Severe acute respiratory syndrome (SARS) first emerged in Guangdong on November 16, 2002, and subsequently spread to Hong Kong, Beijing, Taiwan, and other areas. According to WHO, 8 098 cases were reported in 29 countries.1,2 Although researchers around the world have been carrying out extensive studies for 10 years, the reservoir of SARS coronavirus (CoV) has not been found.1,3-18
At the beginning, a virus sharing 99.8% homology with SARS CoV was found in Himalayan palm civet (Paguma larvata).10 However, it is not the reservoir11-18 and the investigation has focused on bats.19-29 Some researchers believe that Bt-SLCoV (Bat SARS-like coronavirus) Rp3 (DQ71615) of Rhinolophus sinicus is closer to SARS CoV than other strains, but still not the direct ancestor. Rp3 was supposed to emerge first in 1998, 4.08 (1.45-8.84) years earlier than SARS CoV.20,25 It seems that the great grandfather of SARS CoV has been found but not the grandfather and the father. Thus, we define Rp3 as “the parental generation (PG) 3” of SARS CoV, leaving “PG 1 and 2” to be found. To explore the cross-species evolution of SARS CoV, studies have been conducted on its major genes such as ORF1a, ORF1b, S, E, M, N, especially gene S and S-encoded amino acids combined with angiotensin-converting enzyme 2 (ACE2). Although some characteristics have been found, scholars still believe that further studies are needed.1,3,5,6,8-13,18,20,21,22-25,30-39
However, we find that even after a lot of work and achievements, many researchers in a series of academic fields still follow conventional ideas. Therefore, from an innovative macroscopic view, in this article, we restudied and reanalyzed comparatively and comprehensively the findings and evidence of almost all existing researches of SARS and SARS CoV, and other infectious diseases, to reveal whether SARS CoV experiences unnatural evolution.
The very unusual natural history of SARS in epidemiology and debates on the existence of SARS CoV in nature now
As early as on February 20, 2005, Kathryn V. Holmes, an American microbiologist, said at the annual conference of American Association for the Advancement of Science (AAAS) that in the world another outbreak of SARS as in 2003 would not take place and the SARS virus strain could be found only in the experimental specimens.40 But afterwards, she added that perhaps the virus would emerge again due to hazardous incidents or new mutations.31,40 This shows that even an expert like Kathryn V. Holmes in this field harbors reservation. But her words aroused debates and many scholars from China have raised objections (http://scitech.people.com.cn/GB/25893/3196196.html).41
However, it would not be so difficult to answer the question if we reexamine the whole process of SARS epidemic carefully. Based on the most abnormal characteristics in the existing findings on the epidemic, we could now state with certainty that the natural history of SARS is very unusual in epidemiology compared to that of other human infectious diseases, and furthermore, the conclusions should be extraordinary compared with those in the studies on SARS conducted so far.
Unusual natural history of SARS in epidemiology
In China, over 100-150 cases every day, as well as many super spreaders, were reported between mid-April and early May of 2003 but none after June 3.2 And only four mild cases were reported between mid-December 2003 and early January 2004 in the Guangzhou outbreak. It is surprising that nine SARS cases were diagnosed in the laboratory outbreak between March and April 2004 in China (http://www.who.int/csr/don/2004_05_18a/en/index.html), and among them one died and a nurse was super spreader.42 So the manifestation is different from that in Guangzhou outbreak, but similar to that of the 2002-2003 epidemic. The isolated virus in laboratory outbreak presents the same situation. Since then SARS has disappeared. It is exclusively one phenomenon in the natural history of human infectious diseases.
In the history of human beings, only one infectious disease, smallpox, has been eradicated. Its eradication was officially announced by WHO in 1980 after 20 years of effort of medical workers and governments around the world.43 Unlike SARS, acquired immunodeficiency syndrome (AIDS), a variety of viral hepatitis, influenza A (H1N1) that emerged in 2009, and other emerging infectious diseases are still found occurring and spreading extensively and continually around the world. Hendra virus (HV) and Nipah virus (NV), as members of the genus Henipavirus, were found at the end of last century to be of zoonotic origin and can still be found from time to time.44-46 Comparatively, the natural history of SARS is different from the rest in that it is against the general epidemiological pattern of infectious diseases.
Reservoir of SARS CoV still has not been identified
Since April 2003, researchers have endeavored to search for the reservoir which is believed to be a kind of bat. However, in spite of the efforts for almost 10 years, the direct ancestor still remains a mystery till now.21,23,24,26-29 One question may arise: is there a direct ancestor of SARS CoV in nature?
