The Biological Characteristics Of The Pangolin Coronavirus GX＿P2V(Short＿3UTR) And Its Cross-immune Protection To SARS-CoV-2

The coronavirus disease 2019(COVID-19)caused by the severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)is the most serious emergent infectious disease in this century.In the process of tracing the origins of SARS-CoV-2,while multiple coronaviruses related to SARS-CoV-2 have been identified in bats and pangolins,few of these have been successfully isolated and cultivated in practice.Consequently,there is currently a scarcity of research characterizing these coronaviruses related to SARS-CoV-2.Our team previously analyzed the sequences of coronavirus derived from small intestine-lung tissues of pangolins smuggled to Guangxi and obtained a nearly complete whole genome sequence,which was named GX＿P2V.We have studied the complete genome sequence,biological characteristics,infectivity,attenuation and cross-immune protection against SARS-CoV-2 and its variants.The specific research contents are as follows:(1)Whole genome sequence determination and analysis of GX＿P2V(short＿3UTR)isolate from pangolin coronavirusConsidering the original sample GX＿P2V has a small amount of unknown base information and an incomplete 5’ terminal sequence,high-throughput sequencing(NGS)and 5’c DNA terminal rapid amplification(5’RACE)were employed to re-sequence GX＿P2V(short＿3UTR),which had been cultured in Vero cells for eight successive generations.The results showed that the viral genomic contig has 29,729 nucleotides including eight consecutive adenine residues at the 3’-terminus.Compared to the first passage sample,the multiply passaged GX＿P2V(short＿3UTR)isolate exhibits complete 5’/3’-terminus sequences and two mutations: a nonsynonymous alteration in the final amino acid codon of the nucleoprotein gene(GCU→GUU)and a 104-nucleotide deletion in the hypervariable region(HVR)of the 3’-terminus untranslated region(3’-UTR).It was renamed GX＿P2V(short＿3UTR)and its complete whole genome sequence(29,729 nt)was submitted to Gen Bank(entry number MW532698).The HVR region of original sample GX＿P2V is 166-nt long and ranges from 29,611-29,776(Gen Bank: MT072864).The missing region of GX＿P2V(short＿3UTR)range from 29617 to 29720,covering 62.7% of the HVR region.The results showed that only one read in the original sample GX＿P2V(SRR11093271)contained a single nucleotide mutation of N protein,and no reads showed HVR deletion,while all reads covering the two mutation regions in GX＿P2V(short＿3UTR)strain contained the above two mutations.Therefore,we obtained a cellular adaptive mutant,which is also the first report of spontaneous large fragment deletion in the HVR region of pangolin coronavirus.The research reveals that HVR deletion mutations are associated with reduced virulence.These findings provide a scientific basis for further research on the infectivity and attenuation of GX＿P2V(short＿3UTR).(2)Analysis of cell tropism and host factors of pangolin coronavirus GX＿P2V(short＿3UTR)isolateWe analyzed the infectivity of GX＿P2V(short＿3UTR)to a variety of cells and its utilization of different species receptors and potential accessory receptors.The results showed that GX＿P2V(short＿3UTR)showed significant sensitivity to six human organ-derived cell lines(Hep-2,Calu-3,Caco-2,Huh-7,HK-2 and HEK-293T)and two monkey kidney cells(Vero and BGM).However,GX＿P2V(short＿3UTR)lacks susceptibility to five cell lines from mice(BALBc-3T3/,LLC,Hepa1-6,C57 and LA795).We also found that,except human angiotensin-converting enzyme 2(ACE2),ACE2 from rats,monkeys,pigs,cats,but not from mice,can also effectively facilitate GX＿P2V(short＿3UTR)infection.These results indicate that the GX＿P2V(short＿3UTR)spike protein can interact with those ACE2 receptors.In addition,the promotion effect of rat ACE2 on SARS-CoV-2 pseudoviruses was not obvious,which was consistent with reports.Furthermore,in view of the high similarity between GX＿P2V(short＿3UTR)and SARS-CoV-2 receptor binding domains,we screened five functional accessory receptors for SARSCoV-2 and discussed their effects on GX＿P2V(short＿3UTR)infection.The results demonstrated that five potential receptors(AXL,CTSL,CLEC4 G,TMEM30A and LDLRAD3)also promoted GX＿P2V(short＿3UTR)infection to varying degrees,indicating that GX＿P2V(short＿3UTR)and SARS-CoV-2 may have similar infection mechanisms.Subsequently,we evaluated the infectivity of GX＿P2V(short＿3UTR)in rat and C57 mouse models.GX＿P2V(short＿3UTR)could effectively infect rats and induce specific antibodies in rats,but did not cause pathogenic damage,suggesting that this strain was highly attenuated in rat models.The limited infectivity in C57 mice may be attributed to the binding efficiency of mouse ACE2 receptor.Finally,we comparatively analyzed the binding sites and space structures of GX＿P2V(short＿3UTR)and SARS-CoV-2receptor binding domains and human,rat and mouse ACE2,and proposed that binding number of key amino acid residues and intermolecular interaction force at the binding interface likely affect the affinity of GX＿P2V(short＿3UTR)to receptors.