Amplification was performed on original swab samples and allantoic fluids. None of these samples was positive for the 3’-UTR of the avian coronavirus (UTR11−/41+). Three out of 10 swab samples were positive for the N gene coronavirus, but none of them was positive for the S1 IBV gene. However, four out of 10 samples of allantoic fluid tested were positive for the coronavirus N gene. Of the two that tested positive for the S1 gene of IBV, only one of them, which was designated as parrot/Indonesia/BX9/16, was able to be further sequenced. These results are presented in Table-3.

About 81,000 people received national support (120,000 won per person; about 100 USD) for immunoglobulin administration, antigen and antibody tests for hepatitis B to prevent vertical infection from infected mothers. The participation rate was 60% in 2002, 89% in 2003, 96% in 2004, 98% in 2005, and 98% in 2006.

Only one isolate, defined as parrot/Indonesia/BX9/16, was sequenced for the partial S1 gene of IBV using XCE2+/XCE2− primers (Table-1). Nucleotide sequencing of 323 nucleotides from the partial S1 gene showed that there was no difference in the nucleotide sequence of the parrot/Indonesia/BX9/16 gene when compared with IBV 4/91 Israel variant 1 (AF093794.1) and the 4/91 vaccine strain (KF377577.1) (Figure-1). The nucleotide and amino acid pairwise distance also showed 100% homology with the IBV 4/91 Israel variant 1 (AF093794.1) and the 4/91 vaccine strain (KF377577.1). However, differences were observed between the sequenced gene, the H120 (FJ888351) positive control, and the non-chicken IBV-like peafowl/GD/KQ6/2003 virus (AY641576) (Table-4). A phylogenetic tree (Figure-2) of the aligned nucleotide sequence of the partial S1 gene was constructed using the maximum likelihood method with Mega 7 software with 1000 bootstrap value. The tree showed a close relatedness of viral isolate, parrot/Indonesia/BX9/16, to the IBV strain 4/91 variant 1 Israel (AF093794.1), the 4/91 vaccine strain (KF377577.1), CK/CH/YN/SL 1301-1 (KX107779.1), chicken/Attock/NARC-786/2013 (KU145467.1), and gammaCoV/Ck/Poland/G193/2015 (MK576138.1), whereas there were differences observed when compared with the H120 vaccine (FJ888351.1) positive control.

Specimens in viral transport medium were tested by real time reverse transcriptase-polymerase chain reaction (rRT-PCR) analysis and by inoculation into Madin-Darby canine kidney cell (MDCK) culture for viral isolation, including one to three blind passages, as previously described.1

Viral RNA was reverse transcribed into cDNA with SuperScript III First-Strand Synthesis System (Invitrogen, Life Technologies, Carlsbad, CA, USA) with random hexamers. Double strand cDNA was synthesised with Sequenase 2.0 (USB, Affymetrix, Cleveland, OH, USA). Sequencing libraries were prepared with the Nextera XT DNA sample preparation Kit (Illumina, San Diego, CA, USA). Samples from the second patient and the environment were pooled together and then run on Illumina MiSeq platform to generate 150 bp paired end reads. Assembly of viral reads was carried out with influenza A isolate A/Anhui/1/2013(H7N9) as the reference genome (GISAID, accessions EPI439503-EPI439510). In addition, full genomes from the index case were amplified with previously described methods15 and sequenced on ABI 3130 automatic DNA analyser (Life Technologies) with ABI BigDye Terminator v3.1 cycle sequencing kit (Life Technologies). Full genome sequences of the viruses were deposited in GenBank (accession number: KF034916-KF034923 for the father (index case), KF034908-KF034915 for the daughter, and KF150605-KF150612 for the environment).

Sequences were compiled with the Lasergene sequence analysis software package (DNAStar, Madison, WI, USA). Nucleotide BLASTn analysis was used to identify related reference strains, and reference sequences were obtained from GeneBank and GISAID. Pairwise sequence alignments were also performed with the MegAlign program (DNASTAR) to determine similarities between nucleotide and amino acid sequence. Phylogenetic analysis of the aligned sequences for eight genomic segments was performed by the maximum likelihood methods with MEGA4.1 software.16 The reliability of the unrooted neighbour-joining tree was assessed by bootstrap analysis with 1000 replications; only bootstrap values ≥70 % are shown. Horizontal distances are proportional to genetic distance. Alignments of each influenza virus sequence were created with the program ClustalX 1.83.

