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.

Detection of viromes of RNA viruses using the next generation sequencing libraries prepared by three methods.

Virome (viral megagenomics) detection using next generation sequencing has been widely applied in virology, but its methods remain complicated and need optimization. In this study, we detected the viromes of RNA viruses of one mock sample, one pooled duck feces sample and one pooled mink feces sample on the Personal Genome Machine platform using the sequencing libraries prepared by three methods. The sequencing primers were added through random hybridization and ligation to fragmented viral RNA using a RNA-Seq kit in method 1, through random reverse transcription (RT) and polymerase chain reaction (PCR) in method 2 which was developed in our laboratory, and through hybridization and ligation to fragmented amplicons of random RT-PCR using a single primer in method 3. Although the results of these three samples (nine libraries) all showed that more classified viral families and genera were identified using methods 2 and 3 than using method 1, and more classified viral families and genera were identified using method 2 than using method 3, most of the differences were of no statistical significance. Moreover, 11 mammalian viral genera in minks were possibly identified for the first time through this study.

Comparison of three media for transport and storage of the samples collected for detection of avian influenza virus.

Detection of avian influenza viruses (AIVs) is important for diagnosis, surveillance and control of avian influenza which is of great economic and public health significance. Proper transport and storage of samples is critical for the detection when the samples cannot be detected immediately. As recommended by some international or national authoritative entities and some publications, phosphate buffered saline (PBS), PBS-glycerol and brain heart infusion broth (BHIB) are frequently used for transport and storage of the samples collected for detection of AIVs worldwide. In this study, we compared these three media for transport and storage of simulated and authentic swab and feces samples collected for detection of AIVs using virus isolation and reverse transcription-PCR. The results suggest that PBS-glycerol is superior to PBS and BHIB as the sample transport and storage media. The results also suggest that the samples collected for detection of AIVs should be detected as soon as possible because the virus concentration of the samples may decline rapidly during storage within days at 4 or -20°C.

Genomic Analysis and Surveillance of the Coronavirus Dominant in Ducks in China.

The genetic diversity, evolution, distribution, and taxonomy of some coronaviruses dominant in birds other than chickens remain enigmatic. In this study we sequenced the genome of a newly identified coronavirus dominant in ducks (DdCoV), and performed a large-scale surveillance of coronaviruses in chickens and ducks using a conserved RT-PCR assay. The viral genome harbors a tandem repeat which is rare in vertebrate RNA viruses. The repeat is homologous to some proteins of various cellular organisms, but its origin remains unknown. Many substitutions, insertions, deletions, and some frameshifts and recombination events have occurred in the genome of the DdCoV, as compared with the coronavirus dominant in chickens (CdCoV). The distances between DdCoV and CdCoV are large enough to separate them into different species within the genus Gammacoronavirus. Our surveillance demonstrated that DdCoVs and CdCoVs belong to different lineages and occupy different ecological niches, further supporting that they should be classified into different species. Our surveillance also demonstrated that DdCoVs and CdCoVs are prevalent in live poultry markets in some regions of China. In conclusion, this study shed novel insight into the genetic diversity, evolution, distribution, and taxonomy of the coronaviruses circulating in chickens and ducks.

Subclinical highly pathogenic avian influenza virus infection among vaccinated chickens, China.

Subclinical infection of vaccinated chickens with a highly pathogenic avian influenza A(H5N2) virus was identified through routine surveillance in China. Investigation suggested that the virus has evolved into multiple genotypes. To better control transmission of the virus, we recommend a strengthened program of education, biosecurity, rapid diagnostics, surveillance, and elimination of infected poultry.

Evaluation of avian influenza virus isolated from ducks as a potential live vaccine candidate against novel H7N9 viruses.

Recent outbreaks of a novel H7N9 avian influenza virus in humans in China raise pandemic concerns and underscore an urgent need to develop effective vaccines. Theoretically, live influenza vaccines are of multiple advantages over traditional inactivated influenza vaccines to be used in a pandemic, because they can be produced rapidly, safely, and inexpensively. However, studies on live vaccines against the novel H7N9 virus are limited. In this study, we evaluated a potential live influenza vaccine candidate using an H7N3 avian influenza virus isolated from ducks with controls of two recombinant viruses generated through reverse genetics. The potential candidate could be produced efficiently using chicken embryonated eggs, and is homogenous to the novel H7N9 virus in their viral hemagglutinin genes. The potential candidate is likely low pathogenic to birds and mammals, and likely sensitive to oseltamivir and amantadine, as suggested by its genomic sequences. Its low pathogenicity was further supported through inoculation in mice, chicken embryonated eggs and chickens. Specific antibodies elicited in mice were detectable at least during the period between day 14 and day 56 after intranasal administration of the candidate for one time. Titers of the specific antibodies increased significantly with a boost intranasal administration or a higher inoculation dose. The induced specific antibodies were of substantial cross-reactivity with the novel H7N9 virus. These primary but promising evaluation data suggest that the duck influenza virus could be used as a potential live vaccine candidate, favorably through a prime-boost route, to mitigate the severity of the possible pandemic caused by the newly emerging H7N9 virus, and is valuable to be further evaluated.

Identification of a potential novel type of influenza virus in Bovine in China.

