Zhixun Xie

Development and Application of a Multiplex Polymerase Chain Reaction for Avian Respiratory Agents

SUMMARY. A multiplex polymerase chain reaction (PCR) was developed and optimized to simultaneously detect 6 avian respiratory pathogens. Six sets of specific oligonucleotide primers for infectious bronchitis virus (IBV), avian influenza virus (AIV), infectious laryngotracheitis virus (ILTV), Newcastle disease virus (NDV), Mycoplasma gallisepticum (MG), and Mycoplasma synoviae (MS) were used respectively in the test. With the use of agarose gel electrophoresis for detection of the PCR-amplified DNA products, the sensitivity of detection was between 10 pg for IBV, AIV, MG, and ILTV and 100 pg for NDV and MS after 35 cycles of PCR. Similar sensitivity of these primers was achieved with chickens experimentally infected with respiratory pathogens. In experimental infections, the multiplex PCR was able to detect all the infected chickens in each group at 1 and 2 wk postinfection as compared with serologic tests at 2 wk postinfection that confirmed the presence of specific antibodies. The multiplex PCR was also able to detect and differentiate coinfections with two or more pathogens. No specific DNA amplification for respiratory avian pathogens was observed among noninoculated birds kept separately as a negative control group.

Homologous recombination as an evolutionary force in the avian influenza A virus.

Avian influenza A viruses (AIVs), including the H5N1, H9N2, and H7N7 subtypes, have been directly transmitted to humans, raising concerns over the possibility of a new influenza pandemic. To prevent a future avian influenza pandemic, it is very important to fully understand the molecular basis driving the change in AIV virulence and host tropism. Although virulent variants of other viruses have been generated by homologous recombination, the occurrence of homologous recombination within AIV segments is controversial and far from proven. This study reports three circulating H9N2 AIVs with similar mosaic PA genes descended from H9N2 and H5N1. Additionally, many homologous recombinants are also found deposited in GenBank. Recombination events can occur in PB2, PB1, PA, HA, and NP segments and between lineages of the same/different serotype. These results collectively demonstrate that intragenic recombination plays a role in driving the evolution of AIVs, potentially resulting in effects on AIV virulence and host tropism changes.

Detection of Avian Adenovirus by Polymerase Chain Reaction

An avian adenovirus-specific polymerase chain reaction was developed. The origin of primers was from the DNA sequence data of the chicken embryo lethal orphan avian adenovirus virus genome. An avian adenovirus-specific 421-bp DNA product was amplified by these primers from group I of adenovirus containing 12 serotypes and serotypes of adenovirus from group II and group III. The adenovirus-specific DNA product was also amplified from the 19 field isolates of avian adenoviruses but not from the mammalian adenovirus and other avian pathogenic viruses and bacteria. As little as 1 fg of avian adenovirus DNA was detected by gel electrophoresis and Southern blot analysis.

Epidemiological surveillance of low pathogenic avian influenza virus (LPAIV) from poultry in Guangxi Province, Southern China.

Low pathogenic avian influenza virus (LPAIV) usually causes mild disease or asymptomatic infection in poultry. However, some LPAIV strains can be transmitted to humans and cause severe infection. Genetic rearrangement and recombination of even low pathogenic influenza may generate a novel virus with increased virulence, posing a substantial risk to public health. Southern China is regarded as the world “influenza epicenter”, due to a rash of outbreaks of influenza in recent years. In this study, we conducted an epidemiological survey of LPAIV at different live bird markets (LBMs) in Guangxi province, Southern China. From January 2009 to December 2011, we collected 3,121 cotton swab samples of larynx, trachea and cloaca from the poultry at LBMs in Guangxi. Virus isolation, hemagglutination inhibition (HI) assay, and RT-PCR were used to detect and subtype LPAIV in the collected samples. Of the 3,121 samples, 336 samples (10.8%) were LPAIV positive, including 54 (1.7%) in chicken and 282 (9.1%) in duck. The identified LPAIV were H3N1, H3N2, H6N1, H6N2, H6N5, H6N6, H6N8, and H9N2, which are combinations of seven HA subtypes (H1, H3, H4, H6, H9, H10 and H11) and five NA subtypes (N1, N2, N5, N6 and N8). The H3 and H9 subtypes are predominant in the identified LPAIVs. Among the 336 cases, 29 types of mixed infection of different HA subtypes were identified in 87 of the cases (25.9%). The mixed infections may provide opportunities for genetic recombination. Our results suggest that the LPAIV epidemiology in poultry in the Guangxi province in southern China is complicated and highlights the need for further epidemiological and genetic studies of LPAIV in this area.