Serum samples were tested in a modified in house haemagglutination inhibition assay with turkey erythrocytes.17 Antigens for the assays were produced from the A/Nanjing/1/2013(H7N9) strain isolated from the confirmed case in Jiangsu.8

All testing was performed at the BSL-2 or BSL-3 laboratories of Jiangsu Province Center for Disease Control and Prevention, Nanjing, China.

To our knowledge, this is the first time that the iiRT‐PCR was validated for H7N9 detection. The validation design, including a broad set of reference viruses and the inclusion of oropharyngeal swabs of H7N9 infected birds, ensured that the analytical and diagnostic testing was performed according to the guidelines in the OIE Terrestrial Manual12.

Validation results show that the iiRT‐PCR system's performance is equivalent to a widely used laboratory‐based real‐time PCR setup. The analytical detection limits of the H7 and N9 iiRT‐PCR reagents are similar to RRT‐PCR, and excellent specificity to H7 and N9 viruses was observed, respectively. It should be noted that the sensitivity of the N9 protocol is about one log lower than the H7 iiRT‐PCR. A possible explanation is that the H7 primers and probes were optimized for use on the Pockit PCR, while for N9 the exact CNIC China design was used. It was shown before that also in qPCR the CNIC N9 protocol is less sensitive than various H7 protocols tested.13

The diagnostic performance of the H7 iiRT‐PCR with a relatively high sensitivity of 98% and a 100% specificity shows that the system would be performant to be used as a diagnostic platform. Ideally, the diagnostic performance would be tested on a larger set of samples, yet practically it showed difficult to obtain sufficient clinical samples for validation purposes. This current validation was aimed to validate the fitness of the iiRT‐PCR for the early diagnosis of H7 and N9 in animal samples (oropharyngeal swabs). Further validation is needed to show the possibility to use other sample types (environmental samples, human swabs) or to use the system to detect different HA (H5, H9) or NA‐types (N6 or N8).

Recently, other H7Nx were also found to be circulating in China and Southeast Asia. These H7Nx belong to the Eurasian H7‐lineage and are distinct from the Chinese H7N9. The current H7 test will most likely also detect these viruses as the H7 iiRT‐PCR primer/probe design targets a broad spectrum of H7. For public health purposes and early warning, this possible cross‐reactivity can be considered beneficial as any type of H7 is potentially zoonotic and detection of H7Nx in livestock samples can serve as an early warning system to raise public awareness of circulating animal influenza with pandemic potential. Also, the N9 protocol can detect other non‐H7N9 viruses, for example, H1N9 or H12N9 as shown in Table 1. Further steps in these cases should be confirmation of the subtype in the laboratory, followed by full HA and NA (or full genome) sequencing.

The innate characteristics of the iiRT‐PCR system make it a very useful tool for onsite diagnosis. The device is compact (hand‐held), uses lyophilized thermostable PCR reagents, and runs on rechargeable batteries, enabling it to be used in various field conditions (live bird markets, farms, quarantine stations, veterinary stations, road check points, or hospitals) and thus reducing time of sample transportation. Having a quick result onsite will make it possible to initiate a quick response, such as initial movement control and quarantine measures in live bird markets or on farms, reducing the risks of further spread and potential human infections of H7N9. In Vietnam for example, the portable PCR was introduced into the active live bird market surveillance for the early detection of H7N9. Once a case of H7 would be detected in the field through the portable system, immediate movement restrictions will be put in place while waiting for confirmatory diagnosis from the laboratory.

Results from field pilot studies (unpublished data) showed that it was feasible to install the system rapidly at any given sites and that personnel in the flied could be trained in the use of the iiRT‐PCR system within 2 days. One of the challenges of the system is how it will be incorporated into current ongoing active surveillance designs. iiRT‐PCR can be complementary to current surveillance designs, yet it needs to be clear if and how results will be confirmed by standard laboratory‐based tests (real‐time RT‐PCR, virus isolation, or sequencing) and how data sharing will occur from local levels to province or central levels.

In conclusion, this study showed that field‐based portable PCR (iiRT‐PCR) can be used for the early diagnosis of H7N9 as an alternative approach to laboratory‐based real‐time PCR. Using field‐based test will reduce the time to obtain a result and will enable possible quick response measures in the field, reducing the risk of further spread and human infections with H7N9 in currently non‐infected countries.

