Antigenic evolution of H9N2 chicken influenza viruses isolated in China during 2009-2013 and selection of a candidate vaccine strain with broad cross-reactivity.

We previously demonstrated that H9N2 subtype avian influenza viruses (AIVs) isolated from 1994 to 2008 evolved into distinct antigenic groups (C, D, and E) and then underwent antigenic drift from commercial vaccines, causing a country-wide outbreak during 2010-2013. In this study, H9N2 AIVs isolated from chickens during 2009-2013 were antigenically analyzed by performing hemagglutination inhibition and neutralization assays using a panel of polyclonal antibodies. Our findings confirmed the antigenic drift of recent H9N2 viruses from the commercial vaccine and showed that most of these antigenic variants form a novel HI antigenic group, F, with a few belonging to groups D and E. Slight antigenic variation was observed in group F viruses. Genetic analysis of amino acid sequences deduced from hemagglutinin (HA) gene sequences indicated that 9 of 15 mutations predominant in the 2009-2013 viruses can be mapped to known antigenic sites, which might be responsible for the novel antigenicity of group F. These antigenic changes make it necessary to modify the influenza vaccine to ensure efficient protection. A vaccine candidate, Ck/HeB/YT/10, was selected and provided significant protection against viruses from different antigenic groups in terms of reduction in virus shedding, suggesting broad cross-reactivity. Taken together, our results indicate that the H9N2 chicken influenza viruses in China have evolved from distinct antigenic groups into a novel group F that became dominant during the country-wide outbreak and now seems to be undergoing new antigenic divergence. Systematic surveillance and timely updating of vaccine strains are important for viral prevention and control in the future.

Evolution of the H9N2 influenza genotype that facilitated the genesis of the novel H7N9 virus.

The emergence of human infection with a novel H7N9 influenza virus in China raises a pandemic concern. Chicken H9N2 viruses provided all six of the novel reassortant's internal genes. However, it is not fully understood how the prevalence and evolution of these H9N2 chicken viruses facilitated the genesis of the novel H7N9 viruses. Here we show that over more than 10 y of cocirculation of multiple H9N2 genotypes, a genotype (G57) emerged that had changed antigenicity and improved adaptability in chickens. It became predominant in vaccinated farm chickens in China, caused widespread outbreaks in 2010-2013 before the H7N9 viruses emerged in humans, and finally provided all of their internal genes to the novel H7N9 viruses. The prevalence and variation of H9N2 influenza virus in farmed poultry could provide an important early warning of the emergence of novel reassortants with pandemic potential.

Virulent avian infectious bronchitis virus, People's Republic of China.

A virulent avian infectious bronchitis virus (IBV) was isolated from 30-day-old broiler chickens that exhibited respiratory symptoms, nephropathologic lesions, and a high proportion of deaths in the People's Republic of China during 2005. The strain, designated YN, was genetically and pathologically characterized. Phylogenetic analysis showed that YN and most of the previously characterized IBV isolates found in China were phylogenetically classified into 2 main genetic clusters. The YN isolate caused severe lesions and resulted in deaths of 65% in experimental infections of 30-day-old specific-pathogen-free chickens. Tracheal and severe kidney lesions developed in all infected birds, confirming the ability of YN strain to induce both respiratory and renal disease. IBV antigens were detected by immunohistochemical analysis in the trachea, lung, kidney, and bursa, consistent with histopathologic observations, virus isolation, and reverse transcription PCR detection. We showed that YN IBV exhibits severe pathogenicity in chickens, and that similar viruses are prevalent in China.

First reported fatal Morganella morganii infections in chickens.

Morganella morganii, a Gram-negative rod commonly found in the intestines of humans and other animals, is here confirmed to cause a fatal infection in chickens by isolation and identification of the bacteria, 16S rRNA gene sequencing, and experimental infection. This is the first case of M. morganii infection in chickens.

Serologic and virologic survey for evidence of infection with velogenic Newcastle disease virus in Chinese duck farms.

