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Identification and genetic characterization of MERS-related coronavirus isolated from Nathusius’ pipistrelle (Pipistrellus nathusii) near Zvenigorod (Moscow region, Russia) By Speranskaya A.S.Artyushin I.V. Samoilov A.E., Korneenko E.V., Khabudaev K., Ilina E.N., Yusefovich A.P., Safonova M.V., Dolgova A.S., Gladkikh, A.S., Dedkov V.G. Daszak Peter, Creative Commons BY 4.0 International License https://www.biorxiv.org/content/10.1101/2022.06.09.495421v1

British born and educated Peter Daszak of EcoHealth Alliance, who has become infamous for his work with China’s Wuhan lab (WIV), has also co-authored a paper on coronaviruses with Russian government researchers, which received Russian government funding, but which was only recently published (see below). However, the US government was funding Daszak-EcoHealth Alliance in the same period. The US funding period was June 1, 2014 to May 31, 2019, i.e. from shortly after Russia first invaded Ukraine to months before the Covid-19 pandemic. The Moscow area bat research was in 2015. However, the Daszak Russian document wasn’t published until after Russia’s full-scale invasion of Ukraine in 2022. Interestingly, Daszak’s father apparently fought alongside the Nazis in Ukraine, and the father of Putin friend Gerhard Schroeder died fighting for the Nazis. Putin apparently loves the children of Nazi soldiers. In 2016, the Daszak project was updated to include funding for the Wuhan lab.

Recall that Russians, at the US NIH, helped Chinese researchers change coronavirus sequences in GenBank database, or so it appears. https://miningawareness.wordpress.com/2022/03/13/russians-at-us-nih-helped-chinese-researchers-change-coronavirus-sequences-in-genbank-database/

Excerpts from US NIH-NIAID funded project: https://www.nih.gov/sites/default/files/institutes/foia/5R01AI110964-year-3.pdf
Project Title: Understanding the Risk of Bat Coronavirus Emergence/Grant Number: 5R01AI110964-05
Project/Grant Period: 06/01/2014 – 05/31/2019
Reporting Period: 06/01/2018 – 05/31/2019
Requested Budget Period: 06/01/2018 – 05/31/2019
Report Term Frequency: Annual Date Submitted: 08/03/2021
Program Director/Principal Investigator Information: PETER DASZAK , PHD BS
Recipient Organization: ECOHEALTH ALLIANCE, INC. ECOHEALTH ALLIANCE, INC. 520 EIGHTH AVENUE NEW YORK, NY 100181620
Administrative Official: ALEKSEI CHMURA 460 W 34th St., 17th Floor New York, NY 10001
Signing Official: ALEKSEI CHMURA 460 W 34th St., 17th Floor New York, NY 10001
Human Subjects: Yes HS Exempt: NA Exemption Number: Phase III Clinical Trial: NA
Vertebrate Animals: NA
hESC: No Inventions/Patents: No
Interim RPPR Page 1
Page 2:
WHAT ARE THE MAJOR GOALS OF THE PROJECT?
Zoonotic coronaviruses are a significant threat to global health, as demonstrated with the emergence of severe acute respiratory syndrome coronavirus (SARS-CoV) in 2002, and the recent emergence Middle East Respiratory Syndrome (MERS-CoV). The wildlife reservoirs of SARS-CoV were identified by our group as bat species, and since then hundreds of novel bat-CoVs have been discovered (including >260 by our group). These, and other wildlife species, are hunted, traded, butchered and consumed across Asia, creating a largescale human-wildlife interface, and high risk of future emergence of novel CoVs. To understand the risk of zoonotic CoV emergence, we propose to examine 1) the transmission dynamics of bat-CoVs across the human-wildlife interface, and 2) how this process is affected by CoV evolutionary potential, and how it might force CoV evolution. We will assess the nature and frequency of contact among animals and people in two critical human-animal interfaces: live animal markets in China and people who are highly exposed to bats in rural China. In the markets we hypothesize that viral emergence may be accelerated by heightened mixing of host species leading to viral evolution, and high potential for contact with humans. In this study, we propose three specific aims and will screen free ranging and captive bats in China for known and novel coronaviruses; screen people who have high occupational exposure to bats and other wildlife; and examine the genetics and receptor binding properties of novel bat-CoVs we have already identified and those we will discover. We will then use ecological and evolutionary analyses and predictive mathematical models to examine the risk of future bat-CoV spillover to humans. This work will follow 3 specific aims:

Specific Aim 1: Assessment of CoV spillover potential at high risk human-wildlife interfaces. We will examine if: 1) wildlife markets in China provide enhanced capacity for bat-CoVs to infect other hosts, either via evolutionary adaptation or recombination; 2) the import of animals from throughout Southeast Asia introduces a higher genetic diversity of mammalian CoVs in market systems compared to within intact ecosystems of China and Southeast Asia; We will interview people about the nature and frequency of contact with bats and other wildlife; collect blood samples from people highly exposed to wildlife; and collect a full range of clinical samples from bats and other mammals in the wild and in wetmarkets; and screen these for CoVs using serological and molecular assays.

Specific Aim 2: Receptor evolution, host range and predictive modeling of bat-CoV emergence risk. We propose two competing hypotheses: 1) CoV host-range in bats and other mammals is limited by the phylogenetic relatedness of bats and evolutionary conservation of CoV receptors; 2) CoV host-range is limited by geographic and ecological opportunity for contact between species so that the wildlife trade disrupts the ‘natural’ co-phylogeny, facilitates spillover and promotes viral evolution. We will develop CoV phylogenies from sequence data collected previously by our group, and in the proposed study, as well as from Genbank. We will examine co-evolutionary congruence of bat-CoVs and their hosts using both functional (receptor) and neutral genes. We will predict host-range in unsampled species using a generalizable model of host and viral ecological and phylogenetic traits to explain patterns of viral sharing between species. We will test for positive selection in market vs. wild-sampled viruses, and use data to parameterize mathematical models that predict CoV evolutionary and transmission dynamics. We will then examine scenarios of how CoVs with different transmissibility would likely emerge in wildlife markets….
Specific Aim 3: Testing predictions of CV inter-species transmission. The following experiments will be undertaken in Year 2:
– Humanized mice with human ACE2 receptors will be infected with WIV1 and the two rescued chimeric SARS-like coronaviruses to
determine the tissue tropism and pathogenicity of bat SL-CoV
– Isolation of novel bat coronaviruses. Live virus or pseudovirus will be used to infect cells of different origin or expressing different
receptor molecules. Spillover potential for each isolated virus will be assessed.
An infectious clone of full-length MERS-CoV will be constructed using reverse genetic method. Using the S sequence of different
MERS-related viruses identified from Chinese bats, the chimeric viruses with S gene of bat MERS-related coronaviruses and backbone
of the infectious clone of MERS-CoV will be constructed to study the receptor usage and infectivity of bat MERS-related coronavirus.

– Surveillance of infection in human populations by SARS-like CoVs. This work will be performed at locations in Yunnan, Guangxi, and
Guangdong provinces, in previously identified areas with human populations of high risk of exposure to bats. PCR and ELISA will be
used, respectively, for detection of viral replicase gene and antibodies against the viral nucleocapsid protein.

B.6 WHAT DO YOU PLAN TO DO DURING THE NEXT REPORTING PERIOD TO ACCOMPLISH THE GOALS?
Specific Aim 3: Testing predictions of CoV inter-species transmission. We will test our models of host range (i.e. emergence potential) experimentally using reverse genetics, pseudovirus and receptor binding assays, and virus infection experiments in cell culture and humanized mice. With bat-CoVs that we’ve isolated or sequenced, and using live virus or pseudovirus infection in cells of different origin or expressing different receptor molecules, we will assess potential for each isolated virus and those with receptor binding site sequence, to spill over. We will do this by sequencing the spike (or other receptor binding/fusion) protein genes from all our bat-CoVs, creating mutants to identify how significantly each would need to evolve to use ACE2, CD26/DPP4 (MERS-CoV receptor) or other potential CoV receptors. We will then use receptor-mutant pseudovirus binding assays, in vitro studies in bat, primate, human and other species’ cell lines, and with humanized mice where particularly interesting viruses are identified phylogenetically, or isolated. These tests will provide public health-relevant data, and also iteratively improve our predictive model to better target bat species and CoVs during our field studies to obtain bat-CoV strains of the greatest interest for understanding the mechanisms of cross-species transmission.