This study used a panel of 59 virus isolates for analytical specificity that included 28 H7N9 AIVs, 7 H7 AIVs of Eurasian lineage but not in the cluster of recent H7N9 virus in China, 15 non‐H7 AIVs, seven non‐H7 influenza viruses of swine and human origin, two poultry viruses, Newcastle disease virus, and infectious bursal disease virus (Table 1).

While the rate of measles vaccination was under 95% (complete eradication rate), breakouts occurred in every 4-6 years and a major breakout which took place in 2000-2001 affected 55,696 patients between 2-10 years old with 7 mortality cases. In an effort to maintain over 95% vaccination rate, a nationwide measles vaccination program took place in 2001 on 5,700,000 students (elementary 2nd grade to high school 1st grade) and announced eradication of this disease based on WHO standards in November 2006.

The PCR products of all positive samples were purified using PCR Purification Kit (Bioneer Co., South Korea) and were sent for sequencing (Bioneer Co., South Korea). All sequences from a given sample were combined and used to construct alignments. ClustalX (Version 1.83) multiple sequence alignment analysis was performed to calculate the percentage of sequence similarity between our positive samples and sequences of referral strains and other IBV strains. Phylogenetic trees of sequences were constructed by the neighbor-joining method and the Kimura 2-parameter model by MEGA package, Version 5.1 (15). A bootstrap resampling analysis was performed (1000 replicates) to test the robustness of the major phylogenetic groups.

Public health staff interviewed all family members and doctors and nurses who provided service for the two patients on multiple occasions to validate timelines of events and, in particular, to verify possible exposure history before the onset of illnesses, including raising or contact with animals, visiting live poultry markets and purchase of live poultry, and contact with febrile patients. We could not interview the two patients because they were critically ill at the time of investigation.

In addition to the household and surrounding environments (the residential district, which was about 1 million square metres) where the two patients lived, live poultry markets and convenience stores the index patient visited were all inspected to assess potential exposures to poultry and the environment.

We reviewed medical records to determine the time of onset and progression of the illness. All household members and healthcare workers who had contact with the two patients within 1 metre without effective personal protection at any time from the day before illnesses onset to when the patients were isolated in hospitals were treated as close contacts and placed under medical observation for seven days. Paired serum samples (separated by at least three weeks) were collected to ascertain potential person to person transmission as well as asymptomatic and subclinical infections.

Clinical samples for laboratory testing included samples from the two patients on 27 and 31 March, 13 environmental samples including two smears from chicken cages, three smears from pigeon cages, two samples of chicken faeces, one sample of pigeon faeces, one sample of duck faeces, two cloacal swabs from chicken, and two sewer water samples from two live poultry markets on April 2, and two cloacal swabs from swans on 5 April from the residential area where the two patients lived.

Clinical specimens were homogenized in phosphate-buffered saline and added at different dilutions to confluent layers of Vero cells (ECACC, Cat. No 85020206) cultivated with media with 2% fetal calf serum supplemented with penicillin/streptomycin in 24-well cell culture plates. Once cytopathic effects were evident under the microscope, passaging was initiated.

Poultry sera were assayed for IBV antibodies using a commercially available blocking ELISA (Biocheck), and antibody titers obtained from samples were also evaluated. Laboratory results of IBV ELISA were entered and managed using Microsoft Excel (Windows 2010). Descriptive statistics for the ELISA antibody titers were performed using the same program.

All respiratory samples after extraction by either the Maxwell 16-S or QIAamp method exhibited positive RP reactions by RRT-PCR with Ct values less than 37. The differences between the median Ct values of RP obtained with Maxwell 16-S and QIAamp Kit were –2.13 (P<0.001) for throat swabs and –0.95 (P = 0.001) for BALFs (Table 2). All throat swabs after extraction with the two methods also tested positive for flu A or B virus by RRT-PCR. The Maxwell 16-S yielded lower Ct values in 42/49 throat swabs than the QIAamp method, with the median Ct difference being –0.64 cycles (P<0.001) (Table 2). Of the 32 BALFs, the Flu A RRT-PCR detected 31 positives (96.9%) after Maxwell 16-S extraction and 29 positives (90.6%) after QIAamp extraction, and the only one missed after Maxwell 16-S extraction exhibited a weak positive reaction (Ct = 39.52) after QIAamp extraction. There was no statistical difference (P = 0.625) in the detection rates between the two methods. However, the Maxwell 16-S gave lower Ct values in 27/32 BALFs than the manual method, with the median Ct difference being –1.35 cycles (P<0.001) (Table 2). Furthermore, the Ct values of 31 mock-infected BALFs were delayed 0.76 cycles after Maxwell 16-S extraction and 1.74 cycles after QIAamp extraction, respectively, relative to the Ct values of controls containing the same amount of the virus (pH1N1SWL02).

