Control the influenza A (H7N9) virus that causes catastrophic death of poultry and large numbers of human infections. In addition to slaughtering poultry infected with the highly pathogenic H7N9 virus, since September 2017, reassortant AIV (H5 + H7) inactivated vaccines have also been vaccinated throughout China as one of the prevention and control measures . Live poultry in Nanchang City have also fully implemented immunization in accordance with the plan.
Through active monitoring for 4 consecutive years before and after vaccination, we found that after H5 + H7 influenza vaccination, the positive rate of H5 and H7 subtypes showed a downward trend, and even dropped to 0. Some studies have similar results. [8, 10]. This shows that measures taken by the government's health and related departments, such as vaccination of avian influenza, catching and killing infected poultry, and cleaning the live poultry market, can effectively curb the growth of highly pathogenic AIV subtypes. At the beginning of vaccination, H9 subtype may gain a competitive advantage, increase rapidly, and then have a trough in summer, which is related to the characteristics of H9 subtype . But this rise deserves our vigilance. Unlike H5 and H7 subtypes, which cause high mortality and high pathogenicity, low pathogenicity H9 (H9N2) subtypes generally do not cause significant clinical symptoms or death in infected poultry . This will bring challenges to the identification and control of infected poultry, and promote the rapid spread of H9 subtype virus in the LPM. Although the H9 subtype is classified as a low pathogenic AIV, some studies have found that some H9N2 viruses are highly lethal to mice and can spread systemically, similar to the highly pathogenic avian influenza virus (HPAIV) [19, 20]. It is well known that the low pathogenic H9 (H9N2) subtype provides internal genes for H5N1, H7N9 or H10N8 viruses that have caused fatal human infections since 2013 [21,22,23]. For example, a new type of H3N6 virus was isolated from migratory birds in Poyang Lake, Nanchang City, and it was discovered that the H9N2 virus contributed the PB1 gene to the new type of virus . And the H9 (H9N2) subtype can also spread across species [25, 26], and has been confirmed to infect humans [17, 27]. However, patients usually show only mild and typical human influenza like diseases, which is easy to be ignored . So in fact, the number of people infected with H9N2 virus is far greater than the number of confirmed cases. After vaccination, the H5 and H7 subtypes are suppressed, and the H9 subtype gains a competitive advantage, leading to an increase in the positive rate of the H9 subtype. Therefore, this may be the reason why the positive rate of type A avian influenza virus has not decreased after vaccination. If the H9 subtype continues to maintain a high positive rate, it will pose a huge potential threat to public health. However, in order to reduce this threat, we must take certain measures to reduce the high positive rate of the H9 subtype virus.
The determination of the viral load and subtypes of different poultry oropharyngeal swabs and cloacal swabs may help to understand the impact of viruses carried by poultry on the number of viruses released in the environment . Our results show that the positive rates of poultry samples and environmental samples are similar. Therefore, the high positive rate of some subtypes in poultry samples may be the main contributing factor of subtypes detected in the environment, while environmental pollution in turn may pose a great potential threat to healthy poultry and other species, and may cause mixed infection of infected live poultry. This may result in the continued spread of the virus in the LPM, thus increasing the risk of human infection with AIV. The H9 subtype positive rate of environmental sewage samples changed the most after vaccination. Nanchang City is located in southern China, the air is already humid, and the humid environment will affect the survival time of the AIV . During the slaughter process of live poultry, aerosols containing virus particles may be generated and spread in a narrow, poorly ventilated space , which can easily cause environmental pollution in the live poultry market. If the contaminated sewage is not disinfected in time, it is very easy to produce the accumulation effect of AIV. Studies have shown that  subtypes in the air have a good correlation with those in the environment (water, feces and smear samples), so the high concentration of AIV accumulated in the environment will pollute the surrounding air, thus indirectly increasing the possibility of poultry spreading to humans. It has been reported that  some infected patients, without direct or indirect contact with poultry, just went to live poultry market but were infected with AIV. And in the poultry market, people have more opportunities to be exposed to the environment. Therefore, compared to poultry infected with AIV, an environment with a high viral load is more likely to pose a threat to poultry practitioners and customers. Laboratory confirmed cases of human infection with the H9 (H9N2) subtype virus have been reported sporadically from the WHO, and the incidence has been significantly higher in the past few years. In China, only in the first half of 2021, there were 9 human cases of H9N2 infection, while only one case of H5N6 occurred, and H7N9 was zero infection . Therefore, the high detection rate of the H9 subtype should be valued by us, and the monitoring, prevention and control of AIVs at this stage cannot be slackened.
Our test results show that the positive rate of AIV in the live poultry wholesale market (21.76%) is lower than the AIV positive rate in the retail market (24.62%). This confirms that the detection rate of AIV mentioned in some studies  has increased with the increase of the live poultry supply chain, and a large number of AIVses have been spread and accumulated for a long time at the end of the live poultry transaction. Due to the aggregation of different types of live poultry in the retail market, dense stalls, poor air circulation, and untimely treatment of ground pollutants and sewage, poultry raising, slaughtering and sales are not divided into different areas. This may have promoted the spread of AIV in the live poultry retail market , resulting in a higher AIV positive rate in the retail market. In the live poultry wholesale market, different types of live poultry are placed separately, with sufficient space, less dirt and other reasons, which may slow the spread of the virus at a certain level. We understand that poultry workers in the retail market also have higher AIV serum antibodies than poultry workers in other environments . This suggests that more attention should be paid to the daily management of the live poultry retail market, and LPM should be sealed and disinfected regularly to prevent the spread of AIV to live poultry workers and the general population. After vaccination, both the live poultry retail market and the wholesale market, the positive rates of H9 subtypes and untypable HA type have increased significantly. This shows that even if vaccination measures are taken, cleaning and disinfection measures at all levels still need to be improved to reduce virus amplification. And as long as the live poultry trade continues, the role of the live poultry market as a reservoir of AIV and gene pool will not be fundamentally changed. Although the H9 subtype AIV is classified as a low pathogenic AIV, it is widely distributed in the live poultry market, which will pose a continuous challenge to poultry and humans . Studies have shown that after the live poultry market is closed, the number of H9 (H9N2) viruses detected has decreased . The best way to reduce exposure and infection is to close the live poultry market and implement centralized slaughtering and marketing. At the same time, it can also provide additional protection for the poultry supply chain from farm to table and reduce related economic losses.