key: cord-0276554-fwwwtx0v authors: Su, Shuo title: A re-emerging arbovirus in livestock in China: Early genomic surveillance and phylogeographic analysis date: 2022-03-14 journal: bioRxiv DOI: 10.1101/2022.03.11.483952 sha: c4ec28f10568b24e01fc8521068dc8c9c0e5fa41 doc_id: 276554 cord_uid: fwwwtx0v Viruses in livestock represent a great risk to public health due to the close contact of their hosts with humans and the potential long-range transportation though animal trade. Here, we show how to predict outbreaks of potential zoonotic pathogens and use spatially-explicit phylogeographic and phylodynamic approaches to provide estimates of key epidemiological parameters. First, we use metagenomic next generation sequencing (mNGS) to identify a Getah virus (GETV) as the pathogen responsible for a re-emerging swine disease in China. The GETV isolate is able to replicate in a variety of cell lines including human cells and shows high pathogenicity in a mouse model suggesting a potential public health risk. We obtained 16 complete genomes and 78 E2 gene sequences from viral strains collected within China from 2017 to 2021 through large-scale surveillance among livestock, pets and mosquitoes. Moreover, phylogenetic analysis reveals that three major GETV lineages are responsible for the current epidemic in livestock in China. We identify three potential positively selected sites and mutations of interest in E2, which may impact the transmissibility and pathogenicity of the virus. We then reconstruct the evolutionary and dispersal history of the virus and test the impact of several environmental factors on the viral genetic diversity through time and on the dispersal dynamic of viral lineages. Of note, we identify temporal variation in livestock meat consumption as a main predictor of viral genetic diversity through time. Finally, phylogeographic analyses indicate that GETV preferentially circulates within areas associated with relatively higher mean annual temperature and pig population density. Our results highlight the importance of continuous surveillance of GETV among livestock in southern Chinese regions associated with relatively high temperatures, and the need to control mosquitoes in the livestock farms. Our analyses of GETV also provide a baseline for future studies of the molecular epidemiology and early warning of emerging arboviruses in China. large-scale surveillance among livestock, pets and mosquitoes. Moreover, phylogenetic 23 analysis reveals that three major GETV lineages are responsible for the current 24 epidemic in livestock in China. We identify three potential positively selected sites and 25 mutations of interest in E2, which may impact the transmissibility and pathogenicity of 26 the virus. We then reconstruct the evolutionary and dispersal history of the virus and 27 test the impact of several environmental factors on the viral genetic diversity through 28 time and on the dispersal dynamic of viral lineages. Of note, we identify temporal 29 variation in livestock meat consumption as a main predictor of viral genetic diversity Approximately 18% of emerging infectious diseases (EIDs) that affect humans 41 originate from wild animals or livestock [1] [2] [3] [4] . In many of these host reservoir species, 42 emerging viruses appear to be well adapted, with little or no evidence of clinical disease. 43 However, when these viruses spill over into humans, the effects can sometimes be 44 devastating [5, 6] . Because livestock can often act as a conduit for pathogen spillover 45 into susceptible human populations, research on emerging viral diseases is focused on 46 livestock infections that often occur due to contact with wild animals [7] . For example, 47 the swine industry couples high-density farming with international trade, thus 48 generating a high risk of emerging virus transmission and the potential for global spread 49 [8]. Moreover, the swine industry will increasingly represent such risk due to its 50 constant growth to fulfill a high demand for pork. A disease outbreak caused by a new 51 or emerging virus may incur substantial economic burden and also endanger human 52 health due to close human contact with pigs. Pigs have also been shown to be a 53 significant source of zoonotic viruses such as Nipah virus in Malaysia [9] or influenza 54 A (H1N1) virus (IAV) that caused the "swine-origin influenza" pandemic [10] . The 55 2009 A/H1N1 influenza pandemic in Mexico arose from viruses circulating in pigs in 56 central-west Mexico for more than a decade. The virus originated from Eurasia (the 57 landmass containing Europe and Asia) owing to an expansion of influenza A virus 58 diversity in swine resulting from long-distance live swine trade [11] . The importance 59 of pigs as a source of emerging viruses has recently been illustrated by four cases of 60 human acute encephalitis that were associated with a variant strain of pseudorabies Before the piglets died, they showed clinical symptoms such as diarrhea, wasting, 133 panting, skin rash, and some neurological symptoms. To investigate the cause of dead 134 piglets, we collected swabs, feces and tissue samples of dead piglets from these farms. 