Comparatively, the fruit-eating bats (Pteropus spp. bats or flying foxes) are already identified as the reservoirs of HV and NV.44-46 Despite the identification of HIV as early as in 1983, only recently has it been identified that M among three groups of HIV-1 has 13 subtypes, A1, A2, A3, A4, B, C, D, F1, F2, G, H, J, and K, and probably a new subtype P, and has 53 circulating recombinant forms.47-53 Researchers have revealed that tMRCA (the time of the most recent common ancestor) of SIVcpz is around 1492 and M is around 1921, N 1963, and O 1920.50-53 In spite of such a complicated origin, studies have eventually revealed the origin of HIV-1. The global spreading of H1N1 influenza A (S-OIV) in 2009 bears more similarities with that of SARS. CDC of America isolated the same virus from two epidemiologically unrelated cases. The virus was identified as the pathogen in the epidemic and composed of eight genes from mainly four ancestries and much more closely related with the triple-reassortant swine influenza virus spreading in North America.54,55 Although the origin of the H1N1 virus is so complex, studies revealed it within only 2 months.
By comparing the study history above, one may find that it is not because of inadequate efforts or backward methods, but only the one-way thought that makes the identification of the reservoir of SARS CoV a failure. If there is no reservoir for SARS CoV, how can it survive? In fact, SARS CoV has disappeared in nature, so SARS CoV is just a “passenger virus” named by us through the human history. Why is there no reservoir for SARS CoV in nature? To answer the question, we shall first clarify that SARS CoV experiences the reverse evolution.
Reverse evolution in phylogenesis of SARS CoV
When investigating the phylogenetic development of SARS CoV and its relationship with the SARS epidemic, we found SARS CoV experienced “reverse evolution”, which was defined as “the evolution of regaining the ancestral state”.56 Due to some restrictions, the current literature mainly involves experimental studies on the reverse evolution, especially in microorganism, protozoa, and insects.56,57 However, there are still some long-term and extensive studies on the reverse evolution closely related to human living environment.58,59 Interestingly, we also found the reverse evolution in HIV-1 evolution in the literature. Although the researchers did not use the term “reversion evolution”, they did use “retrieve” and even “evolves toward ancestral states” and “frequent reversion”. Three mechanisms may contribute to this “reversion”, among which the “storage and retrieve” of HIV-1 plays the most important role.60-62
The reverse evolution of SARS CoV greatly differs from that mentioned above. We restudied and reanalyzed a massive amount of research papers by scholars worldwide,1,3-16,18-39 although they have already made conclusions through routine analyses. Under the guidance of the theory, we revealed the rules of the reverse evolution in SARS CoV, which lays a scientific foundation for the exploration of the origin of SARS CoV.
SARS CoV in Guangzhou outbreak shows a closer relationship with that at the early phase of epidemic than at the late phase and the ratio of Ka/Ks decreased gradually in epidemic
The researchers chronologically divided the 2002-2003 epidemic into the early, middle, and late phases.1,18 Song et al18 constructed an unrooted phylogenetic tree of 96 SARS-CoV sequences. It was shown that SARS CoV in Guangzhou outbreak is more closely related with that at the early phase in the 2002-2003 epidemic than at the late phase, suggesting the occurrence of the reverse evolution. In the study, Song aimed to prove that the Guangzhou outbreak was an independent event. Coincidentally, it provides evidence for our study on the reverse evolution. Moreover, the phylogenetic trees constructed by Janies et al25 as well as the trees in other literature11,20,24 show similar tendency.
The change of SARS-CoV positive selection pressure in the patients can also illustrate the reverse evolution. Chinese researchers pointed out that the ratio of Ka/Ks (the rates of nonsynonymous to synonymous) decreased gradually over the three phases of the epidemic and the Ka/Ks ratios at late phases were significantly less than 1.0.1
Reverse evolution in some key amino acids at the receptor banding site of SARS CoV and Bt-SLCoV
The bonding sites of SARS CoV to ACE2, closely related to the transmissibility, are among amino acids 318-510 of spike protein, especially amino acids 487 and 479.1,11,12,18,20,25,30,31-34,36,63 Li et al63 indicate that when the variations occur among the four amino acids including 487 and 479 in the binding interface between SARS-CoV RBD (receptor-binding domain) and human receptor ACE2, the charges increase (N479K) and the key methyl radicals miss (T487S), causing a thousand times decrease in the affinity of SARS CoV just like the viral strains isolated from Guangzhou outbreak. These findings objectively provide us with scientific basis for the reverse evolution.
