Once again, Avian influenza and the serious effects of using the inactivated H9N2 vaccine in Iraqi poultry.

 

 

Professor Dr. Salah Mahdi Hassan, expert and consultant in poultry diseases and production

 

On November 16, 2024, a scientific article titled “Avian Influenza in Iraq… Between Fact and Fiction” was published by the Nahrain Veterinary Network on its website and in its prestigious magazine (Nahrain Veterinary). The article noted that the low-pathogenic avian influenza virus (H9N2 AIV) was mentioned by workers in the Iraqi poultry industry in Kurdistan region of Iraq, during the 1990s and was not scientifically documented, despite the fact that the inactivated vaccine was used in poultry farms in that region. It was later used in Iraqi poultry farms through smuggling the vaccine from the Kurdistan Region governorates, which were outside the control of the Iraqi government at the time.

After 2003, the use of the inactivated H9N2 vaccine became common in some layer and parent stock farms. After the H5N1 virus was confirmed in Iraq in 2005, and we learned the scientific facts that the H5N1 virus is a result of genetic proteins from the H9N2 virus, which is produced through its reproduction and presence in the field, I became one of those who called for stopping the use of the vaccine and conducting a field study to determine the specific genetic subtype. This was after scientific information was received confirming that there are at least 13 mutated viruses of the H9N2 virus. However, I was met with the response of many specialists who rejected the idea, saying that the vaccine is dead and is not dangerous to remain in the field. When I responded to those who rejected it, I said that the dead vaccine may be dangerous and contribute to mutation with other viral subtypes of influenza present in the Iraqi environment, thus producing mutated viruses of H5N1, H5N8, and H5N6. Furthermore, some people raised a storm over my interpretation, claiming that I had reached the level of disbelief in science when I stated that the genetic material of the killed virus remains active in the field.

On the other hand, I also mentioned in that article that I personally isolated and diagnosed the H9N2 virus as a single case or as a case associated with a Newcastle virus infection. This was achieved by sending pathological samples from 32 poultry farms from Erbil and Sulaymaniyah to a reference laboratory in Weyberg, United Kingdom, on July 15, 2008. It should be noted that the farms examined suffered from severe respiratory infections accompanied by high mortality rates (41.9%).

Following the official approval of the use of the H5N1 avian influenza vaccine in Iraq in 2021, the article noted that the prevalence of these two subtypes (H9N2 and H5N1) in commercial poultry in Iraq increases the likelihood of reassortment with other avian influenza virus subtypes, which increases the risk of a dangerous pandemic virus, which would naturally pose a significant threat to both the poultry industry and public health. “I reiterate what I said earlier: In 1994, scientific reports indicated that H9N2 had evolved into more than 102 genotypes, and when its proteins were reassorted with H5N1, they produced more than 13 genotypes of new modified viruses. Therefore, it appears that this phenomenon may be normal in terms of the presence of the same subtypes in the same places in Iraq. Scientific reports indicated that the reassortment of these viruses may be direct or indirect, which plays a dangerous role in the entry of strong epidemic viruses, exposing Iraqi people to health risks. Scientific sources stated that parts of the internal H9N2 virus genes contributed to the emergence of many avian influenza viruses that infect humans (for example, H5N6, H7N9).”

I would like the kind and interested reader to follow this article, as it contains comprehensive and accurate information about the risks of avian influenza viruses in the Iraqi environment and Iraqi poultry farms. Therefore, I will focus in this article on the seriousness of the H9N2 virus, both for poultry and for human public health.

Currently, the H9N2 AIV virus is endemic in poultry in at least 60 countries worldwide, with its continued spread in Asia, the Middle East, and North/West Africa, while also being reported periodically in the Americas and Europe. Although the H9N2 AIV virus is considered a weak pathogen in poultry, it causes significant economic losses due to reduced production and cross-infection, which increases its virulence and causes high mortality.