Analysis of fecal extracts by the Flu A RRT-PCR showed that the median Ct value differences between the Maxwell 16-S and QIAamp methods were –0.04 (P = 0.307) in poultry fecal pools and 1.40 (P = 0.234) in human fecal pools (Table 3). Their positive rates were 34/42 (81.0%) vs 39/42 (92.9%) in poultry fecal pools (P = 0.125), 12/18 (66.7%) vs 14/18 (77.8%) in human fecal pools (P = 0.688). There were no statistical differences in the Ct values and detection rates. Nevertheless, the Ct values of mock-infected poultry fecal samples (n = 32) and human fecal samples (n = 18) were increased by 1.55 and 4.72 cycles, respectively, for the Maxwell 16-S and 0.71 and 2.65 cycles, respectively, for the QIAamp method compared to those of viral controls. The results indicate that more RT-PCR inhibitors were co-extracted with viral RNA by Maxwell 16-S than by QIAamp Kit, which led to more false negatives. Of the 14 false-negative samples following Maxwell 16-S extraction, 9 turned positive after a 10-fold dilution of their extracts. To improve the capacity of the Maxwell 16 System to remove inhibitors from fecal matrix in a simple and effective way, Maxwell 16-M (100-µl input and 100-µl output) was adopted for further investigation. With Maxwell 16-M extraction, RRT-PCR detected flu-v in all fecal samples and gave improved Ct values for weak-positive samples found by Maxwell 16-S extraction (Appendix S1).

The effect of automated sample preparation on the precision of RRT-PCR was estimated by extracting 10–2, 10–4 and 10–6 dilutions of the pH1N1SWL02 stock with the Maxwell 16-S compared to the QIAamp method. Quadruplicates per dilution for each of 4 runs were performed on 4 consecutive days. Both methods achieved high precision, i.e., the intra- and inter-run coefficients of variation (CV) ranged from 1.19% to 1.46% and 1.07% to 1.62% for the automated procedure, and from 0.62% to 1.42% and 0.42% to 2.09% for the manual method over the three measured concentrations (Table 1). To address the possibility of cross-contamination between samples within the Maxwell 16 Instrument, 3 batches of 8 reagent-blank samples were co-extracted in an alternating pattern with the high-titer virus samples used in precision determination across the cartridge rack. No false-positive results of RRT-PCR were observed in the blank samples.

Samples taken from the lip and the nasal planum, a skin nodule from the index patient in cluster 1, and the toe wound from the index patient in cluster 2 were fixed in 10% neutral buffered formalin and then embedded in paraffin. Sections of the samples were routinely processed, sectioned at 5 μm, and stained with hematoxylin and eosin for histological examination.

With the aim of improving prevention and control of viral outbreaks, the Chinese government has been investing continually in the advancement of science and technology since 2003, including the appropriation of more than 12 billion RMB for research and development related to combating SARS, influenza, and other major infectious diseases. Meanwhile, China has built 11 national technology platforms, 11 national research centers, 6 national key laboratories, and 2 national engineering laboratories. In 2010, the Chinese National Influenza Centre was designated as a WHO Collaborating Centre for Reference and Research on Influenza. All these laboratories and funding contributed to application of advanced technologies in preventing and controlling infectious diseases.

Above all, quick identification of pathogens is a prerequisite to controlling emerging epidemics. To achieve it, China has developed state-of-the-art pathogen isolation and identification technologies such as high-throughput sequencing method. In contrast to the SARS-Cov debacle, H7N9, H10N8, and H5N6 were identified within China [28–30]. BGI, a Chinese company, helped Germany sequence the pathogen Escherichia coli O104:H4 within a week using high-throughput sequencing technology in 2011. Meanwhile, Chinese researchers exploring the genesis and source of emerging viruses have found that bats are natural reservoirs of SARS-like coronaviruses and have demonstrated that domestic fowl play an important vector role for H5N1 and H7N9 [4, 32, 33].

