Overview of Coronavirus Intermediate Hosts

When the outbreak of pneumonia caused by SARS-CoV-2 was identified in Wuhan, China, in December 2019, it was established that bats were likely the source. Bats are known carriers of coronaviruses, including those responsible for SARS and MERS, as well as other zoonotic diseases.

The closest relative to SARS-CoV-2 identified thus far is the RaTG13 coronavirus, with a genomic similarity of 96.2%, found in horseshoe bats in Yunnan, China. However, a 4% genomic difference represents a significant evolutionary gap—approximately 50 years—prompting scientists to speculate that an intermediate host may have facilitated the virus's evolution.

Historically, coronaviruses that caused major outbreaks, like SARS in 2003 and MERS in 2012, had identifiable intermediate hosts that helped in the transmission from bats to humans. Specifically, SARS-CoV-1 and MERS-CoV were traced back to palm civets and dromedary camels, respectively.

For COVID-19, the most promising lead has been the pangolin coronavirus, which shares about 90% of its genetic material with SARS-CoV-2. Notably, the receptor-binding domain (RBD) of the pangolin virus is 97.4% identical to that of SARS-CoV-2, indicating a possible genetic exchange. However, a 3% difference in the RBD is substantial, particularly as this domain is critical for binding to the ACE2 receptor necessary for infection.

Moreover, pangolins exhibited severe respiratory issues when sampled, which contradicts the characteristics of an ideal intermediate host, as noted in a 2020 research review. The review states that an effective intermediate host should not suffer severe disease upon infection, allowing the virus to replicate and explore other hosts.

Additionally, the RaTG13 coronavirus does not infect cells with the pangolin ACE2 receptor, raising doubts about the possibility of its evolution in pangolins. A bioinformatics study published in Nature Microbiology also refutes the pangolin as an intermediate host for COVID-19, asserting that there is no evidence to suggest that pangolin infections are necessary for bat viruses to infect humans.

Consequently, the identity of the animal that serves as an intermediary in the COVID-19 spillover from bats to humans remains unknown.

The Case Against an Intermediate Host in SARS-CoV-2 Evolution

Returning to the core question: What if no intermediate host is necessary for SARS-CoV-2's evolution? A recent study published in PLOS Biology suggests this could indeed be the case.

Conducted by researchers from the University of Glasgow Centre for Virus Research and Temple University, the study analyzed SARS-CoV-2 genomes sampled over a span of 11 months since the outbreak began in December 2019. The findings indicated only a few key mutations had occurred, which aligns with earlier observations.

This is unexpected because viral evolution typically accelerates when a virus adapts to a new host's biological system. One co-author expressed surprise at the high transmissibility of SARS-CoV-2 from the onset, noting that viruses transitioning to new host species usually require time to adapt and often fail to establish themselves, leading to localized outbreaks.

The study suggests that most adaptive changes in SARS-CoV-2 occurred before it reached the human population, likely while still in bats, possible intermediate hosts, or even humans.

The authors hypothesize that bats are the source based on their analyses. They acknowledge that significant genetic changes may have happened before the isolation and sequencing of SARS-CoV-2 genomes commenced. There's also a theory that SARS-CoV-2 was circulating unnoticed in humans before the outbreak in December 2019.

However, the study reports, "we fail to find evidence of diversifying selection on the terminal branch leading up to the emergence of SARS-CoV-2 in humans," suggesting that the adaptations that enabled SARS-CoV-2 to efficiently replicate in humans were likely minimal or non-existent.

Diversity selection refers to mutations that significantly alter a protein's biological function, often resulting in the emergence of new species. The research indicates that such diversifying mutations were present in earlier bat coronavirus lineages rather than in the more recent lineages leading to SARS-CoV-2.

The authors conclude that SARS-CoV-2 likely transitioned from bats to humans with little to no genetic change, explaining the stability of SARS-CoV-2 genomes since the pneumonia outbreak began in December 2019, and suggesting that an intermediate host may not be necessary for its evolution.

Contextual Insights and Ongoing Questions

The study I referenced was led by David L. Robertson, a professor and head of Viral Genomics and Bioinformatics at the University of Glasgow Centre for Virus Research, who graciously answered my questions about this topic.

#### 1. Differentiating Between Reservoir and Conduit Hosts

One significant point clarified by Professor Robertson is the distinction between an intermediate host serving as an active reservoir versus one merely acting as a conduit for viral transmission.