A serologic and virologic survey was performed to determine the prevalence and distribution of Newcastle disease virus (NDV) infection in Chinese duck flocks. NDV infection was detected in nine of the 12 sampled farms throughout the two geographic regions covered by the survey. The percentage antibody positivity among the 406 serum samples was 35.7%. Three velogenic NDVs were obtained from different duck flocks identified by hemagglutination and hemagglutination-inhibition tests, a pathogenicity test, and reverse transcription-polymerase chain reaction on the fusion (F) genes. Phylogenetic analysis revealed that all three isolates clustered with the class II viruses; two were phylogenetically close to genotype VII NDVs, and the other was more closely related to genotype IX NDVs. These findings suggest that NDV infections were prevalent, and at least two distinct virulent genotypes may be responsible for recent epidemics in Chinese duck flocks.

Phylogenetic and pathotypical analysis of two virulent Newcastle disease viruses isolated from domestic ducks in China.

Two velogenic Newcastle disease viruses (NDV) obtained from outbreaks in domestic ducks in China were characterized in this study. Phylogenetic analysis revealed that both strains clustered with the class II viruses, with one phylogenetically close to the genotype VII NDVs and the other closer to genotype IX. The deduced amino acid sequence of the cleavage site of the fusion (F) protein confirmed that both isolates contained the virulent motif (112)RRQK/RRF(117) at the cleavage site. The two NDVs had severe pathogenicity in fully susceptible chickens, resulting in 100% mortality. One of the isolates also demonstrated some pathogenicity in domestic ducks. The present study suggests that more than one genotype of NDV circulates in domestic ducks in China and viral transmission may occur among chickens and domestic ducks.

Genomic characterization of two avian paramyxovirus type 2 isolates from chickens in China.

The complete genome sequences were determined for avian paramyxovirus type 2 (APMV-2) strains F8 and NK isolated from chickens in China. Both strains had a genome of 14,904 nucleotides (nt) in length, which followed the "rule of six". Each genome consisted of six genes in the order 3'-N-P-M-F-HN-L-5', with a 55-nt leader at the 3' end and a 154-nt trailer at the 5' end. Sequence alignment and phylogenetic analysis showed that APMV-2 strains F8 and NK shared the highest sequence identity with APMV-2 prototype strain Yucaipa, being classified in the same subgroup as strains Yucaipa, England and Kenya, while strain Bangor represented another subgroup of APMV-2. Among the APMVs, APMV-2 strains F8 and NK exhibited a closer evolutionary relationship with APMV-7 and APMV-8 representative strains.

Isolation and characterization of a reovirus causing spleen necrosis in Pekin ducklings.

High rates of mortality for Pekin ducklings have been recorded in several duck farms in China since 2006. Dead ducklings were characterized by spleen necrosis, suggesting microbial infection as a cause of disease. Laboratory investigations led to the isolation of a virus strain from the spleen tissues of dead ducklings, designated DRV-HC. Subsequent experimental infections with DRV-HC resulted in marked spleen necrosis in the ducklings similar to those observed in the natural outbreaks. Electron microscopy of the cultured DRV-HC revealed viral particles that were non-enveloped and icosahedral with a mean diameter of approximately 72 nm. Agar gel precipitating tests showed that the isolate shared a common group-specific antigen with chicken reovirus S1133. DNA sequencing revealed that this isolate was closely related to Muscovy duck reoviruses. Experimental infection with DRV-HC resulted in death of young chicks with necrotic foci in the liver and spleen. To the best of our knowledge, this is the first report of the isolation of a duck reovirus with high virulence in Pekin ducklings and SPF chickens.

Isolation and analysis of two naturally-occurring multi-recombination Newcastle disease viruses in China.

Two Newcastle disease viruses (NDV), designated QG/Hebei/07 and XD/Shandong/08, were isolated from infected chicken flocks in China in 2007 and 2008, respectively. The results of phylogenetic and recombination analyses on complete NDV genome sequences (excluding terminal segments) show that the QG/Hebei/07 isolate had evidence of recombination in the M and F genes, and recombination in the XD/Shandong/08 isolate in the F, L genes and the non-coding region between the HN and L genes. These two naturally-occurring recombinants we found to be descended from at least three putative parents from vaccine and circulating virus lineages. Moreover, we found that evidence that homologous recombination also occurred between NDV viruses of chicken and swine lineages, while the major putative parent is likely to have been derived from the chicken avirulent vaccine lineage. This study suggests that homologous recombination can occur in all coding and non-coding regions of the NDV genome and a live vaccine strain is capable of recombination with circulating viruses resulting in significant genetic change. The potential role of swine-origin viruses in the evolution of virulent NDV warrants further investigation.