Interim RPPR
SECTION IV – Al Special Terms and Conditions – 5R01A/110964-03 REVISED: The Research Performance Progress Report (RPPR), Section G.9 (Foreign component), includes reporting requirements for all research performed outside of the United States… REVISED AWARD: This Notice of Award is revised to provide approval for collaboration with the Wuhan University School of Public Health (CHINA) in accordance with the request submitted by Aleksei Chmura, Ecohealth Alliance, Inc. on October 6. 2016.
Supersedes previous Notice of Award dated 7/26/2016. REVISED AWARD: This Notice of Award is revised to provide approval for collaboration with the Wuhan University School of Public Health (CHINA) in accordance with the request submitted by Aleksei Chmura, Ecohealth Alliance, Inc. on October 6. 2016. Supersedes previous Notice of Award dated 7/26/2016.
*******************
No funds are provided and no funds can be used to support gain-of-function research covered under the October 17. 2014 White House Announcement (NIH Guide Notice NOT-OD-15-011). Per the letter dated July 7, 2016 to Mr. Aleksei Chmura at EcoHealth Alliance, should any of the MERS-like or SARS-like chimeras generated under this grant show evidence of enhanced virus growth greater than 1 log over the parental backbone strain you must stop all experiments with these viruses and provide the NIAID Program Officer and Grants Management Specialist, and Wuhan Institute of Virology Institutional Biosafety Committee with the relevant data and information related to these unanticipated outcomes.
This Notice of Award (No) includes funds for consortium activity with:
Wuhan Institute of Virology – CHINA awarded in the Total Costs amount of $159,122
($147,335 Direct Costs + $11,787 F&A Costs). Future year commitments are as follows:
Year 4 Total Costs: $159,122 and Year 5 Total Costs: $159,122
East China Normal University – CHINA awarded in the Total Costs amount of $54,117
($50,108 Direct Costs + $4,009 F&A Costs). Future year commitments are as follows:
Year 4 Total Costs: $42.300 and Year 5 Total Costs: $32,454

Specific Aim 3: Testing predictions of CoV inter-species transmission.
•Using the full-length infectious cDNA clone of MERS-CoV, chimeric viruses with the spikes of newly identified MERSr-CoVs will be constructed. The pathogenesis of these MERSr-CoVs will be tested on the human DPP4-expressing mouse model that has already been developed and validated in Y4. •To conduct a population genetics study of Rhinolophus sinicus ACE2s, including the amplification of ACE2 genes from R. sinicus samples of different origin, test of the usage efficiency of R. sinicus ACE2s of different origins by SL-CoVs and kinetics study on the binding of SL-CoV RBD to different R. sinicus ACE2s. •In collaboration with South China Agrricultural University, gather data on the spatial structure and barn-level mortality records to parameterize our mathematical model of virus spread that incorporates a meta-population structure in individual and use this to fit the model on a training set of farms and validate it on a hold-out set. •Using the intra-farm transmission model, we will (a) determine the characteristics of a farm that determine the likelihood and size of an outbreak given a spillover event, and (b) determine whether SADS and PEDV outbreaks on farms can be distinguished by differing dynamics, as measured by transmission parameters in our intra-farm transmission model
”. SEE THE ORIGINAL HERE. THE PAGES NUMBERS ARE A BIT JUMBLED: https://www.nih.gov/sites/default/files/institutes/foia/5R01AI110964-year-3.pdf

Putin Decreed that All Russians Have Genetic Certification-Genetic Passports By 2025

According to federal government data, EcoHealth received tens of millions in federal research dollars. EcoHealth has partnered with the WIV since 2004.” https://reschenthaler.house.gov/media/press-releases/reschenthaler-uncovers-11-million-taxpayer-funding-sent-wuhan-institute https://miningawareness.wordpress.com/2021/06/11/usaid-predict-nih-gave-1-9-million-to-the-wuhan-wiv-lab-through-daszak-ecohealth-alliance-daszak-talks-china-partners-work-on-killer-viruses-biden-admin-plans/

The Moscow area funded project tries to implicate European hedgehogs. However, bats live in closer proximity to humans, because they live in dwellings. And, when roofs of old buildings leak, bat droppings can (and do) fall into living areas. Recall that the first European appearance of Covid-19 was in northern Italy. The bat migration appears to be a possible vector, and a known vector, to spread disease, including genetically manipulated diseases. Additionally, bat migration could be blamed when humans have intentionally (or unintentionally) spread a genetically altered (or non-genetically altered) coronavirus (or other disease).

