key: cord-0880359-wn9l4wtc
authors: Wong, Yik Chun; Lau, Siu Ying; To, Kelvin Kai Wang; Mok, Bobo Wing Yee; Li, Xin; Wang, Pui; Deng, Shaofeng; Woo, Kin Fai; Du, Zhenglong; Li, Cun; Zhou, Jie; Chan, Jasper Fuk Woo; Yuen, Kwok Yung; Chen, Honglin; Chen, Zhiwei
title: Natural transmission of bat-like SARS-CoV-2(ΔPRRA) variants in COVID-19 patients
date: 2020-07-10
journal: Clin Infect Dis
DOI: 10.1093/cid/ciaa953
sha: 80a6cd4349e060e346b51ca31217b82a2c714120
doc_id: 880359
cord_uid: wn9l4wtc

BACKGROUND: SARS-CoV-2 contains the furin cleavage PRRA motif in the S1/S2 region, which enhances viral pathogenicity but is absent in closely related bat and pangolin coronaviruses. It remains unknown if bat-like coronaviral variants without PRRA (ΔPRRA) can establish natural infection in humans. METHODS: Here, we developed a duplex digital PCR assay to examine ΔPRRA variants in Vero-E6-propagated isolates, human organoids, experimentally infected hamsters and COVID-19 patients. RESULTS: We found that currently transmitting SARS-CoV-2 contained a quasispecies of wildtype, ΔPRRA variants and upstream variants that have mutations upstream the PRRA motif. Moreover, the ΔPRRA variants were readily detected despite at a low intra-host frequency in transmitted founder viruses in hamsters and in COVID-19 patients including acute cases and a family cluster with a prevalence rate of 52.9%. CONCLUSIONS: Our findings demonstrate that bat-like SARS-CoV-2(Δ)(PRRA) not only naturally exists but remains transmissible in COVID-19 patients, which have significant implications to zoonotic origin and natural evolution of SARS-CoV-2.

M a n u s c r i p t 4

A novel beta-coronavirus, now recognized as SARS-CoV-2, led to the global coronavirus disease 2019 (COVID-19) outbreak [1, 2] . The rapid spreading of SARS-CoV-2 is clearly due to aggressive person-to-person transmission with early evidence in hospital and family settings [3, 4] .

Based on viral genome analysis, early studies have indicated that SARS-CoV-2 is similar to bat coronaviruses with 96% identity, but is relatively distant from SARS-CoV [5] [6] [7] [8] . In spite of only 40% amino acid identity in the external subdomain of receptor binding domain (RBD), SARS-CoV-2 uses the same cellular receptor angiotensin-converting enzyme 2 (ACE2) as SARS-CoV to initiate infection [6, 9] . Since many bat coronaviruses use ACE2 as cellular receptor but have not easily caused human outbreaks [10, 11] , other viral factors may contribute to more efficient zoonotic and person-toperson transmission besides ACE2 usage [7, 12] .

One such viral factor has been associated with an insertion of the polybasic cleavage motif PRRA in the S1/S2 cleavage site of SARS-CoV-2 spike protein, which has not been found in either bator pangolin-derived coronaviruses [10, 11, [13] [14] [15] . Protease-mediated viral entry is one of the determinants of success in SARS-CoV infection [16] . Similarly, furin and serine protease TMPRSS2 are essential for SARS-CoV-2 infection of human target cells [9, 17] . Interestingly, our team has recently discovered that a series of variants, which contain animal-like PRRA deletions (PRRA) in the S1/S2 cleavage region through plaque purification of SARS-CoV-2 in Vero-E6 cells [18] . Since conventional methods failed to detect these variants in clinical samples [18] , we sought to develop a highly sensitive digital PCR assay to investigate this viral variant among experimentally infected animals and naturally infected COVID-19 patients. Although several studies have indicated that SARS-CoV-2 is a naturally occurring virus through zoonotic transmission, we aimed to investigate the critical missing link whether or not SARS-CoV-2 with bat-or pangolin-like PRRA deletion (SARS-CoV-2 PRRA ) can be found in COVID-19 patients.