Full genome sequences of two reticuloendotheliosis viruses contaminating commercial vaccines.

Reticuloendotheliosis virus (REV) fragments are a common contaminant in some commercial vaccines such as fowl poxvirus (FPV) and Marek's disease virus. However, only those strains integrating or containing a near-intact REV provirus are more likely to cause problems in the field. We confirm here, by PCR assays and animal experiments, that vaccines against FPV and herpes virus of turkeys were contaminated with full genome sequences of REV. Further, we determined the complete proviral sequence of two REV isolates from contaminated vaccines. Two REV isolates (REV-99 and REV-06) present in the vaccines were both replication competent, and their proviral genome was 8286 nucleotides in length with two identical long terminal repeats (LTR). The complete genome in these two REV isolates shared 99.8% identity to APC-566 and fowl poxvirus REV proviral inserts (FPV-REV). REV-99 and REV-06 LTR showed over 99% identity to chicken syncytial virus (CSV), but an identity of only 75.8% and 78.0%, respectively, to SNV. Alignments with other available REV gag, pol, and env sequences revealed high similarity at the nucleotide level. The results further indicated that the prototype CSV may be the most-important REV contaminant in the commercial vaccines, and distinct genotypes of REVs may cocirculate in chicken flocks of China at the present time.

Complete genome sequence analysis of a predominant infectious bronchitis virus (IBV) strain in China.

Infectious bronchitis (IB) is one of the major diseases in poultry flocks all over the world caused by infectious bronchitis virus (IBV). In the study, the complete genome sequence of strain A2 was sequenced and analyzed, which was a predominant IBV strain in China. The results indicated that there were mutations, insertions, and deletions distributed in the whole genome. The A2 virus had the highest identity to S14 and BJ in terms of full genome, whereas had a further distance to Massachusetts strains. Phylogenetic analysis showed that A2 isolate clustered together with most Chinese strains. The results of this study suggest that strain A2 may play an important role in IBV's evolution and A2-like IBVs are predominant strains in China.

Production and characterization of monoclonal antibodies against chicken secretory IgA.

Abstract Two monoclonal antibodies (MAbs) against chicken secretory immunoglobulin A (SIgA) were generated and their binding specificities were characterized by enzyme-linked immunosorbent assay (ELISA), SDS-PAGE, and Western blotting. Analysis revealed that the subtypes of two MAbs were both IgG2b, with the light chain belonging to the kappa configuration. The affinity constant (K(aff)) of the two MAbs was 5.0 x 10(10) M(-1) and 9.7 x 10(9) M(-1), respectively. The MAbs are directed against the heavy chain domains of chicken SigA, and no cross-reactivities to IgG were observed. These results indicate that the MAbs are specific for SIgA and may be a useful tool for investigating issues regarding mucosal immunity and in the development of a good diagnostic kit for detection of specific IgA in chicken.

[Preparation and identification of the monoclonal antibody specific to hemagglutinin of avian paramyxovirus type 2].

A hybridoma cell line 1G4A7 secreting monoclonal antibody (McAb) specific to hemagglutinin of avian paramyxovirus type 2 (APMV-2) was developed by fusing the spleen cells of APMV-2 immunized BAlb/c mice with SP2/0 myeloma cells. The immunoglobulin subclass of 1G4A7 was IgG1 with light chain kappa and the affinity constant against APMV-2 was 1.02 X 10(10). Identified by HI and indirect ELISA, the McAb titers in ascities were 10 log 2 and 1 : 10(6) respectively. The McAb did not cross react with the common avian viruses, showing good specificity. There existed obvious differences in antigenitic relationship among APMV-2 viruses analyzed by HI and indirect ELISA using McAb 1G4A7.