The number of bats sampled is statistically insufficient. However, the report that half are infected is compelling.

Russian funded project co-authored with Daszak:
Identification and genetic characterization of MERS-related coronavirus isolated from Nathusius’ pipistrelle (Pipistrellus nathusii) near Zvenigorod (Moscow region, Russia) Speranskaya A.S.1,2,3*, Artyushin I.V.3, Samoilov A.E.1,4, Korneenko E.V.1, Khabudaev K.1, Ilina E.N.1, Yusefovich A.P.3, Safonova M.V.5, Dolgova A.S.4, Gladkikh, A.S.4, Dedkov V.G.4,6, Daszak Peter7
1Scientific Research Institute for Systems Biology and Medicine, Federal Service on Consumers’ Rights Protection and Human Well-Being Surveillance, Moscow, Russia;
2Department of Molecular Diagnostics and Epidemiology, Central Research Institute for Epidemiology, Federal Service on Consumers’ Rights Protection and Human Well-Being Surveillance, Moscow, Russia;
3Lomonosov Moscow State University, Biological Department, Moscow, Russia;
4Saint-Petersburg Pasteur Institute, Federal Service on Consumers’ Rights Protection and Human Well-Being Surveillance, Saint-Petersburg, Russia;
5Department of Particularly Dangerous Diseases, Anti-Plague Center, Federal Service on Consumers’ Rights Protection and Human Well-Being Surveillance, Moscow, Russia;
6Martsinovsky Institute of Medical Parasitology, Tropical and Vector Borne Diseases, Sechenov First Moscow State Medical University, Moscow, Russia;
7EcoHealth Alliance, New York, USA
Keywords: Bat-CoV, MERS-related coronaviruses, Pipistrellus nathusii, bats, hedgehogs, humans, camels, DPP4, Spike protein, molecular docking
CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted June 10, 2022. ; https://doi.org/10.1101/2022.06.09.495421 doi: bioRxiv preprint

Abstract

The majority of emerging infectious diseases are caused by pathogens with zoonotic origin, and most of these emerged from wildlife reservoirs. Bats are diverse, and widely distributed globally, and are the known or hypothesized reservoir of a series of emerging zoonotic viruses. Analyses of bat viromes have been used to identify novel viruses with potential to cause human infection. We characterized the fecal virome of 26 samples collected from six bat species captured during 2015 in Moscow Region. Of these 13/26 (50%) samples were found to be coronavirus positive. We sequenced and assembled the complete genome of a novel MERS-related Betacoronavirus from Pipistrellus nathusii, named MOW-BatCoV strain 15-22. Of P. nathusii 3/6 samples were found to carriers of MOW-BatCoVs.

The genome organization of MOW-BatCoV/15-22 was identical to other known MERS-related coronaviruses. Phylogenetic analysis of whole genomes suggests that MOW-BatCoV/15-22 falls into a distinct subclade closely related to human and camel MERS-CoV, and MERS-related CoVs from the bat species Hypsugo savii and Pipistrellus kuhlii (from Italy) and Neoromicia capensis (from South Africa).

Unexpectedly, phylogenetic analysis of the novel MOW-BatCoV 15-22 spike genes showed the closest similarity to a bat CoV Neoromicia/5038 and CoVs from Erinaceus europaeus (the European hedgehog), thus MOW-BatCoV could arise as result of recombination between ancestral viruses of bats and hedgehogs. Computer molecular docking analysis of MOW-BatCoV 15-22 Spike glycoprotein binding to DPP4 receptors of different mammal species predicted highest binding interaction with DPP4 of the bat M. brandtii (docking score -320.15) and the European hedgehog, E. europaeus (docking score -294.51). Hedgehogs are widely kept as pets, and are commonly found in areas of human habitation. Our finding of a novel bat-CoV likely able to infect hedgehogs suggests the potential for hedgehogs to act as intermediate hosts for bat-CoVs between bats and humans.

Introduction

Coronaviruses (CoVs) have been responsible for three high impact outbreaks in the past 2 decades, including severe acute respiratory syndrome (SARS), the Middle East respiratory syndrome (MERS) and the ongoing coronavirus disease 2019 (COVID-19) pandemic. Each of these diseases affects the human respiratory system, causing a spectrum from asymptomatic and mild respiratory illness to severe pneumonia, acute respiratory failure and death.