A c c e p t e d M a n u s c r i p t 5

To detect SARS-CoV-2 PRRA variants amongst COVID19 patients, we established a duplex digital PCR assay that included the amplification of a 162bp sequence spanning the viral S1/S2 cleavage site and the detection of this sequence with two fluorescent oligonucleotide probes, namely PRRA and upstream probes ( Figure 1A ). The PRRA probe specifically targeted the PRRA motif within the S1/S2 cleavage region. This probe would bind to amplicons generated from wildtype SARS-CoV-2 sequence, but not from PRRA variants, including Del-Mut-1 and Del-Mut-2 isolates, which carry deletion in the S1/S2 site [18] [19] [20] . For viral variants containing deletion directly at the 5'flank of the PRRA site, such as Del-Mut-3 [18] [19] [20] , the PRRA probe would still detect their sequences.

The probe upstream the PRRA site acted as a reference probe for the identification of SARS-CoV-2 sequence. In general, wildtype viral sequences should be detected by both the upstream and PRRA probes while PRRA variants should be detected by the upstream probe only, but not the PRRA probe.

We firstly validated the specificity of the digital PCR assay using cDNAs generated from plaque-purified wildtype and Del-Mut-1 isolates. Our assay successfully identified the presence of 100% PRRA in the Del-Mut-1 isolate, without detection of wildtype sequence ( Figure 1B ). In comparison, the majority of viral cDNAs from the wildtype isolate contained the wildtype PRRA cleavage site. Unexpectedly, our assay also detected a low frequency of PRRA variants (1.4% of total detected viral copies) within the wildtype isolate, which had undergone plaque purification in Vero-E6 cells ( Figure 1B) . Furthermore, another unexpected population of viral sequences from the wildtype isolate was readily detected by the PRRA probe, but not the upstream probe. These findings suggested that the wildtype isolate from Vero-E6 cells actually contained a mixture of wildtype, PRRA and upstream variants.

A c c e p t e d M a n u s c r i p t 6 We next examined the sensitivity of our digital PCR assay using serially diluted cDNA samples generated from the wildtype isolate. Focusing on the upstream probe, our assay consistently produced measurable signals from the diluted cDNA samples equivalent to 0.15 plaque forming unit ( Figure 1C ). We then evaluated the ability of the digital PCR assay to distinguish PRRA variants from wildtype sequences using cDNA mixtures containing fixed amount of wildtype viral cDNAs with serially diluted Del-Mut-1 cDNAs ( Figure 1D ). Our assay readily detected the presence of a higher frequency of PRRA mutants (averaged 2.96% ±0.09% SD) in the cDNA sample containing 1:0.01 ratio of wildtype to Del-Mut-1 cDNAs, as compared to the wildtype only sample (averaged 1.88% ±0.46% SD). Overall, our assay is sensitive for detecting the presence of low abundance PRRA mutants.

Our team recently demonstrated that SARS-CoV-2 replicates in human intestinal organoids [21] . We sought to determine if wildtype SARS-CoV-2 and SARS-CoV-2 PRRA could establish infection in human intestinal organoids using plaque-purified wildtype or Del-Mut-1 isolates. The presence of S1/S2 cleavage site variants were tested from culture supernatants at 48 hours post-infection.

Although both upstream and PRRA variants could be detected from the wildtype viral inoculum, as shown in Figure 1B , the presence of these variants was suppressed to minimal level after propagation in the intestinal organoids ( Figure 2A) . A single copy of PRRA variant was detected from one of the triplicate organoid samples. In organoids infected with Del-Mut-1, only PRRA variants were detected (Figure 2A ). These findings suggest that the viral genomic region of the S1/S2 cleavage site remains stable after prorogation in human intestinal organoids. Furthermore, due to the low PRRA frequency in organoids infected with the wildtype isolate, PRRA variants have no A c c e p t e d M a n u s c r i p t 7 growth advantage to outcompete wildtype virions in human organoids, as opposed in Vero-E6 cells [18] .

