Classic Serial Passage Experiment
#16,774
A little less than a month ago alarm bells went off after avian H3N8 was identified in a 4-year-old boy admitted to a hospital for severe pneumonia in China (see NHC Confirms Human Avian H3N8 Infection In Henan Province).
While this was the first documented human H3N8 infection, the avian, equine, and canine versions of this virus have been on our radar for years.
PLoS One: Evidence of Subtype H3N8 Influenza Virus Infection among Pet Dogs in China
J.Virol.: Experimental Infectivity Of H3N8 In Swine
Nature Communications: Respiratory Transmission of Avian H3N8 In Ferrets
mBio: A Mammalian Adapted H3N8 In Seals
Seven months ago, in CCDC Weekly: Epidemiological and Genetic Characteristics of the H3 Subtype Avian Influenza Viruses in China, we looked at a rare, detailed, and highly informative overview of avian H3 viruses detected in wild birds and poultry across China. One which highlighted both H3N2 and H3N8 as growing threats.
All of which brings us to a new study, published in the journal Viruses on May 19th - written by Chinese researchers - that investigates the transmissibility and pathogenicity of two H3N8 LPAI viruses (GZA1 & XJ47) isolated from wild birds.
While both strains could infect mice and guinea pigs, they produced few symptoms and very limited transmissibility. But, after conducting a serial passage study (see graphic at the top of this blog), both strains had much higher virulence, and had picked up known mammalian adaptations ( PA T97I and D701N in PB2).
I've only posted some excerpts from a much longer paper, so follow the link to read it in its entirety.
Adaptation of Two Wild Bird-Origin H3N8 Avian Influenza Viruses to Mammalian Hosts
Jianpeng Liang 1,2,3,4,†,Qian Li 1,2,3,4,†, Linlin Cai 1,2,3,4, Qingli Yuan 1,2,3,4, Libin Chen 1,2,3,4, Qiuyan Lin 1,2,3,4, Chencheng Xiao 5,6, Bin Xiang 7,* and Tao Ren 1,2,3,4,*
Academic Editors: Chao-Nan Lin and Peck Toung Ooi
Viruses 2022, 14(5), 1097; https://doi.org/10.3390/v14051097
Received: 25 April 2022 / Revised: 13 May 2022 / Accepted: 17 May 2022 / Published: 19 May 2022
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Browse FiguresAbstractWild birds play an important role in the emergence, evolution, and spread of zoonotic avian influenza viruses (AIVs). However, there are few studies on the cross-species transmission of the H3N8 AIV originating from wild birds.
In this study, we investigated the transmissibility and pathogenicity of two H3N8 low pathogenic avian influenza viruses (LPAIVs) isolated from wild birds, GZA1 and XJ47, to mammals. The HA genes of both strains belonged to Eurasian isolates, while the other genes were derived from a variety of other subtypes of AIVs. Both strains can infect specific-pathogen-free (SPF) chickens, BALB/c mice, and guinea pigs.
The XJ47 strain spread horizontally in SPF chickens and guinea pigs. The GZA1 strain did not spread horizontally but caused higher weight loss and mild lung inflammation in mice.
P12-GZA1- and P12-XJ47-adapted strains obtained after 12 passages in the lung of mice showed enhanced pathogenicity in mice, which led to obvious clinical symptoms, lung inflammation, and 100% death. Both adapted strains have the reported mutation T97I in the PA, and the reported mutation D701N in PB2 has been found in the P12-GZA1-adapted strain. This study provides an important scientific basis for the continuous monitoring of wild AIVs and the mechanism underlying AIV cross-species transmission.(SNIP)
3.5. Molecular Characteristics of the Two Adapted Strains
After 12 passages, sequencing results show that multiple amino acid sites were mutated in both strains (Table 4). Among them, the P12-GZA1 adaptive strain mutated at the T235I, D408E, N448S, and D701N sites of the PB2 protein, the T413I and V609I sites of the PB1 protein, the T97I and P534S sites of the PA protein, and the A27V site of the NP protein. It has been reported that the D701N mutation of the PB2 protein can promote replication of the influenza virus in mammals and enhance its pathogenicity [19,29]. The mutation T97I in PA was also found in the P12-XJ47 strain, and it has been reported that the T97I could enhance the pathogenicity of H6N1 AIV [30]. Additional mutations occurred at the R17C and T35A of PB2 in the P12-XJ47 strain. The enhanced pathogenicity of the adaptive strain in mice may be related to mutations at these sites.
3.6. Pathogenicity of the Two Adapted Strains in Mice
To study the pathogenic changes of the two H3N8 isolates after transmission in mice, the pathogenicity of the two H3N8 strains in mice after adaptation was also evaluated. The adapted strains, P12-GZA1 and P12-XJ47, showed higher virulence in mice (Figure 4). At 2 dpi, the mice in the two inoculated groups showed obvious clinical symptoms such as depression, shortness of breath, trembling, and significantly reduced drinking of water and eating. In the P12-GZA1-inoculated group, three mice died at 3 dpi, and the others at 4 dpi. All mice infected with P12-XJ47 lost more than 30% of their initial body weight at 5 dpi, which was recorded as death.Compared with the wild-type strain, the pathological changes in mouse lungs caused by the adapted strains P12-GZA1 and P12-XJ47 were more serious. The lungs of mice infected with P12-GZA1 exhibited more eosinophilic fibrin exudation in the alveoli, hyaline membrane formation, small-scale hemorrhage, alveolar cystic dilatation, extensive incomplete bronchial epithelium, large epithelial nucleus, clear nucleolus, basophilic cytoplasm, and small focal infiltration of inflammatory cells around many blood vessels (Figure 2c). There was more intra-alveolar hemorrhage, focal infiltration of perivascular inflammatory cells, and alveolar cystic dilatation in the lung tissues of mice infected with P12-XJ47 (Figure 2d).Discussion
In this study, two H3N8 AIV strains isolated from wild birds were recombinant from different subtypes of AIVs. In HA and M1, both strains have mutations that can increase the virulence of mice, while the GZA1 strain has an additional mutation in NS1. Two wild-type strains replicated effectively in mice and guinea pigs.
After 12 passages, the adapted strains caused more weight loss and death in mice. Several mutations were found in the adapted strains, and D701N in PB2 had been reported. In PA, the substitution T97I was found in both adapted strains, P12-GZA1 and P12-XJ47.
(SNIP)
In summary, our study provides two adapted H3N8 AIV strains to mice, and identifies several mutations that may increase the pathogenicity of H3N8 in mice.