The government encourages the development of diagnostic reagents, vaccines, and medicines as well as prophylactic equipment (e.g., infrared thermometers). China's national vaccine regulatory system was confirmed to meet WHO standards in 2011. China has developed SARS, H5N1, H1N1, and H7N9 vaccines (Table 1) and became the first country to use an H1N1 vaccine. China now produces oseltamivir (like Tamiflu®) and peramivir (like Rapivab®), obviating the need to import antivirals.

China's improvements in research funding and technical capabilities have led to a series of important findings. For example, Chinese researchers have revealed the crystal structures of key viral proteins (e.g., SARS-Cov protease, H1N1 neuraminidase N1, and H5N1 polymerase PAC-PB1N complex) [36–38], which is useful for drug design, and discovered an oseltamivir-resistance mechanism in H7N9. A traditional Chinese medicine (TCM) herbal formula was confirmed to reduce H1N1 influenza-associated fever safely and with efficacy similar to that of oseltamivir in a randomized clinical trial.

This approach uses viral RNA, amplified either directly (one-step RT-PCR) or following cDNA synthesis (two-step RT-PCR). An RT-PCR assay was designed and introduced in 1991 for detecting the IBV-S2 gene. Subsequently, general and serotype-specific RT-PCR assays were designed to target different regions and/or fragments (Figure 10) in the IBV viral genome [71–73]. The UTR and N-gene-based RT-PCR are used for universal detection, because of the conserved nature of the target region in many IBV serotypes [68, 71]. A pan-coronavirus primer, targeting a conserved region of different coronavirus isolates, could also be used in one-step RT-PCR amplification of IBV strains. However, amplification and sequencing of the S1 gene provide a reliable means for genotypic classification of new IBV strains. A serotype-specific PCR assay has been designed to enable differentiation of Massachusetts, Connecticut, Arkansas, and Delaware field isolates.

Air sampling for viruses is a difficult undertaking and the literature on the subject remains sparse in comparison with that for bacteria and fungi (Sattar and Ijaz, 2002). Only three attempts to detect airborne variola were published. The earliest attempt used highly inefficient methods and was negative (Meiklejohn et al., 1961). In a subsequent study, Downie and colleagues used short duration, low volume air sampling with liquid impingers and obtained 5 positive samples out of 47 attempts to sample exhaled breath of patients (Downie et al., 1965). Assuming that each positive sample represented a single infectious particle, the concentration of airborne infectious particles was 0.85/m3; higher concentrations were observed close to shaken bed sheets. Concentrations were likely to have been underestimated because of several frequently encountered problems with air sampling for viruses including failure of impingers to retain particles less than 1 μm in diameter that represent the majority of particles in exhaled breath, culture of only a portion of the impinger fluid, uncertain suitability of sampling fluid for virus survival, and loss of infectivity due to sampling trauma (Spendlove and Fannin, 1982).

In the 1970s, Thomas adapted Andersen samplers (capable of colleting submicrometer particles) and slit samplers (with lower efficiency for submicrometer particles) for long duration large air volume viral sampling (Thomas, 1970a). He showed that 23% of naturally airborne rabbit pox particles were ≤2.5 μm and 71% were between 2.5 and 10 μm (Thomas, 1970b). Both Thomas and Westwood et al. (1966) measured concentrations of natural rabbit pox aerosols. Thomas observed 12 pock forming units (PFU) per m3 in a room supplied with six air changes per hour (ACH) containing 27 ill rabbits. Westwood et al. observed 44 PFU/m3 in a room supplied with 10 ACH containing 7–9 infected rabbits. Westwood et al. probably obtained higher concentrations because they used an electrostatic precipitator allowing higher efficiency collection of submicrometer particles compared with Thomas's slit sampler.

Thomas also studied convalescent cases of variola minor (Thomas, 1974). One patient with relatively active lesions produced an average concentration of approximately 1 PFU/m3. Unfortunately the samples were collected late in the disease when the patient was probably minimally infectious, based on comparison with epidemiological data (Rao et al., 1968; Eichner and Dietz, 2003). The airborne virus observed appears to have been due to resuspension and is unlikely to be representative of the airborne concentration of respirable variola earlier in the course of the infection. The method used would also not have been able to collect submicrometer viral aerosol particles.