To directly test for the transmissibility of SARS-CoV-2 PRRA in vivo, the digital PCR assay was conducted with various airway tissue samples from hamsters at 4 days post-infection with either plaque-purified wildtype or Del-Mut-1 isolates ( Figure 2B ). There were no detectable levels of both upstream and PRRA variants within the nasal turbinates and lung tissues from hamsters infected with the wildtype viral isolate. Both upstream and PRRA variants, however, could be identified within the tracheal tissues. The frequencies of these two variants within tracheal tissues were lower than those in the viral inoculum. In hamsters infected with Del-Mut-1 , only PRRA variants could be detected from all airway tissues examined, with no evidence of wildtype virus ( Figure 2B ). The finding of a mixture of wildtype, PRRA and upstream variants in tracheal tissues of both hamsters challenged with wildtype SARS-CoV-2 isolate at 4 days post-infection indicates a lack of genetic bottleneck for single virus mucosal transmission. The absence of PRRA and upstream variants in nasal turbinates and lungs suggested that they probably have a reduced in vivo fitness when compared to the wildtype virus. Moreover, the lack of wildtype sequences in hamsters infected with

Del-Mut-1 demonstrated that the PRRA-to-wildtype reversion did not happen during the acute 4day infection period.

We next determined the prevalence and frequencies of S1/S2 cleavage site viral variants in clinical samples collected from COVID-19 patients. In the digital PCR assay, 51 patients' samples Figure 1) . Although the remaining sample showed the presence of PRRA variants without any upstream variants, it should be taken into account that only a low viral copy level was detected from this sample.

Overall, while three types of viruses have been transmitted in humans, the wildtype virus has greater advantages than upstream and PRRA mutants for infection at both inter-and intra-host levels.

A c c e p t e d M a n u s c r i p t 9

We here demonstrated that bat-like SARS-CoV-2 PRRA and upstream variants exist naturally and are currently transmitting in COVID-19 patients, as revealed by our duplex digital PCR assay. Although these variants only consisted of a very small fraction in the wildtype viral challenge stock, they were consistently detected in intranasally inoculated hamsters. PRRA and upstream variants were also readily detected among acute patients, including a family cluster. These results indicate that person-to-person mucosal transmission of SARS-CoV-2 is unlikely a genetic bottleneck allowing infection only by single transmitted founder viruses, but rather by viral quasispecies. PRRA is unlikely an overwhelming restriction factor for human transmission by zoonotic bat-like SARS-CoV-2 PRRA , which may support zoonotic origin and evolution of SARS-CoV-2 in humans. It is, therefore, necessary to implement stringent measures to prevent human infection by animal SARS-CoV-2 PRRA variants including handling field and laboratory specimens derived from wild bats and pangolins.

Due to mutations and ability to undergo genomic recombination [23] , genetic variations of different coronaviruses, including SARS-CoV [24, 25] , MERS-CoV [26, 27] , and other animal coronaviruses [28] , are readily identified at both population and intra-host levels. SARS-CoV-2 variants have also been reported among COVID-19 patients, mainly by next generation sequencing methods [2, 19, 29, 30] . In particular, viral variants carrying PRRA in SARS-CoV-2 isolates passaged in vitro in Vero-E6 cells have been recently reported by our team [18] and others [19, 20] . Using the duplex digital PCR assay, we detected PRRA and upstream variants from clinical isolate propagated in Vero-E6 cells, suggesting that this genomic region is instable and dispensable in SARS-CoV-2 during viral replication in Vero-E6 cells. An initial minor viral variant population carrying PRRA mutations would be generated and selected for, becoming the dominant strain after further propagation in Vero-E6 cells [18, 19] . The spike protein of coronaviruses needs to be activated via A c c e p t e d M a n u s c r i p t 10 sequential proteolysis at the S1/S2 and S2' sites for viral entry into target cells [31] . For MERS-CoV and SARS-CoV-2, spike protein activation is achieved either via initial cleavage at the S1/S2 site by furin, followed by S2' site cleavage by TMPRSS2 on cell surfaces or via protolytic processing by cathepsin B/L after endocytosis [9, 17, 32] . Vero cells express low level of furin [33] but high level of cathepsin B/L, and they support furin-independent MERS-CoV and SARS-CoV-2 viral entry [17, 32] .

