Genetic Evolution Analysis of AKAV Gc gene

Xiaolin Lan¹Fang Liang¹Ruqing Zeng¹Qipeng Zhang¹Jiaman Li¹Gan Li¹Feng Li²Yaqiong Ye^1*Mengmeng Zhao^1*

• ¹Foshan University, Foshan, China
• ²Center of animal epidemic disease prevention control, Long zhouxian County, Chongzuo City, Guangxi Zhuang Autonomous Region, China;, Guangxi, China

Akabane disease (AKAD), also known as Akabane encephalomyelitis, is an arthropod-borne infectious disease caused primarily by Akabane virus (AKAV), characterized by abortion, premature birth, stillbirth, congenital joint contractures, and arthrogryposis-hydranencephaly syndrome (AH) in cattle and sheep (1,2). Additionally, AKAV can infect avian embryos, mice, and hamsters, leading to death. AKAV is widespread in Africa, the Middle East, East Asia, Southeast Asia, and Australia (3)(4)(5). Between 1972 and 1975, a serious outbreak of Akabane disease broke out in Japan, with the original strain JaGAr39 isolated from mosquitoes in Japan in 1959, resulting in over 31,000 cases of abortion, stillbirth, congenital joint diseases, and AH syndrome cases (6). In 1974, AKAV-induced AH syndrome was prevalent in Australia, when more than 8,000 confirmed cases were reported (7).In 1998, AKAV was first isolated from mosquitoes in Shanghai, China, and named the SH-1 strain (8).The global prevalence of AKAD exhibits distinct regional, periodic, and seasonal patterns (9) and can spread across species, causing significant economic losses to livestock industries in endemic regions and posing a long-term threat to animal health and sustainable farming practices. As the envelope glycoprotein of AKAV, Gc not only serves as a crucial target for the immune system of vertebrate hosts, eliciting strong specific immune responses, but also plays a decisive role in various important biological properties of AKAV, such as pathogenicity, neutralizing activity, hemagglutination ability (10)(11)(12). It is crucial to study the key aspects of the AKAV Gc gene to improve the economic viability of livestock farming and ensure its stable development. From the GenBank database on the NCBI website, a total of 147 AKAV Gc strains sequences were selected, comprising 61 strains from Japan, 39 from the United States, 35 from China, 2 from Israel, 9 from South Korea, and 1 from Turkey, spanning lineages I-V (see Supplementary Table 2). These strains encompass different years from 1959 to 2023, including the majority of AKAV strains from China. The genetic variation of the AKAV Gc gene sequence over the past 64 years has helped to analyze the evolution of AKAV Gc and provide a theoretical basis for AKAV prevention and control. Out of the 147 AKAV strains, 45 strains were carefully selected for further Gc sequence analysis, representing strains from lineages I-V, including reference strains, vaccine strains, and epidemic strains, to ensure a comprehensive analysis of genetic variations in the AKAV Gc gene (Table 1). The nucleotide similarity of 45 strains selected from 147 strains of AKAV Gc was analyzed. These 45 AKAV strains cover classical strains and vaccine strains in different countries and different periods. The nucleotide homology of Gc gene was analyzed from NCBI website (https://www.ncbi.nlm.nih.gov/) by Clustal W method in MegAlign function of DNAStar software (version 7.0, Madison, WI). The phylogenetic analysis of 147 AKAV Gc sequences (Table 2, submitted as supplementary materials) was carried out by using the maximum likelihood method (ML) with 1000 Bootstrap repeated sampling of MEGA software (7th edition, Mega Limited, Auckland, New Zealand). Subsequently, these sequences were annotated with a network tool called interactive tree of life. Descriptive resultsIn this study, 45 representative isolates were systematically selected from the initial 147 strains. These isolates covered a wide range of time (1959-2023) and geography, including Japan, China, South Korea, Australia, and Israel. They also represented five known genetic families (I to V). Key reference strains like the prototype strain JaGAr39, vaccine strain OBE-1, and genetically distinct isolates such as MP496 were included for a comprehensive analysis. The phylogenetic tree, based on Gc protein sequences, displayed significant genetic diversity. Amino acid homology similarity ranged from 69.2% to 100% (refer to Figure 1). The maximum likelihood phylogenetic tree was utilized to analyze the relationship between these strains further, highlighting conservation and variation (refer to Figure 2).Notably, the Kenyan strain MP496 formed a unique and deeply branched pedigree (pedigree IV), showing stark differences from all isolates in Southeast Asia and Australia. Its nucleotide homology with other AKAV isolates ranged from 69.2% to 69.8%, with the highest similarity to R7949 at 69.8% and the lowest with OBE-1 and TJ2016 at 69.2%.In contrast, pedigree I included strains like CY-77 from Taiwan Province, Iriki, and ON-1/E/98 from Japan, while pedigree II encompassed vaccine strains OBE-1, JaGAr39, and KT3377, forming a major Asian branch. Australian isolates B8935 and R7949 clustered together into lineage III, underlining the impact of geographical isolation on AKAV evolution. Isolates from the same country and year displayed high nucleotide homology (>99%). For instance, Chinese isolates 52 and 55 exhibited a 99.8% homology, while Japanese isolates JaGAr39 and JaLAB39 showed 100% similarity, suggesting a shared ancestral origin. Australian isolates B8935, CS0016, and CS231, collected from 1968 to 1982, formed a closely related phylogenetic group with 97.1%-99.5% nucleotide homology. The Korean strain AKAV-7 shared 83.1%-85.7% nucleotide homology with the Chinese strain PT-1-AKA-C-12-TW.Between 2010 and 2023, East Asian isolates from China, South Korea, and Japan formed a highly homologous group, with nucleotide homology exceeding 97%. For example, the Korean strain AKAV-7 had a 99.5% homology with Japanese strains EH-3-Br-11 and MZ-1-Br-11, indicating recent cross-border transmission of closely related virus populations. These results underscore the combined impacts of geographical isolation and time evolution on AKAV genetic diversity. linked to various factors such as host population structure, environmental influences, and viral transmission pathways. Combining the results of amino acid similarity and phylogenetic analysis allows for a more comprehensive understanding of the genetic diversity and transmission pathways of AKAV. This helps predict the transmission potential of different subtypes or strains, assess the risk of virus epidemics, and provide guidance for future disease surveillance and control measures. Through in-depth exploration of genetic variations and evolutionary mechanisms of the AKAV Gc gene, better preparation can be made for potential epidemic threats, safeguarding the health and sustainable development of the livestock industry. These research findings provide crucial scientific foundations for understanding the transmission, pathogenesis, and epidemiology of AKAV, offering robust support for future disease prevention and control efforts.