Overall, the air sampling studies suggest that animals and people infected with poxviruses generated respirable aerosols, but that air concentrations may have been low, or airborne virus was present in submicrometer particles that could not be collected the instruments available. Because detection of virus aerosols is subject to potentially large losses in sampling equipment, especially when sampling dilute natural aerosols over extended periods, and because plaque assays may not accurately represent the infectivity of virus deposited in human airways at 100% relative humidity, (Spendlove and Fannin, 1982; Sattar and Ijaz, 1987, 2002) the available data can be considered a lower limit on concentration of infectious natural poxvirus aerosols.

Experimental aerosol data suggested that poxvirus, which survived the trauma of artificial aerosolization, remained infectious for significant periods of time. Aerosols of vaccinia demonstrated a half-life of about 6 h at 22°C and relative humidity ≤50% with reduced stability at higher relative humidity and temperature (Harper, 1961). Variola appeared to have a similar half-life and not to be affected by relative humidity at 26.67°C (Mayhew and Hahon, 1970). Other experiments demonstrated that airborne vaccinia is highly sensitive to inactivation by germicidal ultraviolet light (Edward et al., 1943; Jensen, 1964).

This is an IBV genotyping method carried out to differentiate different known strains of IBV and to identify new variants following RT-PCR amplification. Full-length sequence of IBV S1 glycoprotein could be targeted for amplification and enzymes analysis [72, 76]. RFLP allows differentiation of various known IBV strains, based on their unique electrophoresis banding patterns defined by restriction enzyme digestion [72, 77]. The assay was found to be comparable with traditional virus neutralization assay, although some strains such as the Gray and JMK strains were reportedly difficult to differentiate using arrays of restriction enzymes, thus limiting the universal application of this method.

The swab and homogenized tissue samples were stored at -80°C until titration into SPF eggs. The values for titration were calculated as described by Reed and Muench and were expressed as EID50/mL and EID50/g, respectively.

PK-15 cells were seeded at a concentration of 2.5 × 105 cells/well into 6-well tissue culture plates (Corning Costar Co., Cambridge, MA, USA) and transduced with purified baculovirus particles at an MOI of 10. After 48 h incubation, the cells were fixed with absolute methanol for 5 min at -20°C, rinsed with PBS, and blocked with 2% bovine serum albumin for 30 min at 37°C. The cells were then incubated with the primary anti-body (anti-HA mAb) for 1 h at 37°C, followed by 3 PBS washes. The cells were then incubated with the secondary antibody (FITC-conjugated rabbit anti-mouse IgG; Sigma) for 1 h at 37°C, followed by 3 PBS washes. Fluorescence images were examined under an inverted fluorescence microscope (Olympus IX70).

Sera were collected from all chickens before inoculation and from virus-infected and mock-inoculated chickens at 10 days post infection, and the sera were treated with receptor destroying enzyme to remove any nonspecific inhibitors. The HI test was performed using treated sera, chicken red blood cells, and four hemagglutination units of H5N1 HPAIV. Pre-inoculation sera from all chickens used in this study were determined to be serologically negative for H5-specific HI antibodies.

Inactivated vaccines are safer than live vaccines because they cannot replicate at all in a vaccinated host, resulting in no risk of reversion to a virulent form capable of causing diseases. However, they generally provide a shorter length of protection than live vaccine and generally elicit weak immune responses, in particular cell-mediated immunity, as opposed to live viral vaccines. For this reason, inactivated vaccines are administered with potent adjuvant, and require boosters to elicit satisfactory and a long-term immunity. Vaccines of this type are generally created by inactivating propagated viruses by treatment with heat or chemicals such as formalin or binary ethyleneimine. This procedure can destroy the pathogen's ability to propagate in the vaccinated host, but keeps it intact so that the immune system can still recognize it. Although inactivated virus vaccines have been used for preventing various types of viral diseases over the decades, they need further development for controlling newly emerging diseases.

For examples, influenza virus vaccines are continually improved to contain all serotypes because many new serotypes emerge in new outbreaks. As with other approaches, many studies have been focused on searching for better adjuvants which enhance immune responses in accordance with inactivated vaccines as well as help to overcome the inhibitory effects of maternal antibody. For live AIV vaccines, the possibility of reassortment between live vaccine strain and field isolates and of back mutation from low-pathogenic to highly pathogenic viruses lead to serious concerns for vaccine safety. Thus, prior stimulation of the immune system using some immunomodulators followed by vaccination with inactivated vaccines may be needed to confer better protective immunity within a short period of time and may be promising in controlling LPAI H9N2.