He noted that civet cats in the case of SARS were primarily conduits for the virus's transmission from bats to humans, with bats being the actual reservoir species. In contrast, camels for MERS serve as true reservoirs, allowing repeated camel-to-human spillover. Civets, however, do not function as reservoirs for SARS-CoV-1.

Regarding SARS-CoV-2, Professor Robertson remarked that since the virus is a generalist capable of infecting various animals such as pangolins, minks, and cats, an intermediate species might not be necessary, though it cannot be entirely ruled out as a pathway to humans. He emphasized that the virus's emergence in humans likely required little to no adaptation to achieve its success.

In summary, while an intermediate host is not essential for the evolution of SARS-CoV-2, the possibility of such a host facilitating the transition from bats to humans remains uncertain.

#### 2. The Elusive SARS-CoV-2 Progenitor in Yunnan

Despite the insights gained, we still lack definitive proof. Bioinformatics studies like the one led by Professor Robertson only present the most plausible scenarios.

Crucially, the direct jump of SARS-CoV-2 or its progenitor from bats to humans remains unsupported by crucial evidence—a coronavirus with approximately 99% genomic similarity to SARS-CoV-2 has yet to be isolated from a bat species.

To resolve the origin of SARS-CoV-2, further sampling and research on bat coronaviruses in Yunnan, the primary geographic area of interest, is necessary. Both RaTG13 and RmYN02, coronaviruses closely related to SARS-CoV-2, have been isolated from bats in Yunnan.

Scientists speculate that RmYN02 may shed light on the origins of the furin cleavage site in SARS-CoV-2, which enhances its spike protein's efficiency in binding to the human ACE2 receptor, though this topic is complex and heavily debated.

#### 3. Is Yunnan the True Origin?

In a pertinent preprint released this month, researchers collected various samples from multiple bat species in Yunnan from May 2019 to November 2020. They successfully assembled 24 novel coronavirus genomes, including four that are similar to SARS-CoV-2.

This discovery indicates that bat coronaviruses could potentially jump to humans directly without an intermediate host, reinforcing the idea that the progenitor of SARS-CoV-2 may have done the same.

However, it is also possible that the SARS-CoV-2 progenitor was not present among the collected bat samples. The preprint authors noted that SARS-CoV-2 related viruses continue to circulate within bat populations, suggesting the progenitor could still exist elsewhere.

Ultimately, whether the SARS-CoV-2 progenitor—a coronavirus with around 99% genomic similarity—exists among the bats in Yunnan remains an open question requiring extensive further investigation.

If it turns out that the progenitor is absent in Yunnan, it raises the possibility that it could have evolved in: - An intermediate host, though this seems unlikely. - A laboratory, which recent WHO investigations have largely ruled out, though there are ongoing debates regarding the validity of these investigations. - Other bats in neighboring regions of China or Southeast Asia.

#### 4. Beyond Yunnan

Professor Robertson also directed me to a preprint authored by his team, which utilized bioinformatics to analyze coronavirus genomes. The findings suggest that a horseshoe bat species known as Rhinolophus affinis is the most probable reservoir for the SARS-CoV-2 progenitor. This bat species is also home to RaTG13, the closest relative of SARS-CoV-2.

Notably, R. affinis has a wide geographical range across China and Southeast Asia, including Thailand and Cambodia. The complexity increases as at least 20 different Rhinolophus species inhabit these regions, leaving many species with unknown viruses.

The preprint also included a map illustrating that several Rhinolophus species coexist in overlapping geographic areas, presenting numerous opportunities for bat coronaviruses to co-infect and recombine among various bat species. This process likely contributed to the evolution of SARS-1.

Thus, Yunnan is not the sole location where spillover events can occur; this can happen throughout China or other Southeast Asian regions where R. affinis is found. Discovering the progenitor of SARS-CoV-2 will be a challenging endeavor that may take time.

As noted in the conclusion of Professor Robertson's preprint, “The only way, however, to identify the animal progenitor of SARS-CoV-2 and its close relatives, which could pose similar emergence threats to humans and other animals, is by significantly increasing our sampling efforts” of bat coronaviruses.

Special thanks to Professor Dr. David L. Robertson for his valuable insights for this article.

I appreciate your attention in reaching this conclusion. If you found this article informative, consider subscribing to my Medium email list. For unlimited access to Medium, you can use my referral link, which provides me with a small commission.