The current COVID-19 pandemic is now in its third year and continues to be a global health emergency, with more than 500 million confirmed cases, including more than six million deaths, reported to WHO by the date of submitting this manuscript [1]. The earlier outbreak of MERS was caused by zoonotic virus, MERS-CoV, transmitted to humans from infected dromedary camels, with secondary human-to-human transmission mainly nosocomial [2]. MERS was first identified in Saudi Arabia in 2012 [3] and has now been reported in 27 countries, with the largest outbreaks in Saudi Arabia, United Arab Emirates, and the Republic of Korea leading to 858 known deaths due to the infection and related complications The disease has a high fatality rate of up to 35%[4,5]. The origin of the virus is not fully understood yet but phylogenetic analysis of different virus genomes suggest it originated in bats and passed to humans in 2012 after circulating endemically in dromedary camels for around 30 years [6,7].

Bats are the known or putative reservoirs of several viruses that can cause severe human disease, including rabies, Hendra, Nipah, Marburg, SARS, MERS and Ebola viruses [8–10]. Bats also carry diverse coronaviruses, some of which are able to bind to human cells in vitro, suggesting that bats are the likely reservoirs of potential future zoonotic CoVs [11]. In addition, bats are a diverse group of mammals (representing around 1/5th of all mammalian biodiversity), have wide geographic distribution, long life span and are known to feed and roost near to human communities, suggesting they are an important reservoir of other potential emerging diseases [12].

Two genera of CoVs infect mainly mammals: Alphacoronavirus (α-CoV) and Betacoronavirus (β-CoV) [11,13,14]. Phylogenetic analysis shows that β-CoVs are grouped into several clades (from A to D). These four Betacoronavirus lineages have been reclassified into five subgenera: Embecovirus (A), Sarbecovirus (B), Merbecovirus (C), Nobecovirus (D), and an additional lineage Hibecovirus [15],[16]. MERS CoV, which causes fatal pneumonia in humans and has a dromedary camel reservoir, along with MERS-related (MERSr-) CoVs form clade C (or Merbecoviruses) within the Betacoronaviruses and includes CoVsdiscovered in bats and hedgehogs [14–17]. MERSr-CoVs have been reported from South Africa (NeoCoV from Neoromicia capensis) [18], Mexico (Mex_CoV-9 from Nyctinomops laticaudatus) [19], Uganda (MERSr-CoV PREDICT/PDF-2180 from Pipistrellus cf. hesperidus) [20], Netherlands (NL-VM314 from Pipistrellus pipistrellus) [21], Italy (BatCoV-Ita1 strain 206645–40 from Hypsugo savii and BatCoV-Ita2 strain 206645-63 from Pipistrellus kuhlii) [17] and China (BatCoV/SC2013 from Vespertilio superans [22] and strains of HKU4- and HKU5-CoVs from Tylonycteris and Pipistrellus bats [23–26]. To date, few surveys for CoVs have been conducted in Russia, and no MERSr-CoVs have been reported.

Coronavirus cell tropism and ability to infect hosts is determined primarily by the spike protein which is part of the receptor binding domain (RBD) of the sCoV genome [27–29]. Unlike SARS-CoV and SARS-CoV-2, which bind to Angiotensin-converting enzyme 2 (ACE2), MERS-CoV targets the cell surface receptor Dipeptidyl peptidase 4 (DPP4, also known as CD26). DPP4 is relatively conserved among mammalian species, so that MERS-CoV is capable of infecting a wide range of cell lines derived from humans, non-human primates, bats, swine, horse, rabbit, civet, and camel but not from mice, hamster, dog, ferret, and cat [10,27]. The MERS-CoV spike protein undergoes adaptive evolution when inoculated onto normally non-permissive hamster cells transfected to express DPP4 from different bat species [27,30]. HKU4 MERSr-CoVs from bats in China have RBDs that potentially can bind human DPP4 [31] with even low affinity for human cells suggesting potential to infect humans and adapt to more efficient cell entry [17].