It is worth noting that the H9N2 AIV virus is also capable of infecting mammals, including pigs, dogs, cats, foxes, pikas, and humans. The increased binding capacity of human receptors and the occurrence of mammalian-adapted mutations in H9N2 influenza viruses facilitate viral susceptibility to infection in humans. Scientific studies have indicated that the H9N2 serological positivity rate in humans ranges between 10% and 37%, and other scientific studies have shown that recent years have witnessed a significant increase in the number of human cases. Infected with the H9N2 virus. A 2024 WHO report indicated that more than 130 human cases of H9N2 AIV infection had occurred by November 2024, including 84 cases since 2020.

Furthermore, H9N2 influenza viruses have been shown to overlap with genes from several emerging human influenza viruses, including H5N6, H7N9, H10N8, H3N8, H10N3, and H10N5. Therefore, controlling avian influenza viruses of the H9N2 subtype is important for both the poultry industry and public health.

The field reality of Iraqi poultry farms indicates that despite the use of inactivated vaccines to control avian influenza viruses in poultry for long periods of time, avian influenza viruses of the H9N2 type are still widespread in Iraq in general, regardless of the intensive vaccination program which has been implemented for a long time, even for broiler flocks, and we have always advised and warned against the use of inactivated vaccines in broiler flocks due to their lack of immunological benefit, in addition to their field danger.

The same situation exists in China, a country that has adopted a strategy of inactivated vaccines to control influenza viruses in poultry since 1998. However, the H9N2 virus is still prevalent in China. What’s more, H9N2 influenza viruses, which contain little or no antigenic differences from the vaccine virus strain, are regularly isolated from vaccinated chickens with high levels of antibodies. It’s worth noting that numerous scientific reports from different countries have repeatedly indicated the low effectiveness of vaccination against H9N2 viruses in poultry.

It has not been clear what the reason for the continued spread of the H9N2 AIV virus in chickens vaccinated with inactivated vaccines is, nor the impact of its continued spread in vaccinated poultry on the evolution and mutation of the virus.

A recent study published in npj Vaccines, a journal of the Nature Publishing Group, on April 4, 2025, examined the effects of inactivated H9N2 vaccines on virus replication, transmission, genetic evolution, and the potential for interspecific adaptation. Other species, using in vivo virus transmission models and deep sequencing methods. The study also evaluated alternative vaccination strategies capable of inducing broader immune responses.

Impact of inactivated vaccine on transmission and evolution of H9N2 avian influenza virus in chickens. npj Vaccines, 10, 67 (2025)DOI https://doi.org/10.1038/s41541-025-01115-y

Controlling H9N2 avian influenza viruses in chickens is essential to mitigate disease outbreaks in birds and the emergence of new animal avian influenza viruses. However, the long-term use of inactivated H9N2 vaccines has not reduced the prevalence of H9N2 avian influenza viruses in chickens. On the contrary, H9N2 virus replication appears to have increased in chickens and several mammalian species.

In the current study, it was found that the use of inactivated H9N2 vaccine did not prevent or reduce virus shedding, but rather selected for virus strains with increased replication capacity and specific DIP production, facilitating the transmission of H9N2 AIVs in vaccinated chickens. The transmission of H9N2 AIVs in vaccinated chickens raises another problem: increased viral genetic diversity in the circulating virus, which could lead to the generation of more novel strains. Therefore, there appears to be an urgent need to review vaccination policies, along with changes to the current use of inactivated H9N2 virus vaccines in poultry.

Although H9N2 AIVs do not exhibit significant antigenic drift like H5 or H7 AIVs in the field, the emergence of H9N2 variants belonging to a new antigenic group has been shown to reduce the effectiveness of inactivated vaccines. Importantly, in this study, the H9 AIV subtype was found to have a greater ability to replicate in the upper respiratory tract than the H5 or H7 subtypes. However, IgG antibody levels in upper respiratory lavage fluid were only 3.65% of those observed in the circulating blood of chickens vaccinated with the inactivated H9N2 vaccine. Therefore, the humoral immune response induced by the inactivated vaccine in the upper respiratory tract is insufficient to neutralize the highly replicating H9N2 virus at this site. This may explain why H9N2 continues to circulate in vaccinated flocks.