Development of a real-time multiplex PCR assay for detection of viral pathogens of penaeid shrimp

A real-time multiplex polymerase chain reaction (rtm-PCR) assay was developed and optimized to simultaneously detect three viral pathogens of shrimp in one reaction. Three sets of specific oligonucleotide primers for white spot syndrome virus (WSSV), infectious hypodermal and haematopoietic necrosis virus (IHHNV) and Taura syndrome virus (TSV), along with three TaqMan probes specific for each virus were used in the assay. The rtm-PCR results were detected and analyzed using the Light Cycler 2.0 system. Forty-five PCR-positive samples and four negative samples were used to confirm the sensitivity and specificity of the rtm-PCR. The rtm-PCR identified and differentiated the three pathogens. With one viral infection of shrimp, a specific amplified standard curve was displayed. When samples from shrimp infected with two or three pathogens were analyzed, two or three specific standard curves were displayed. The sensitivity of the rtm-PCR assay was 2,000, 20, and 2,000 template copies for WSSV, IHHNV and TSV, respectively. No positive results (standard curves) were displayed when nucleic acid from Vibro spp., and Streptococcus spp. DNA were used as PCR templates. The results indicate that real-time multiplex PCR is able to detect the presence of and differentiate each pathogen in infected shrimp. This real-time multiplex PCR assay is a quick, sensitive, and specific test for detection of WSSV, IHHNV and TSV and will be useful for the control of these viruses in shrimp.

Amplification of Avian Reovirus RNA Using the Reverse Transcriptase-Polymerase Chain Reaction

A reverse transcriptase-polymerase chain reaction method was developed for the detection of avian reovirus. The origin of primers was from the S1 gene of the avian reovirus genome. A reovirus-specific 532-base pair cDNA product was amplified by these primers from six reference strains and 23 field isolates of avian reoviruses, but not from seven different avian pathogenic viruses and bacteria. As little as 1 pg of avian reovirus RNA was detected using gel electrophoresis and Southern blot hybridization.

Phylogenetic and pathogenic analysis of Newcastle disease virus isolated from house sparrow (Passer domesticus) living around poultry farm in southern China

House sparrow (Passer domesticus) is one of the most widely distributed wild birds in China. Five Newcastle disease virus (NDV) strains were isolated from house sparrows living around the poultry farms in southern China. These isolates were characterized by pathogenic assays and phylogenetic analysis. The results showed that all NDV isolates except one were velogenic and virulent for chickens. These four virulent strains for chickens possess the amino acid sequence 112R/K-R-Q-K/R-R-F117 in the F0 cleavage site which is typical of velogenic NDV. Phylogenetic analysis indicated that these isolates belong to genotype VII and were closely related to the strains which were isolated from NDV outbreaks in chickens since 2000. One isolate of NDV from house sparrow belong to genotype II and was proved to be vaccine strain (Chicken/U.S./LaSota/46). The result of this study proved that house sparrow can carry the virulent NDV strains and the same genotype of viruses that are circulating in poultry are existing in house sparrows living around poultry farm in southern China.

Visual detection of H3 subtype avian influenza viruses by reverse transcription loop-mediated isothermal amplification assay.