In this paper, we describe a survey of bats in Russia for CoVs, report a novel MERSr-CoV, describe its genome organization and relationship with known coronaviruses

Materials and methods

Sample collection

In summer 2015 fecal samples were collected from 26 bats of the following species: Myotis dasycneme (n=5), Myotis daubentonii (n=5), Myotis brandtii (n=3), Nyctalus noctula (n=4), Pipistrellus nathusii (n=6), Plecotus auritus (n=2), Vespertilio murinus (n=1), inhabiting the Zvenigorodsky district of the Moscow region (Sharapovskoe forestry, coordinates N55.69, E36.70). No bats were killed for this study and all bats were captured in mist nets and released at the site of capture. Bat capture and sampling was conducted by professionally trained staff of the biological department of Lomonosov Moscow State University. Fecal samples, rectal swabs and ectoparasites were collected after capture, and species, sex, reproductive and health status visually determined by trained field biologists. Swab samples were kept in a transport media for transportation and storage of swabs with mucolytic agent (AmpliSens, Russia) at 4°C during transport to the laboratory, and were then stored at −80 °C before processing….

Discussion

A large number of β-CoVs has been identified from bats globally. The MERSr-CoV described here is the first to be identified in Russia. Twenty-six animals of six different bat species which wide distributed in the central European part of the Russian Federation (five of Myotis dasycneme, three of Myotis brandtii, five of Myotis daubentoniid, four of Nyctalus noctule, six of Pippistrellus nathusii, two of Plecotus auritus and single Vespertilio murinus) were analysed in the study.

The products of amplificaton of RdRp genes was detected in 50% RNA samples extracted from rectal swabs of animals. Sequencing confirms the presence of merbecoviruses in three of six analysed samples from P. nathusii only (semi-adult animals, one of them was female and two were males). The animals were caught at the same time, in the same geographic location, so we believe they were from the same colony. BLAST against GenBank records as well as topology of the phylogenetic tree based on the fragments of the RdRp genes revealed that the bats were infected with same virus, MOW-BatCoV/15-22.

The complete genome of MOW-BatCoV 15-22 showed the highest similarity (88% identity) to MERS-related viruses from bats (Bat-CoV/H.savii/Italy/206645-40/2011 and Bat-CoV/P.khulii/Italy/206645-63/2011) which has been reported in Italy [17]. P. nathusii is a migratory bat, which habitats the major parts of Europe:
from Fennoscandia and British Isles in the north to the Mediterranean areas in the south. The breeding areas of this species are regions of north-eastern Europe. As a result of low abundance of aerial insects during winter, Nathusius’ pipistrelles from Central European (Germany and Poland) and northeastern populations (Fennoscandia, the Baltic countries, Belarus, and Russia) perform long-distance flights migration in the late summer (during approximately two months with stopping for mating) in the Switzerland, Benelux countries, France, Spain, Italy, and Croatia [52,53]. The longest migration record of this species was documented at 2224 km, between S Latvia and N Spain [53]. During migration, P. nathusii may come into contact with bats of the same species when mating. And they can also come into contact with bats of other species in roosting areas. Migration routes explain the fact that very similar viruses have been found in bats from Italy and Russia.

According to phylogenetic analysis of the complete genomes the MOW-BatCoV 15-22 falls into clade of human/camel’s MERS viruses together with a few bat viruses and due to its distinct phylogenetic position and amino acid differences should be considered as a novel MERSr-CoV. The replicase polyprotein of new virus (MOW-BatCoV 15-22) showed more than 90% homology for six of seven domains, the only NSP3 (ADRP) is shown 68,7% of homology to sequences of the other members of Merbecovirus. According to demarcation criteria of ICTV [51] we believe MOW-BatCoV 15-22 represents the same species of Merbecovirus as NeoCoV because of phylogenetic analysis of RdRp showed, MOW-BatCoV 15-22 and NeoCoV together are closest to MERS-CoV. Of known bat viruses, the NeoCoV from Neoromicia capensis (S. Africa) is the closest to Middle East respiratory syndrome coronavirus (MERS-CoV) which could infect humans and dromedary camels and it is considered that NeoCoV shares sufficient genetic similarity in the replicase genes to be part of the same viral species with MERS-CoV [20,54].

Phylogenetic analysis of complete genome sequences as well as N- and RdRp-sequences suggest that ten viruses from bats found in distinct geographic regions (MOW-BatCoV 15-22 from Russia, MG596802.1 and MG596803.1 from Italy, NeoCoV Neoromicia/5038 from South Africa, and multiple strains from bats in China – MG021452.1, MG021451.1, MG987420.1, MG987421.1, KX442565.1, KX442564.1) form a distinct phylogenetic clade with MERS-CoVs from humans and camels, with high bootstrap support. However, phylogenetic analysis of spike protein encoding genes demonstrated similarity of two of these ten bat viruses – the novel MOW-BatCoV 15-22 and NeoCoV – to CoVs from Erinaceus europaeus (the European hedgehog).

