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. 2023 Dec;12(2):2225932.
doi: 10.1080/22221751.2023.2225932.

Assessment and sero-diagnosis for coronaviruses with risk of human spillover

Xiao-Shuang Zheng  1   2 , Qi Wang  1   2 , Ting-Ting Xie  1   2 , Hao-Rui Si  1   2 , Wei Zhang  1 , Yan Zhu  1 , Ang Li  1   2 , Jia Su  1   2 , Zheng-Li Shi  1 , Peng Zhou  1   3
Affiliations

Assessment and sero-diagnosis for coronaviruses with risk of human spillover

Xiao-Shuang Zheng et al. Emerg Microbes Infect. 2023 Dec.

Abstract

Zoonotic coronaviruses (CoVs) caused major human outbreaks in the last two decades. One of the biggest challenges during future CoV disease is ensuring rapid detection and diagnosis at the early phase of a zoonotic event, and active surveillance to the zoonotic high-risk CoVs appears the best way at the present time to provide early warnings. However, there is neither an evaluation of spillover potential nor diagnosis tools for the majority of CoVs. Here, we analyzed the viral traits, including population, genetic diversity, receptor and host species for all 40 alpha- and beta-CoV species, where the human-infecting CoVs are from. Our analysis proposed 20 high-risk CoV species, including 6 of which jumped to human, 3 with evidence of spillover but not to human and 11 without evidence of spillover yet, which prediction were further supported by an analysis of the history of CoV zoonosis. We also found three major zoonotic sources: multiple bat-origin CoV species, the rodent-origin sub-genus Embecovirus and the CoV species AlphaCoV1. Moreover, the Rhinolophidae and Hipposideridae bats harbour a significantly higher proportion of human-threatening CoV species, whereas camel, civet, swine and pangolin could be important intermediate hosts during CoV zoonotic transmission. Finally, we established quick and sensitive serologic tools for a list of proposed high-risk CoVs and validated the methods in serum cross-reaction assays using hyper-immune rabbit sera or clinical samples. By comprehensive risk assessment of the potential human-infecting CoVs, our work provides a theoretical or practical basis for future CoV disease preparedness.

Keywords: Coronavirus; bats; risk assessment; serology; spillover; zoonosis.

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Conflict of interest statement

No potential conflict of interest was reported by the author(s).

Figures

Figure 1.
Figure 1.
Genetic diversity of alpha- and beta-CoVs. (A) A phylogeny of alpha- and beta-CoV species according to the latest ICTV classification. We uniformly named the CoVs with host species (e.g. BtCoV) plus their unique identity (e.g. SC2013). Abbreviations: BtCoV, Bat CoV; HCoV, Human CoV; RCoV, Rodent CoV; MCoV, Mink CoV; EriCoV, Erinaceus CoV (hedgehog). Notably, a human representative was used for some zoonotic viral species, includes SARS-related, MERS-related, 229E-related and OC43-related, albeit viruses can also be found in bats (SARSr, MERSr and 229Er) or other mammals (OC43). (B-C) Violin plots show the genetic diversity of each alpha (B) or beta (C) CoV species. A 440 bp RdRp sequence was collected from public database and blasted to the reference CoV sequences (Supplementary table 1). Data shown as percentage to reference, each plot represents a virus species. We matched the sequences to HCoV-SARS-1 and HCoV-SARS-2, respectively, without repeated sequence counting.
Figure 2.
Figure 2.
The zoonotic probability of alpha- and beta-CoVs. (A) Proposed CoV zoonotic events, particularly in humans. The proximate year of emerging, the number of viruses in that viral species and host range for each virus were shown. The year of emerging refers to literatures, and the host range details can be found in 2D. Notably, it is widely accepted that human CoVs, such as HCoV-SARS-1 or HCoV-SARS-2, have animal origins, albeit more direct evidence is needed. (B) CoV disease phylogeny. The most important CoV disease agents, as well as the related viruses from their natural hosts or intermediate hosts were analyzed. The important viral sub-genus or species were highlighted, and the receptors were also indicated. The eight CoVs that infected humans were shown in red. (C) The wildlife hosts of CoVs. The number of continents distributed for a certain host species, the number of CoVs found in that species and the viral species carried by this species were shown. As the most important natural host of CoVs, the details of the bats in genus level were indicated, whereas the details for rodents were not shown due to limited information available. The host species that may harbour SARSr-CoVs were indicated. The details for geographical distribution of each species can be found in Redlist (www.iucnredlist.org) (D) The spillover risk of CoVs and their respective host species. Four categories of risk levels were proposed using four different colors, and each viral species was connected to their natural or intermediate host species. The viral species and the sub-genus were shown on the right, while the host species in genus level (except rodents) and the host families were shown on the left. Four important host species, including Rhinolophus bat, Hipposideros bat, civet and camel were highlighted in brown, as they showed high probability to become a zoonotic source.
Figure 3.
Figure 3.
Sero-diagnosis for zoonotic high-risk CoVs. (A) Sequence alignment of the selected CoV NP based on nucleic acid or amino acid. The sequence information can be found in Supplementary table 5. (B) Expression analysis of NP-based LIPS by WB using antibody against the tag in pREN2 vector. (C) LIPS performance using corresponding rabbit hyper-immune serum. For each antigen, antibody was tested at a 10-fold dilution, ranging from 10−2 to 10−5. Eight naïve rabbit serum samples were used as negative control. The highest dilution that showed up as positive (fold change to cut-off > 5 or 10) was chosen as the optimal dilution for cross-reaction with other antigens, shown as red in the figure. (D) Sero cross-reaction between a panel of rabbit sera against alpha-CoV and 15 NP in LIPS. (E) Cross-reaction between a panel of rabbit sera against beta-CoV NP and 15 NP in LIPS. For D and E, the antigen used in LIPS was shown on the x-axis, while the tested antibody was indicated on the figure. The y-axis indicated fold change to cut-off. Values between 1 and 3 were defined as “grey area” (between two dotted lines), indicating possible cross-reactive. Red dots indicate NP reaction to rabbit serum raised against self (F) Overview of the sero-cross reactivity.
Figure 4.
Figure 4.
Sero-cross reactivity between CoV-LIPS and SARS-CoV-2 or MERS-CoV clinical samples. (A) Reaction between 15 NP and serum samples collected from patients with COVID-19. Eight negative (blue) and 5 positive samples (red), defined using ELISA kit, were used in the LIPS. (B) Reaction between 8 beta-CoV NP and HCoV-MERS NP positive camel serum. Eight negative (blue) and 8 positive samples (red) were used for detection in LIPS. The detection cut-off was shown for each antigen.
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Grants and funding

This work was supported by National Key R&D program of China: [Grant Number 2021YFC2300901]; R&D Program of Guangzhou Laboratory: [Grant Number SPRG22-001]; the Strategic Priority Research Program of Chinese Academy of Sciences: [Grant Number XDB29010204]; the Strategic Priority Research Program of Chinese Academy of Sciences: [Grant Number XDB29010101].