(3)Infectivity and pathogenicity analysis of pangolin coronavirus GX＿P2V(short＿3UTR)isolateWe systematically analyzed its infectivity and pathogenicity in in vitro and in vivo infection models.The results showed that GX＿P2V(short＿3UTR)induced similar cytopathic effects(CPE)in Vero and BGM cells,with only minor damages and small plaques at 5 days post-infection(dpi).However,GX＿P2V(short＿3UTR)did not cause significant CPE and plaques in human Calu-3 cells.In contrast,SARS-CoV-2 caused severe cell damages and large plaques in Vero cells as early as at 2 dpi.Therefore,GX＿P2V(short＿3UTR)is highly attenuated in terms of causing cell damage and plaque formation.Secondly,we established a golden hamster infection model.The results showed that,contrary to the reported results of SARS-CoV-2 infection in golden hamsters,the body weight of golden hamsters infected with of GX＿P2V(short＿3UTR)continued to increase,and no obvious clinical symptoms,organic injuries on the lung surface were observed.Different levels of virus titers were detected in turbinate,trachea and lung at 2 dpi,and viral titers were still measured in turbinate and lung at 5 dpi,indicating that GX＿P2V(short＿3UTR)could infect golden hamsters through respiratory tract.Although different levels of viral loads were detected in extrapulmonary tissue and feces,none were infectious.However,no severe pathological damages were detected in the respiratory system of the golden hamster infection models throughout the entire infection period,indicating that GX＿P2V(short＿3UTR)was highly attenuated.At the same time,we detected specific Ig G antibodies against itself receptor binding domain in golden hamsters,suggesting that this virus has strong immunogenicity.In addition,we also established young and old BALB/c mouse models,and GX＿P2V(short＿3UTR)did not cause pathogenic damage in mice.We have fully demonstrated that GX＿P2V(short＿3UTR)is highly attenuated and non-pathogenic to the hosts,and retains strong immunogenicity.Therefore,GX＿P2V(short＿3UTR)is a potential attenuated live vaccine candidate.(4)Immunogenicity of pangolin coronavirus GX＿P2V(short＿3UTR)isolate and its cross-immunity protection against SARS-CoV-2Considering that GX＿P2V(short＿3UTR)is highly homologous to SARSCoV-2 and highly attenuated,we propose that GX＿P2V(short＿3UTR)may be a candidate attenuated live vaccine against SARS-CoV-2.We found that sera from patients infected with wild-type SARS-CoV-2 and from golden hamsters infected with GX＿P2V(short＿3UTR)exhibited cross-reactivity with key proteins of both viruses.Moreover,both sets of sera were also effectively capable of cross-neutralizing pseudoviruses of two viruses.The above results indicate that GX＿P2V(short＿3UTR)and the original SARS-CoV-2 strain share cross-neutralization epitopes,which supports the possibility of GX＿P2V(short＿3UTR)as a live attenuated vaccine candidate against SARSCoV-2.Furthermore,we found that individuals who received the standard SARS-CoV-2 vaccine but subsequently experienced breakthrough infection with the Omicron BF.7 or XBB.1.16 variant developed potent cross-neutralizing antibodies against GX＿P2V(short＿3UTR).The sera from golden hammers infected with GX＿P2V(short＿3UTR)also had a certain neutralization effect on XBB.1.16,suggesting that GX＿P2V(short＿3UTR)and SARS-CoV-2 variants also shared cross-neutralization epitopes.The results showed that golden hamsters inoculated with two doses of GX＿P2V(short＿3UTR)intranasally produced long-term sterilizing immunity against the virus itself.In the golden hamsters,two doses of GX＿P2V(short＿3UTR)not only effectively reduced the infection and replication of wild-type SARS-CoV-2 and variants XBB.1.16 and EG.5.1 in respiratory tissues,but also effectively alleviated the clinical symptoms and pathological damage.These results indicated that GX＿P2V(short＿3UTR)had effective cross-immunity protection against SARSCoV-2 and variants.The hybrid immunization combined the inactivated wildtype SARS-CoV-2 vaccine with GX＿P2V(short＿3UTR)produced different degrees of protection against SARS-CoV-2 and variants,but the protection effect was not as robust as that achieved with two doses of GX＿P2V(short＿3UTR)immunization.The above results convincingly demonstrate that the GX＿P2V(short＿3UTR)provides effective protection against SARS-CoV-2 and its variants and is a potential broad-spectrum COVID-19 vaccine.In summary,we found spontaneous large fragment deletion mutations in the HVR region of pangolin coronavirus.GX＿P2V(short＿3UTR)is both immunogenic and highly attenuated,and has no pathogenicity to the hosts,which makes GX＿P2V(short＿3UTR)have the potential of attenuated live vaccine.We have also confirmed that infection with this strain can induce crossimmune protection against SARS-CoV-2 and variants,suggesting that GX＿P2V(short＿3UTR)may be used as a broad-spectrum COVID-19 vaccine.