We believe that the proven close relationship between Spike genes of viruses from bats and hedgehogs which live in the same geographic regions (namely Europe) raises the question of the possibility of interspecies transmission in the present time.

Middle East syndrome Coronavirus (MERS-CoV) likely originated in bats and passed to humans through dromedary camels. Previous work suggests that MERS-CoV originated from an ancestral virus in a bat reservoir and spilled over into dromedary camels around 40 decades ago, where it circulated endemically before emerging in humans in 2012 [6,7]. Presently camels play an important role as a constantly reservoir of MERS-CoV and transmit virus to people [55–57], while the bats are widely considered to be the evolutionary, disposable source of the virus [20].

But, besides dromedaries which are the proven source of human MERS and bats, MERS-related viruses have also been discovered in hedgehogs (Erinaceous).

Hedgehog carriers of betacoronaviruses have been found in China [16], Germany [58], France [59] and Poland [60]. Phylogenetic analysis carried previously showed that betacoronaviruses from Chinese and Germanies hedgehogs (Ea-HedCoV HKU31 and BetaCoV Erinaceus/VMC/DEU/2012) were closely related to NeoCoV and BatCoV from African bats in the spike region. Therefore, authors suggested that the bat viruses arose as a result of recombination between hedgehogs and bat viruses [16]. Our independent finding of one more virus from European bat Pipistrellus nathusii which was found closely related to viruses from hedgehogs in the Spike region but not in N- and RdRp regions support the idea of recombination between ancestral viruses of bats and hedgehogs. Our independent finding of a novel CoV from a European bat with spike protein encoding sequences closely related to those from hedgehog MERSr-CoVs also suggests recombination between ancestral viruses of bats and hedgehogs. The overlap in geographic range between P. nathusii and the natural distribution of E. europaeus raises the possibility that this recombination represents an ancestral interspecies transmission. Our findings support the need for wider surveillance of MERSr-CoVs in both bats and hedgehogs MERS-CoV targets a cell-surface receptor, the Dipeptidyl peptidase 4 (DPP4, also known as CD26). A receptor-binding domain (RBD) on the viral spike glycoprotein (S) mediates this interaction and is bound to the extracellular domain of human DPP4 [31,61]. The HKU4 and HKU5 merbecoviruses from Chinese bats are closely related to MERS-CoV in spike protein genes. The HKU4 viruses can use the MERS-CoV receptor DPP4, but not HKU5. Another MERS-CoV-related betacoronavirus, Hp-BatCoV HKU25 occupies a phylogenetic position between that of HKU4 and HKU5 and can binding of DPP4 protein for entry to DPP4-expressing cells, although with lower efficiency than that of MERS and HKU4 viruses [26]. At the same time, at least in some merbecoviruses (namely, Bat-CoV-PREDICT/PDF-2180 and NeoCoV) the domain RBDs of S-protein have been shown to use the ACE2 receptor, their RBDs amino acid composition and sequence differ greatly from RBD of SARS-CoV-2 (slightly more than 18% of identical aa) [62]. In the novel MERSr-CoV (MOW-BatCoV 15-22), the RBD region appears likely to interact with DPP4 across amino acids 366–624 within the S1 subunit. The amino acid composition of the RBD domain of the MOW-BatCoV/15-22 virus differs by approximately the same range from the RBD domains of those viruses that interact with DPP4 receptors (32-36,6% the same amino acids) and of viruses that interact ACE2 (33-33,7% the same amino acids). Thus, while it cannot be ruled out that MOW-BatCoV/15-22 binds to other cell receptors (e.g. ACE2), it is more likely it binds to DPP4.

