, and conducted a large-scale surveillance using the assays which shed novel insights into APMV epidemiology.

Recombinant Newcastle disease virus (NDV) La Sota expressing the haemagglutinin-neuraminidase protein of genotype VII NDV shows improved protection efficacy against NDV challenge.

Intensive vaccination strategies against Newcastle disease (ND) have been implemented in many countries for a long time, but ND outbreaks still occur frequently, with most isolates belonging to genotype VII of Newcastle disease virus (NDV). Many researchers have revealed that vaccines closely matched to epidemic viruses provide better protection. Therefore, using a previously established reverse genetics system, we generated a recombinant NDV vaccine strain (rLa Sota-HN) based on the La Sota vaccine strain expressing the haemagglutinin-neuraminidase (HN) protein of genotype VII NDV. The pathogenicity of the recombinant virus was confirmed by the mean death time in 9-day-old specific-pathogen-free embryonated chicken eggs and the intracerebral pathogenicity index in 1-day-old specific-pathogen-free chickens. Subsequently, 1-day-old chickens were immunized with commercial vaccine La Sota and recombinant virus rLa Sota-HN and then challenged with virulent genotype VII NDV strain. The results indicated that recombinant virus rLa Sota-HN provided increased protection of vaccinated chickens from morbidity and mortality, and inhibited the shedding of virulent virus after challenging with genotype VII virus, compared with the conventional vaccine La Sota. Our findings indicated that rLa Sota-HN is a promising vaccine candidate to improve the protection efficiency against ND in chickens, thereby preventing frequent outbreaks of this disease.

Different Origins of Newcastle Disease Virus Hemagglutinin-Neuraminidase Protein Modulate the Replication Efficiency and Pathogenicity of the Virus.

To investigate the exact effects of different origins of Newcastle disease virus (NDV) hemagglutinin-neuraminidase (HN) protein to the biological characteristics of the virus, we systematically studied the correlation between the HN protein and NDV virulence by exchanging the HN of velogenic or lentogenic NDV strains with the HN from other strains of different virulence. The results revealed that the rSG10 or rLaSota derivatives bearing the HN gene of other viruses exhibited decreased or increased hemadsorption (HAd), neuraminidase and fusion promotion activities. In vitro and in vivo tests further showed that changes in replication level, tissue tropism and virulence of the chimeric viruses were also consistent with these biological activities. These findings demonstrated that the balance among three biological activities caused variation in replication and pathogenicity of the virus, which was closely related to the origin of the HN protein. Our study highlights the importance of the HN glycoprotein in modulating the virulence of NDV and contributes to a more complete understanding of the virulence of NDV.

Roles of the Polymerase-Associated Protein Genes in Newcastle Disease Virus Virulence.

The virulence of Newcastle disease virus varies greatly and is determined by multiple genetic factors. In this study, we systematically evaluated the roles of the polymerase-associated (NP, P and L) protein genes in genotype VII NDV virulence after confirming the envelope-associated (F and HN) proteins contributed greatly to NDV virulence. The results revealed that the polymerase-associated protein genes individually had certain effect on virulence, while transfer of these three genes in combination significantly affected the chimeric virus virulence, especially when the L gene was involved. These results indicated that the L protein was a major contributor to NDV virulence when combined with the homologous NP and P proteins. We also investigated viral RNA synthesis using NDV minigenome systems to assess the interaction between the NP, P, and L proteins, which showed that the activity of the polymerase-associated proteins were directly related to viral RNA transcription and replication.

Pathogenicity of virulent infectious bronchitis virus isolate YN on hen ovary and oviduct.

Avian infectious bronchitis is an economically important poultry disease caused by avian infectious bronchitis virus (IBV). IBV isolate YN is a virulent strain, which is genetically similar to most of the prevalent strains in China. In this study, 21-day-old commercial laying hens were infected with IBV strain YN. The damaging effects of the virus on the reproductive organs were evaluated with clinical observations, gross autopsy and histopathological examinations during the 100-day monitoring period post infection. IBV strain YN infection caused a death rate of 40.5%. Microscopic lesions were observed on the ovary post-infection, but were restricted to the acute infection period. The pathological damage to the cystic oviducts were observed throughout the surveillance period. This study provides detailed information on the pathological changes in the hen ovary and oviduct after challenge with IBV strain YN, which could provide a better understanding about the pathogenicity of IBV.

Evolution of infectious bronchitis virus in China over the past two decades.

Avian infectious bronchitis is a highly contagious disease caused by infectious bronchitis virus (IBV) that affects poultry production worldwide. The absence of vaccine cross-protection and the frequent emergence of new variant strains complicate control of IBV. Here we designed a study to measure the evolution dynamics of IBV strains in China. One hundered and seven complete sequences and 1022 S1-region sequences of Chinese IBVs isolated between 1994 and 2014 were analysed by using MEGA 5.0 software and the Bayesian analysis sampling trees (BEAST) method, and selection pressure on different proteins was assessed. The phylogenetic dissimilarity of different gene trees in the data set indicated possible recombination. Fourteen isolates were identified as recombinants, possibly generated from vaccines of the Massachusetts serotype in recombination with circulating viruses. The earliest IBV in China was found to have existed in the early 1900s, and continues to evolve at a rate of approximately 10-5 substitutions per site per year. We found that purifying selection was the main evolutionary pressure in the protein-coding regions, while the S1 gene bears the greatest positive selection pressure. The proportion of QX-like genotype strains increased over time. These results indicate that the genotypes of Chinese IBVs have undergone a remarkable transition during the past 20 years.

Characteristics of very virulent infectious bursal disease viruses isolated from Chinese broiler chickens (2012-2013).

The objective of this study was to characterize the infectious bursal disease viruses (IBDVs) circulating in broiler chicken farms in China between 2012 and 2013. The VP2 gene sequences of nine newly isolated IBDVs, obtained using reverse transcriptase polymerase chain reaction, were determined and compared with worldwide reference isolates, which have been previously well characterized. Phylogenetic analysis revealed that the nine broiler IBDV isolates are closely related to very virulent IBDV (vvIBDV) strains. Analysis of the predicted amino acid sequences of VP2 from the nine vvIBDVs isolated from the broilers revealed that they share 99.2 to 100% sequence similarity. Additionally, amino acids A222, I242, I256, I294 and S299 of VP2 that are conserved among previously characterized vvIBDV strains are also encoded by the nine isolates. This study confirms the circulation of vvIBDVs in Chinese broiler chicken farms experienced slow evolution and was relatively stable in China.