In the study, we comprehensively reanalyzed the variations of amino acids 487 and 479 from Bt-SLCoV to SARS CoV in the whole SARS epidemic course (Table 1). As shown in Table 1, 479 in Bt-SLCoV (PG 3) is serine (S). In the 2002-2003 epidemic and/or laboratory outbreak, three types coexisted in civet SARS CoV: arginine (R), lysine (K), and asparagine (N), while only N in human. Surprisingly, during Guangzhou outbreak, only R and K existed in civet, while three types coexisted in human just as those in civet during 2002-2003. The evolution process of amino acid 487 is similar during these two periods (Table 1). Obviously, amino acids 487 and 479 went through reverse evolution in a series of strains. Janies et al25 constructed separate phylogenetic trees for 487 and 479, which more clearly support our standpoint.
Reverse evolution of characteristic 29-nucleotide
There is an accessory gene unique to the SARS-CoV ORF8, which contains the characteristic 29-nt sequence, CCTACTGGTTACCAACCTGAATGGAATAT (nt27869-27897), which was absent in most SARS cases except in very few patients of the very early epidemic and all civets. Researchers believed the deletion was related to the adaptation from animals to human.1,13,15,18,20,21,25,33,64-67 Surprisingly, scholars also found the characteristic 29-nt, CCAATACATTACTATTCGGACTGGTTTAT (nt27866-27894), in Bt-SLCoV. To clearly distinguish three conditions of the characteristic 29-nt, we propose here that the 29-nt of Bt-SLCoV, the 29-nt of SARS CoV, and the 29-nt deletion are named “original 29-nt (O 29-nt),” “variation 29-nt (V 29-nt),” and “deletion 29-nt (D 29-nt),” respectively. We found that the reverse evolution also occurs to 29-nt, which is very similar to amino acids 487 and 479 in SARS CoV and Bt-SLCoV (Table 1).
The three aspects analyzed above are our main findings of the reverse evolution in SARS CoV, and there should be other aspects as well. The characteristic coding sequence (CDS) in the nsp3 of ORF1a only exists in SARS CoV and Bt-SLCoV, which is named SARS-CoV unique domain (SUD).9,18,68,69 The researchers found that SUD may regulate the viral replication and resist immune response of the host.68,69 Song et al18 indicated that mutation occurred in ORF 1a nt6295 in SARS CoV of Guangzhou outbreak, leading to a stop codon in the nsp3 CDS. The decrease or loss of SUD function shows inadaptation of SARS CoV to the human, and its genes are retrieving.
Based on the unusual characteristics above and the reverse evolution in table 1 of SARS CoV, we believe that SARS CoV has an origin from the unusual (“unnatural”) evolution of Bt-SLCoV Rp3 or similar various strains. Therefore, we should point out the mode of the reverse evolution of SARS CoV may be unprecedented even in the biological evolution history of human infectious diseases.
Cause for the reverse evolution of SARS CoV and diagram on its unnatural origin and disappearance
The reverse evolution is a normal and common phenomenon in the organic evolution, but our knowledge and studies on it are far from enough. The reverse revolution is caused directly by the changes of landscapes and environments in which organisms live. We try to summarize them into three aspects effecting on the degree and rate of the reverse evolution. (1) Colony is likely to maintain the standing genetic variations derived from its ancestors’ living environments when it moves to a new environment for a short time. (2) The frequency of selecting the variations of existing genotypes adaptive to their ancestors’ living environments would increase when organisms reverse to their ancestors’ state. (3) In a new environment, fewer adaptive variants reflect the poor adaptive conditions, which may contribute to the reverse revolution.
Therefore, since SARS CoV has an unnatural origin and transmits itself unusually to new hosts including human and animals, it has always been under the pressure of “reverse evolution.” Based on the above discussion and scientific reasoning, we draw a diagram on the unnatural origin and evolution of SARS CoV (Figure 1). Though Figure 1 needs to be improved, added, and modified in some details, it can in general provide reasonable answers to the questions unanswered.
With the principles of molecular epidemiology, phylogenetics, and the reverse evolution as the guidance, we have historically and comprehensively restudied and reanalyzed most of the literature about SARS and related diseases. We propose a new explanation about the origin and evolution of SARS CoV. SARS CoV was unnaturally produced and transmitted. It was continuously subjected to the great pressure of inadaptation and returned back gradually to its ancestor’s state through the reverse evolution. According to the research, we put forward the possibility of a global spreading of a zoonosis caused by the pathogen of unnatural origin. The theory of the reverse evolution has played an important role in guiding people to understand the origins of infectious diseases.
We thank professors/lecturers Chen Jingyuan, Yan Yongping, Xia Jielai, Huang Yuan, Fan Jiayong, Zhou Yumei, Su Chunping, Shi Jian, Yu Xiaohan, and Tan Yahui for their encouragement.
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