In contrast, the live vaccine rHVT-H9, which also induces a cellular immune response, and the live attenuated vaccine H9N2-LAIV, which induces both a cellular immune response and a local mucosal immune response in the upper respiratory tract, both effectively prevented H9N2 AIV replication in chickens. Scientific sources have reported that a similar phenomenon has also been observed for SARS-CoV-2 (the virus that causes COVID-19). Although vaccines are effective in reducing the severity and mortality rate of SARS-CoV-2 infection, these vaccines do not effectively prevent virus replication and transmission in the upper respiratory tract. However, live vector vaccines, which induce strong cellular immunity, and vaccines that induce local mucosal immunity are effective in preventing the replication and/or transmission of multiple variants of the 2019 novel coronavirus in the respiratory tract. Therefore, inactivated vaccines are not sufficient to effectively control viruses that replicate efficiently in the upper respiratory tract. This study shows that vaccines capable of stimulating cell-mediated and/or local mucosal immunity in the upper respiratory tract, such as rHVT-H9 vaccine and live attenuated vaccines, exhibit significant protective efficacy against H9N2 infection in chickens.

The same researchers previously demonstrated that the rHVT-H9 vaccine, which can be used in 18-day-old brooded chick embryos or in newly hatched chicks, does not effectively cross-react with maternally acquired antibodies, regardless of their specificity. Furthermore, the fact that only a single dose is required for rHVT-H9 facilitates vaccination and reduces costs.

The main challenge in developing an HVT vaccine is the ability to introduce multiple foreign genes without affecting the replication capacity of the HVT virus itself. Because the influenza virus genome is fragmented, the potential risk of reassortment between the attenuated live vaccine and the field virus remains a concern. Therefore, a previous study by Chen, S. et al. (2020) proposed a strategy for rearranging the target gene to prevent the recombination of specific RNA segments. Although there are currently no commercial vaccines that induce cellular or upper respiratory mucosal immunity against the H9N2 influenza virus, increased awareness of the risks of using inactivated vaccines could accelerate the development of vaccines. And the use of such vaccines. Understanding the evolution of H9N2 influenza viruses in vaccinated chickens can shed light on the mechanisms controlling their genetic and biological diversity, thus providing insight into the development of more effective vaccines or control methods.

Our current understanding of the evolution and mutation of influenza viruses is based primarily on studies of strains isolated during epidemic outbreaks. Although these studies are important, they are influenced by several factors. Recent studies have focused on the viral diversity present within infected individuals using transmission experiments and quantitative analysis of host-pathogen interactions, or how transmission bottlenecks mediate the structure and extent of viral genetic diversity in the recipient host. Such studies are key to understanding how viral and host-associated traits influence the genetic diversity and biological properties of the virus, such as its antigenicity, virulence, host range, and the epidemiological consequences of viral evolution within the host.

In the current study, H9N2 AIV was transmitted by contact between both vaccinated and unvaccinated chickens. The study found that the time between transmission events (serial passage) was initially longer in vaccinated chickens compared to those isolated from unvaccinated chickens, but by the fourth passage (P4), the time had decreased to that found in unvaccinated and infected chickens. Notably, antigenic drift was not detected in virus colonies taken from vaccinated chickens, but the proportion of quasispecies with increased replication capacity was higher in these vaccinated chickens compared to unvaccinated chickens. While it is common in the field for the antigenicity of H5 or H7 subtype influenza viruses to change significantly through single amino acid mutations, a similar phenomenon has not been detected in H9N2 subtype influenza viruses. The study results indicate that H9N2 influenza viruses from vaccinated chickens showed a rapid increase in replication due to the lack of neutralizing antibodies, unlike H5 and H7 influenza viruses, which evade vaccine-induced immunity through antigenic mutation.

The results of the passage sequencing experiments in this study indicated that the genetic diversity in the whole genomes of viruses isolated from vaccinated chickens was significantly higher than that of the unvaccinated group. Previous research has demonstrated a role for glycosylation in facilitating immune evasion by influenza viruses, which is similar to the genetic mutations that enable immune evasion. Although influenza viruses primarily evade replication inhibition by antibodies due to antigenic drift caused by mutations in the HA protein, these mutations may also lead to the emergence of mutations associated with or synergistic with other gene segments.