This might create a selection pressure against the furin cleavage site. Moreover, Vero cells are interferon defective [34] . PRRA variants might have enhanced fitness in cell lines with suboptimal innate immune responses to SARS-CoV-2 infection. This is supported by our previous findings which demonstrated that the PRRA Del-Mut-1 isolate grew well in Vero-E6, but replicated poorly in interferon competent cells [18] . Interestingly, another study showed that propagation of a SARS-CoV-2 isolate in another Vero-derived cell line, Vero-76, did not lead to PRRA site mutation [30] .

Acquiring the PRRA insertion in SARS-CoV-2 might enhance its host adaptation with increased growth capacity and pathogenicity. One hypothesis is that after zoonotic transmission, SARS-CoV-2 with an intact PRRA motif at the S1/S2 cleavage site has probably selected from bat-or pangolin-derived SARS-CoV-2 PRRA to become the most prevalent viral type in both inter-and intrahost levels in patients. Although PRRA variants could be detected in about 52.9% of the patients in this study, these variants were only present in very low frequency in individual patients.

Experimentally, in the hamsters challenged with the wildtype SARS-CoV-2 strain, a lower frequency of PRRA variants were always detected in the airway tissues than from the challenge inoculum.

Thus, in contrast to a better replicative fitness in Vero-E6 cells in vitro, PRRA variants were likely selected against in vivo during the natural course of viral evolution.

We identified a new type of viral variants containing mutations at the 5' upstream region of the S1/S2 site. These variants were more prevalent than the PRRA variants in both intra-and interhost levels in our COVID-19 patient cohort. It is common for the fixation of coronavirus variants in a A c c e p t e d M a n u s c r i p t 11 global epidemiological scale. Founder effect might be involved in selecting orf8 deletion during the early stage of SARS-CoV human transmission [35] . Mutations in viral lineages specific to certain geological regions have also been documented for SARS-CoV and MERs-CoV infections [25, 36, 37] .

Using whole genome phylogenetic analysis, a recent report identified three SARS-CoV-2 variant lineages clustered in distinct geological regions and suggested a possibility of founder events or selective pressures for viral variants specific to certain environmental and/or host backgrounds [2] .

In our patient cohort, SARS-CoV-2 with intact PRRA motif remained the dominant strain in both intra-and inter-host levels. Wildtype virions have a better selection advantage in vivo than S1/S2 cleavage site variants, arguing against the possibility for the fixation of S1/S2 cleavage site variants in a population level.

There are some limitations in this study. We could not determine whether or not the event of acquiring the PRRA motif took place in humans. Interestingly, a partial insertion of a similar polybasic motif as SARS-CoV-2 has been recently reported in a newly identified bat coronavirus strain at the S1/S2 cleavage site [7] . This type of viral strains might also serve as ancestral viruses for SARS-CoV-2. The analytical power of our digital PCR assay would be enhanced by coupling with sequencing analysis. It should also be noted that variants with a small deletion at the direct 5'-upstream of the PRRA site, the same deletion as of Del-Mut-3 [18] , have been reported in another patient cohort, with a prevalence rate of 4.4% 

The SARS-CoV-2 wildtype and Del-Mut-1 viral isolates were plaque purified and passaged in Vero-E6 cells obtained from ATCC, as described previously [18] . Human intestinal organoids derived from normal human small intestinal tissues, collected according to the ethical approval by the Institutional Review Board of the University of Hong Kong/Hospital Authority Hong Kong West Cluster (UW13-364), were generated and maintained, as previously described [21] . Organoids were infected with SARS-CoV-2 wildtype or Del-Mut-1 viral isolates using a similar protocol recently published [21] . All viral culture experiments were performed in biosafety level-3 facilities.

This study included clinical specimens, including saliva, nasopharyngeal secretions, throat swabs, or endotracheal aspirate, from 51 COVID-19 patients (Table 1) A c c e p t e d M a n u s c r i p t 13 Nucleic acid isolation and cDNA synthesis cDNA generated from plaque purified wildtype and Del-Mut-1 viral isolates were obtained from our previous study [18] . RNA from infected organoids and tissues from infected hamsters were extracted 

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M a n u s c r i p t 14 

Statistical analyses were performed using SPSS version 26 (IBM) or Prism version 7 (GraphPad). For patient characteristics shown in Table 1 The corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publication.

The funding sources had no role in study design, data collection, analysis, interpretation, or writing of the report. The authors declare no competing interests.