Bovine influenza virus was first identified in the USA in 2013, and the virus represents a potential novel type of influenza viruses. However, the distribution and evolution of the virus remain unknown. We conducted a pilot survey of bovine influenza virus in China, and identified three bovine influenza viruses which are highly homogenous to the ones identified in the USA, suggesting that the bovine influenza virus likely circulates widely and evolves slowly in the world.

Identification and survey of a novel avian coronavirus in ducks.

The rapid discovery of novel viruses using next generation sequencing (NGS) technologies including DNA-Seq and RNA-Seq, has greatly expanded our understanding of viral diversity in recent years. The timely identification of novel viruses using NGS technologies is also important for us to control emerging infectious diseases caused by novel viruses. In this study, we identified a novel duck coronavirus (CoV), distinct with chicken infectious bronchitis virus (IBV), using RNA-Seq. The novel duck-specific CoV was a potential novel species within the genus Gammacoronavirus, as indicated by sequences of three regions in the viral 1b gene. We also performed a survey of CoVs in domestic fowls in China using reverse-transcription polymerase chain reaction (RT-PCR), targeting the viral nucleocapsid (N) gene. A total of 102 CoV positives were identified through the survey. Phylogenetic analysis of the viral N sequences suggested that CoVs in domestic fowls have diverged into several region-specific or host-specific clades or subclades in the world, and IBVs can infect ducks, geese and pigeons, although they mainly circulate in chickens. Moreover, this study provided novel data supporting the notion that some host-specific CoVs other than IBVs circulate in ducks, geese and pigeons, and indicated that the novel duck-specific CoV identified through RNA-Seq in this study is genetically closer to some CoVs circulating in wild water fowls. Taken together, this study shed new insight into the diversity, distribution, evolution and control of avian CoVs.

Complete genome sequence of a hemagglutination-negative avian paramyxovirus type 4 isolated from china.

We report here the complete genome sequence of a nonpathogenic and hemagglutination-negative avian paramyxovirus type 4 isolated from a duck in southern China. Phylogenetic analysis of the genome sequence indicated that the waterfowl virus possibly has evolved into the Eastern and Western Hemisphere lineages.

Genome sequence determination and analysis of a Chinese virulent strain, LMS, of Gallid herpesvirus type 2.

Marek's disease (MD) is a neoplastic and neurodegenerative disease of chickens, which is caused by the Gallid herpesvirus type 2 (GaHV-2). Although vaccination has been used widely in China, MD still occurs frequently. Some molecular epidemiologic studies have shown that Chinese GaHV-2 isolates seem to constitute a separate clade from strains isolated from other regions. However, more of a genomic background of the Chinese strains is necessary. In 2007, a virulent GaHV-2 field strain, named LMS, was isolated from diseased chicken flocks in the southwest of China. The whole genome sequence of LMS was determined to evaluate its genetic property. The genome of LMS is 177,526 bp long, and 197 open reading frames (ORFs) were predicted. Most of the ORFs have high sequence identity with homologous ORFs of reference strains. Two regions in the LMS genome are grossly different from other strains: the α-like region and the latency-associated transcripts (LATs) promoters. Evolutionary analysis demonstrated that LMS has a larger phylogenetic distance from most American isolated strains but a closer relationship with 648Ap80 and the European pC12/130 strain. The characterised genome of LMS provides further insight into the genetics of the Chinese GaHV-2 field strains, which is useful for the control of MD in China.

Molecular epidemiological surveys of H5 subtype highly pathogenic avian influenza viruses in poultry in China during 2007-2009.

To investigate the prevalence and evolution of the H5 subtype highly pathogenic avian influenza (HPAI) viruses circulating in poultry in China during 2007-2009, five molecular epidemiological surveys were carried out. A total of 21, 591 swab samples were collected, and from them 55 H5 HPAI viruses were isolated. None of the 55 viruses carried any known mutations, which can render the virus binding to human SAa2,6Gal receptors. The surveys indicated that live-bird markets, backyard flocks and slaughtering sites were at greater risk of being infected with the viruses during winter, and Clades 2.3.2, 2.3.4 and 7 of the viruses co-circulated in poultry in China during 2007-2009. Viruses within Clades 2.3.2 and 7 have become genetically distinguishable from the viruses isolated before 2007 and antigenically distinguishable from the vaccine strains used in China. Viruses within Clade 2.3.2 have been circulating widely in China and caused a new wave of cross-continental spreading from Asia to Europe.

Characterization of the hemagglutinin gene of subtype H9 avian influenza viruses isolated in 2007-2009 in China.

Subtype H9 avian influenza viruses (AIVs) circulating in China have aroused concerns for their impact on poultry and risk to public health. In this report, three surveys of the viruses were reported, and the hemagglutinin gene of 55 strains of the viruses isolated in China in 2007-2009 was sequenced and analyzed. The results indicated that the prevalence of the viruses was rising in China, and most of the H9 AIVs circulating in the past decade in China belonged to sublineage h9.4.2. The viruses isolated in China in 2007-2009 were a little different from previous strains (genetic distances >7.1%). Meanwhile, a presumably predominant clade of the viruses circulating in China in 2007-2009 was identified. Mutation analysis suggested that the viruses have become of greater risk to public health in recent years.