135 During the transportation of samples, sufficient cryogenic ice packs were added to the 136 carrying case to maintain a low temperature environment. Collection of all animal Temporal signal in our data set was visually assessed using TempEst (1.5.1) [58] . Time-267 scaled phylogenetic inference was performed using the program BEAST 1.10.4 [59] 268 with high-performance computing library BEAGLE [60] . We assessed the best fitting 269 molecular clock model through marginal likelihood estimation (MLE) using path- that is based on genetic sequence data as well as the covariate data (in contrast to 296 standard coalescent-based approaches that reconstruct the demographic history 297 exclusively from genetic sequence data). We performed a separate analysis for each of 298 the following five covariates: annual mean temperature, annual precipitation, forest are also depicted in the trimeric E1/E2 spike, which is shown as a top view (Fig. 2B) . Residue 323, which is characterized by a conservative Asp to Glu substitution, is factor values >20, which correspond to strong statistical support [70] . Nevertheless, we 521 found that the Henan province in central China and eastern region in China should be 522 the one of hubs for GETV spread (Fig. S5) . The continuous phylogeographic reconstruction does not allow us to trace the precise 524 origin of the spread of GETV lineages because the uncertainty associated with the 525 location inferred for the root of the tree is relatively pronounced (Fig. 4) . However, the 526 reconstructed dispersal history of GETV lineages clearly highlights that some southern 527 and eastern Chinese provinces (Guangxi, Guangdong, Jiangxi, Fujian, Zhejiang) were 528 more recently colonized (>2015; cfr. yellow nodes in Fig. 4) branch durations for similar geographic distances travelled by those branches; Fig S6) . 539 We further tested whether lineage dispersal locations tended to be associated with 540 specific environmental conditions. In practice, we started by computing the E statistic, sequences from swine have been rare [26, 73, 74] . In this study, we perform a state-of-569 the-art genomic surveillance using metagenomic next-generation sequencing coupled 570 with phylodynamic analyses. We find a high abundance of GETV in dead pig samples 571 and identify its link to an outbreak among pig herds in China. We show also that GETV 572 has a broader host range as previously anticipated, which complicates prevention and 573 control because of its diverse reservoir and multiple hosts. We analyze the genetic 574 diversity, dispersal history, and the external factors that may impact the spatial spread 575 of the virus in the early stage of an outbreak/re-emergence in the Chinese pig herd. 576 We highlight that the current emergence GETV can be divided into three main lineages 577 that primarily evolved and spread in livestock and are geographically widespread. Moreover, our study has enabled a more robust analysis of GETV evolutionary history 617 and revealed a more extensive genetic diversity compared to previous analyses [28] . 618 We estimate that the overall genetic diversity of GETV has increased through time since 619 the first report, which increased the possibility of a large-scale GETV outbreak [80] . 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The relative abundance of each virus in 957 each library was estimated and normalized by the number of mapped reads per million 958 total reads (RPM) Red squares represent new sequences obtained in this study. The sampling location and 963 the host are color-coded. (D) GETV was successfully isolated and verified by agarose 964 gel electrophoresis. (E-F) Vero were infected with GETV-GX or GETV-HN 965 (MOI=0.001) and PK15 cells were Cytopathic changes was observed at 12, 24, 36, 48, 60 and 72 hpi Nuclei are stained blue with DAPI. All fluorescent images were taken at 969 20×magnification. (I-J) Growth of GETV-GX or GETV-HN in Vero (I) and PK15 (J) 970 cultures. Viral titers were determined for samples (only medium) between 12 and 72 971 hpi in Vero cells. Data are expressed as mean ±S.D. of viral titers (lg10 TCID50 per 972 0.1ml) derived from three infected cell cultures. 973 974 R250