Seroprevalence of avian reovirus in egg-laying chicken flocks in China.

In the present study, the epidemiologic status of avian reovirus (ARV) infections was investigated from egg-laying chicken flocks in China with an enzyme-linked immunosorbent assay (ELISA) kit. Because the chickens were not vaccinated against ARV, antibodies were attributed to the infection. Antibodies specific to ARV were found in more than 92% (542/587) of the average positivity and ranged from 30% to 100% in different chicken population. A virus, designated HB06, was isolated from flocks with suspicious ARV infections. Sequence analysis of the S1 gene revealed that strain HB06 was closely related with the most ARVs with less than 2% nucleotide divergence, and the homology was highest with the vaccine strain S1133, with a 98.97% nucleotide identity. The potential significance of vaccination against ARV in egg-laying chicken flocks in China is also discussed.

Serological survey on prevalence of antibodies to avian paramyxovirus serotype 2 in China.

We report the prevalent status of avian paramyxovirus serotype 2 (APMV-2) in China. Between 2003 and 2005, 9156 sera in total were collected and screened for APMV-2 antibodies by using the hemagglutination inhibition assay. The averaged seropositivity ofAPMV-2 for chickens, ducks, peacocks, ostriches, and partridges was 42.9%, 25.1%, 45.8%, 47.6%, and 80.0%, respectively. The results of this survey indicate that the distribution of APMV-2 is very widespread in China and that more attention should be paid to the influence of APMV-2 on poultry production.

Isolation and identification of four infectious bronchitis virus strains in China and analyses of their S1 glycoprotein gene.

Between 2003 and 2005, four strains of infectious bronchitis virus (IBV) were isolated from the vaccinated chicken flocks in China. The results from chicken embryo cross-neutralization assays showed that all the four isolates were relative to strain A2 of IBV, which was isolated in 1996 in Beijing and related to strain 4/91. The S1 gene of the spike protein was amplified and sequenced. The nucleotide and amino acid sequence of the S1 gene had a similar degree of identity (88.98-99.28%) among the four Chinese IBV isolates. The identity of the S1 protein gene between the four Chinese IBV isolates and 14 strains of other IBVs varied from 70.06 to 81.59%. Phylogenetic analysis suggested that there are at least four groups of IBVs circulating in China and the disease outbreaks might have been caused by infection of multiple strains of IBV.

Isolation, identification, and comparison of four isolates of avian paramyxovirus serotype 2 in China.

Four Yucaipa-like viruses of avian paramyxovirus serotype 2 (APMV-2) were isolated in China from the imported Gouldian Finch (Chloebia gouldiae) and broilers in 1998-2002, and were named F4, F6, F8, and NK, respectively. Examined under electron microscope, the isolates were found to be round in shape and varying in size. The results of the hemagglutination inhibition test and indirect enzyme-linked immunosorbent assay (using monoclonal antibodies) showed some differences between the isolates and the reference strain Yucaipa. The isolates derived from chickens had a closer relationship to Yucaipa virus than did those of finches. Sequence comparison of the fusion gene and the haemagglutinin-neuraminidase gene showed similar results, although the variations were lesser among APMV-2 viruses in nucleotide and amino acid sequence. By sequence comparison, it was also revealed that at the molecular level the four virus strains belong to APMV-2, and that two of the strains were isolated from the same group of imported Gouldian Finches.

Structure and function study of paramyxovirus fusion protein heptad repeat peptides.

Paramyxovirus might adopt a molecular mechanism of membrane fusion similar to that of other class I viruses in which the heptad repeat (HR) regions of fusion protein (F) HR1 and HR2 form a six-helix bundle structure inducing membrane fusion. In this study, we examined the structure and function of HR1 and HR2 from the avian paramyxovirus-2 (APMV-2) F protein. The study showed that APMV-2 HR1 and HR2 formed a stable six-helix bundle. Only a soluble APMV-2 HR2 peptide showed potent and specific virus-cell fusion inhibition activity. Cross-inhibiting activity with APMV-1 (Newcastle disease virus, NDV) was not found. A possible mechanism of membrane fusion inhibition by the paramyxovirus HR2 peptide is discussed.