Previous studies have shown that vaccine-induced immune pressure can also lead to mutations in other gene segments. In the current study, the NS1-R140W mutation, identified as a promoter mutation, was identified in 35% of first-passage P1 viruses isolated from vaccinated chickens and was fixed in the original vaccinated chickens by 99%. Using reverse genetic manipulation, the NS1-R140W mutation significantly increased the virus’s ability to replicate. Co-occurrence of mutations in the NP genes (M1-V219I and M (NP-N417D) was also observed, suggesting that these sites are under positive selection in this system. This variable combination facilitated rapid fixation after transmission through chickens and significantly increased virus replication and transmissibility in chickens. These findings contribute to a broader understanding of the mechanisms of virus adaptation to vaccinated hosts.

Virus research focuses on producing an infectious viral strain from the original virus, which in turn represents the primary source of viral pathogenesis. The majority of influenza virus progeny are non-infectious because defective interfering particles (DIPs) possess a large internal deletion in at least one gene segment of the viral genome, thus impeding viral replication and interfering with viral replication. In this study, the diversity of DIPs in viruses isolated from the inactivated vaccine group was found to be lower than in those isolated from unvaccinated chickens, suggesting that the H9N2 virus restricts the expression of DIPs to facilitate virus replication under the immune pressure caused by inactivated vaccines. Since the phenomenon of viruses enhancing their replication by altering DIP production in vaccinated hosts has not been reported in previous scientific studies, the results of this study provide new insights into the adaptive evolution of influenza viruses under vaccine-induced immune pressure.

The study also indicates that a combination of three stable mutations (NP-N417D, M1-V219I, and NS1-R140W) was fixed in vaccinated chickens, resulting in increased replication of H9N2 influenza viruses. This is despite the fact that strong genetic barriers typically lead to decreased compatibility.

In this study, the genetic diversity of the H9N2 virus in the vaccinated group was found to be higher than in the unvaccinated group, and more adaptive genetic mutations, previously reported in previous scientific studies, were observed among the viruses in the vaccinated group. Meanwhile, a large number of monoclonal viruses were present in the vaccinated chickens, which showed an increased ability to replicate and cause disease in mice. These findings suggest that when vaccines fail to completely prevent virus shedding and transmission, mutated virus strains may pose a potential risk of interspecies transmission. It should be noted that the proportion of most adaptive mutations in mammals in this study was less than 10%, and therefore the role of these mutations in mammalian adaptation needs to be further investigated in the future.

Furthermore, it remains scientifically uncertain whether viral adaptation in mice fully reflects the situation in humans. It is worth noting that the long-term and widespread use of inactivated vaccines in China, as previously mentioned, has failed to control the H9N2 virus. As a result, the H9N2 virus has become the dominant subtype in poultry, along with an increase in the number of human cases of H9N2. Furthermore, an increase in the adaptability of mammals to newer strains has been observed. Therefore, it is important to consider whether the failure of vaccination against H9N2 AIV may increase the viral threat to public health.

Of particular concern to us in the Iraqi poultry industry is this study, which demonstrated greater genetic diversity and mutation accumulation in virus populations isolated from vaccinated chickens, leading to increased pathogenicity. This is a persistent problem facing the Iraqi poultry industry, given the ineffectiveness of the currently used inactivated H9N2 vaccine. Faced with the continued increase in the replication capacity and potential antigenic diversity of H9N2 avian influenza viruses in chickens, the current vaccination policy in Iraq to control H9N2 requires urgent review by veterinary and scientific authorities. It is imperative to consider the use of H9N2 AIV vaccines that stimulate cellular and/or local mucosal immunity in the upper respiratory tract, as they are less risky and more effective than the currently used inactivated vaccine. Finally, it must be emphasized that the development of multipotent rHVT vaccines and improving the safety of attenuated live vaccines represent key directions for future vaccine research.

 

Reference

• Chen,S.et al. A live attenuated H9N2 avian influenza vaccine prevents the viral reassortment by exchanging the HA and NS1 packaging signals. Front.Microbiol. 11, 613437 (2020).