BackgroundRecent epidemiological investigation of different HA subtypes of avian influenza viruses (AIVs) shows that the H3 subtype is the most predominant among low pathogenic AIVs (LPAIVs), and the seasonal variations in isolation of H3 subtype AIVs are consistent with that of human H3 subtype influenza viruses. Consequently, the development of a rapid, simple, sensitive detection method for H3 subtype AIVs is required. The loop-mediated isothermal amplification (LAMP) assay is a simple, rapid, sensitive and cost-effective nucleic acid amplification method that does not require any specialized equipment.ResultsA reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed to detect the H3 subtype AIVs visually. Specific primer sets target the sequences of the hemagglutinin (HA) gene of H3 subtype AIVs were designed, and assay reaction conditions were optimized. The established assay was performed in a water bath for 50 minutes, and the amplification result was visualized directly as well as under ultraviolet (UV) light reflections. The detection limit of the RT-LAMP assay was 0.1pg total RNA of virus, which was one hundred-fold higher than that of RT-PCR. The results on specificity indicated that the assay had no cross-reactions with other subtype AIVs or avian respiratory pathogens. Furthermore, a total of 176 clinical samples collected from birds at the various live-bird markets (LBMs) were subjected to the H3-subtype-specific RT-LAMP (H3-RT-LAMP). Thirty-eight H3 subtype AIVs were identified from the 176 clinical samples that were consistent with that of virus isolation.ConclusionsThe newly developed H3-RT-LAMP assay is simple, sensitive, rapid and can identify H3 subtype AIVs visually. Consequently, it will be a very useful screening assay for the surveillance of H3 subtype AIVs in underequipped laboratories as well as in field conditions.

Reverse Transcriptase-Polymerase Chain Reaction to Detect Avian Encephalomyelitis Virus

Abstract A reverse transcriptase-polymerase chain reaction (RT-PCR) was developed and optimized for the detection of avian encephalomyelitis virus (AEV). A pair of primers was prepared based on the VP2 gene of the structural protein P1 region of the AEV genome. An avian encephalomyelitis virus-specific 619-base pair cDNA product was amplified by these primers from five reference/field strains of AEVs but not from 10 other avian pathogenic viruses and bacteria. The RT-PCR assay developed in this study was found to be sensitive and specific with as little as 10 pg of avian encephalomyelitis virus RNA detected using gel electrophoresis. Furthermore, AEV-RT-PCR was able to detect AE virus from chicken embryo brain at 3 days postinoculation as compared with the AE agar gel precipitation test (AGP), which required up to 11 days of incubation in the embryos.

Simultaneous typing of nine avian respiratory pathogens using a novel GeXP analyzer-based multiplex PCR assay.

A new, rapid, and high-throughput GenomeLab Gene Expression Profiler (GeXP) analyzer-based multiplex PCR method was developed for simultaneous detection and differentiation of nine avian respiratory pathogens. The respiratory pathogens included in this study were avian influenza subtypes H5, H7, and H9, infectious bronchitis virus (IBV), Newcastle disease virus (NDV), infectious laryngotracheitis virus (ILTV), Mycoplasma gallisepticum (MG), Mycoplasma synoviae (MS) and Haemophilus paragallinarum (HPG). Ten pairs of primers were designed using conserved and specific sequence genes of AIV subtypes and respiratory pathogens from GenBank. Single and mixed pathogen cDNA/DNA templates were used to evaluate the specificity of the GeXP-multiplex assay. The corresponding specific DNA products were amplified for each pathogen. The specific DNA product amplification peaks of nine respiratory pathogens were observed on the GeXP analyzer. Non-respiratory avian pathogens, including chicken infectious anemia virus, fowl adenovirus, avian reovirus and infectious bursal disease virus, did not produce DNA products. The detection limit for the GeXP-multiplex assay was determined to be 100 copies/μl using various pre-mixed plasmids/ssRNAs containing known target genes of the respiratory pathogens. Further, GeXP-multiplex PCR assay was 100% specific when 24 clinical samples with respiratory infections were tested in comparison with conventional PCR method. The GeXP-multiplex PCR assay provides a novel tool for simultaneous detection and differentiation of nine avian respiratory pathogens.