A Live Attenuated H9N2 Avian Influenza Vaccine Prevents the Viral Reassortment by Exchanging the HA and NS1 Packaging Signals.

Sujuan Chen,Tao Qin,Gencheng Fan,Dianfeng Chu,Keji Quan,Jing Xue,Daxin Peng,Hui Wang,Shi Li,Yuanzhao Du

doi:10.3389/fmicb.2020.613437

Abstract

The H9N2 avian influenza virus is not only an important zoonotic pathogen, it can also easily recombine with other subtypes to generate novel reassortments, such as the H7N9 virus. Although H9N2 live attenuated vaccines can provide good multiple immunities, including humoral, cellular, and mucosal immunity, the risk of reassortment between the vaccine strain and wild-type virus is still a concern. Here, we successfully rescued an H9N2 live attenuated strain [rTX-NS1-128 (mut)] that can interdict reassortment, which was developed by exchanging the mutual packaging signals of HA and truncated NS1 genes and confirmed by RT-PCR and sequencing. The dynamic growth results showed that rTX-NS1-128 (mut) replication ability in chick embryos was not significantly affected by our construction strategy compared to the parent virus rTX strain. Moreover, rTX-NS1-128 (mut) had good genetic stability after 15 generations and possessed low pathogenicity and no contact transmission characteristics in chickens. Furthermore, chickens were intranasally immunized by rTX-NS1-128 (mut) with a single dose, and the results showed that the hemagglutination inhibition (HI) titers peaked at 3 weeks after vaccination and lasted at least until 11 weeks. The cellular immunity (IL-6 and IL-12) and mucosal immunity (IgA and IgG) in the nasal and trachea samples were significantly increased compared to inactivated rTX. Recombinant virus provided a good cross-protection against homologous TX strain (100%) and heterologous F98 strain (80%) challenge. Collectively, these data indicated that rTX-NS1-128(mut) lost the ability for independent reassortment of HA and NS1-128 and will be expected to be used as a potential live attenuated vaccine against H9N2 subtype avian influenza.

Highlights

In China, H9N2 subtype low pathogenicity avian influenza virus (LPAIV) outbreak was first reported in Guangdong province in 1992 (Gu et al, 2017), which mainly causes mild respiratory symptoms, egg-drop symptoms, and immunosuppression (Li et al, 2005; Chen et al, 2013; Sun and Liu, 2015)
Chicken embryo fibroblasts (CEF) cell were grown in M199 medium (HyClone, United States) with 4% fetal bovine serum (FBS)
We found that all of the plaques contained both wild-type HA and NS genes, indicating the inability of NSHAmut-NS or HA-NSmut-HA to reassort freely, which illustrates the selection of wild-type virus rTX over reassortant virus rTX-NS1-128 (Figure 2C)

Summary

Introduction

In China, H9N2 subtype low pathogenicity avian influenza virus (LPAIV) outbreak was first reported in Guangdong province in 1992 (Gu et al, 2017), which mainly causes mild respiratory symptoms, egg-drop symptoms, and immunosuppression (Li et al, 2005; Chen et al, 2013; Sun and Liu, 2015). The inactivated vaccines only induce humoral immunity, which is not sufficient to completely control H9N2 LPAI. As the continued evolution of H9N2 viruses, differences of virulence and antigenicity resulted in vaccination failure despite a high level of antibody produced in immunized chickens (Zhang et al, 2008; Sun et al, 2012; Liu et al, 2016; Gu et al, 2017). It is necessary to develop other vaccine types that can induce both humoral and cellular immunity at mucosal sites

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Results

Conclusion