Promptly after the SARS epidemic, the Chinese government accelerated the establishment of an effectual and national unified management system for public health emergencies and enacted two laws: the Regulation on Public Health Emergency and the Measures for the Administration of Information Reporting on Monitoring Public Health Emergencies and Epidemic Situation of Infectious Diseases [18, 19]. In addition to defining the standards and grades of public health emergencies, these laws support the construction of command systems and clarify the responsibilities and the leadership role of the chief executive of central and local governments previously held by the Centers for Disease Control and Prevention (CDC). Accordingly, the executive capacity of the command systems has been much improved. Importantly, China also established an emergency information dissemination system to enable timely (within 2 hours), accurate, and comprehensive release of information. Moreover, both central and local governments are now expected to be prepared for a public health emergency response (e.g., techniques, personnel, materials, and management preparedness).

In 2004, China revised the Prevention and Treatment of Infectious Diseases Law, adding SARS and avian flu as notifiable diseases and revising the law to comply with the principal rules of infectious disease prevention and control (i.e., infection source control, interruption of route of transmission, and susceptible people protection). The law adheres to the “five early” principle of early detection, diagnosis, reporting, isolation, and treatment. Early isolation can restrain contagion. The law applied China's experience in emerging epidemics to prevent and control 37 infectious diseases.

The Chinese Ministry of Health (CMH) issued a technical guide for avian flu prevention and control in 2004. It requires that suspected and confirmed cases be handled quickly at designated hospitals with the equipment to prevent nosocomial infection. The epidemiological and etiological data of patients should be acquired to enable determination of human-to-human transmission capacity. The guide suggests that persons exposed to dead poultry infected by avian flu virus be isolated and observed for 7 days. In order to control zoonotic infectious diseases, China revised its Law on Animal Disease Prevention in 2007, adding an animal epidemic surveillance and reporting system for timely disclosure of animal epidemics and providing compensation to farmers for economic loss due to culling infected or potentially infected poultry.

Guided by the aforementioned laws, a series of social innovations enacted after the SARS epidemic have improved China's ability to combat emerging diseases. The CMH issued a swine flu prevention guide on April 29, 2009, 12 days before the first reported H1N1 case. On April 3, 2013, 4 days after the first H7N9 confirmed case, the CMH also issued a nosocomial H7N9-infection prevention guide. Administrative reforms resulted in better handling of H5N1, H1N1, and H7N9 relative to SARS. Importantly, in keeping with its move toward greater transparency, after confirmation of the first H1N1 case on May 11, 2009, China posted patient zero's travel information publicly on the same day. Likewise, after confirming the first H7N9 avian flu case on March 30, 2013, China published detailed information about the patient's medical consultation. Transparency helps to subdue rumors and maintain social stability.

The sequence information obtained between s2m and the poly(A) tail allowed for designing of two partly overlapping specific reverse primers for the newly identified viruses, and further sequence information was obtained using a Rapid Amplification of cDNA Ends (5′ RACE) and primer walking strategy. The PCR products were sequenced using the ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction kit v3.1.,,.

Six-week-old BALB/c female mice were randomly divided into four groups, each containing 12 mice. Three groups were vaccinated intramuscularly (i.m.) with 109 PFU of BV-Dual-HA, 109 PFU of wild-type AcMNPV (AcMNPV-WT), and 100 μg of pc-HA, respectively. On days 0 and 21. The final group was used as the control and injected with 100 μL PBS. Serum samples were collected on days 20 and 42 for serological tests. On day 42, the mice were challenged intranasally (i.n.) with 50 MLD50 (50% mouse lethal dose) of influenza A/Chicken/Guangzhou/V/2008(H9N2) and observed for clinical signs over a 14-day period. Mice were weighed daily and examined for disease. Mice that lost more than 20% body weight were humanely euthanized. Six days after challenge, 3 mice from each group were sacrificed and the lungs, brains, livers, kidneys, and spleens were harvested to examine virus replication in SPF embryonated eggs. The viral titer, expressed as EID50 (50% egg infection dose), was calculated by the Reed-Muench method.