Previous work demonstrated that only two mutations in the HKU4 coronavirus spike protein encoding region can make it infectious for human cells [61]. These are changes in one amino acid in two motifs each hPPC (recognized by furin proprotein convertase) and hECP (recognized by endosomal cysteine protease Cathepsin L). In MERS-CoV which causes the Middle East respiratory syndrome hPPC is Arg748- Ser749-Val750-Arg751-Ser760, hECP MERS Ala763-Phe764-Asn765 while in HKU4 hPPC is Ser746-Thr747-Phe748-Arg749-Ser750 hECP Asn762-Tyr763-Thr764. When Ser746 was changed on Arg (to make motifs recognizable by protease) and Asn762 on Ala (to destroy a potentially existing N-linked glycosylation site) fully mediates viral entry into human cells. In the MOW-BatCoV/15-22 virus hPPC motif is Pro758-His759-Ser760-Arg761 (based on MERS and HKU4). If compared with the ACE2 interacting S proteins of Human Sars-CoV-2, Bat CoV PREDICT/PDF-2180 and NeoCoV, then it is probably Ser760-Arg761-Thr762-Asn763. It is possible that substitution of either Pro758 or Asn763 the mentioned constitutions can lead to the furin cleavage site formation and it’s subsequent recognition by furin and could result in an increased ability to infect human cells….

Funding and support
ASS, AIV, SAE, KEV, YAP, SMV: The zoology, molecular virology, sequencing and bioinformatics works, supported by RFBR Grant № 20-04-60561.
ASD, ASG, VGD: The biochemistry analysis and comparative virology analysis works supported by RSF Grant № 20-64-46014
“ CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted June 10, 2022. https://doi.org/10.1101/2022.06.09.495421 doi: bioRxiv preprint Read the entire paper here: https://www.biorxiv.org/content/10.1101/2022.06.09.495421v1

Click to access 2022.06.09.495421v1.full.pdf

Another interesting Lomonosov Moscow State University connection. Malone and Iranian American Bavari co-author, V. Soloveva, who is currently with Merck and previously with Pfizer, as well as Fort Detrick bio lab:
Zika virus: Accelerating development of Medical Countermeasures by re-purposing licensed drugs Published Apr 16, 2016
By R.W. Malone* 1,2, V. Soloveva 3,4, S. Bavari 3,4
1. Atheric Pharmaceutical, LLC, Scottsville, VA, USA, 2. Class of 2016, Harvard Medical School Global Clinical Scholars Research Training Program, Boston, MA
3. United States Army Medical Research Institute of Infectious Diseases, Frederick, MD, USA. 
4. United States Army Medical Research Institute of Infectious Diseases, Therapeutic Development Center, Frederick, MD, USA.

https://web.archive.org/web/20220104110753/https://www.linkedin.com/pulse/zika-virus-accelerating-development-medical-licensed-jill
V. Soloveva:
Education: University of Illinois at Chicago 
Ph.D biochemistry 1990 – 1996

Northwestern University, Evanston, IL postdoctoral fellow
Cell/Cellular and Molecular Biology 1996 – 2002


Lomonosov Moscow State University (MSU) 
Ms.Molecular Biology 1986 – 1990 

Moscow State University 
Master in Biochemistry Biology
Molecular Biology diploma cum laude 
1985 – 1990
Principal Scientist, High Content Imaging, Quantitative Biosciences 
Merck Nov 2018 – Present Boston, MA 

PI Hanry M.Jackson Foundation for Advancement of Military Medicine; BCSAI,
USAMRIID 
Jul 2013 – Nov 2018 Fort Detrick, MD 

Director of CCT (Center of Core Technologies)
IP-Korea Mar 2010 – Jun 2013

Institut Pasteur Korea 

Principal Research Scientist II 
Pfizer Pharmaceuticals 
Jan 2009 – Mar 2010
Principal Scientist 
Wyeth Research 
2002 – 2009

Russians at US NIH Helped Chinese Researchers Change Coronavirus Sequences in GenBank Database

New UC Davis VP for “Grand Challenges” JK Mazet Connected to Wuhan Institute of Virology-Daszak (EcoHealth)

USAID (PREDICT) & NIH Gave $ 1.9 Million to the Wuhan (WIV) Lab Through Daszak-EcoHealth Alliance; Daszak Talks China Partners’ Work on “Killer” Viruses; Biden Budget Requests More USAID Money for Similar Projects

Future of Genetically Modified Babies May Lie in Putin’s Hands” By Bloomberg Sep. 29, 2019 https://www.themoscowtimes.com/2019/09/29/future-of-genetically-modified-babies-may-lie-in-putins-hands-a67492 (Putin’s geneticist daughter)