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Uncanny similarity of unique inserts in the 2019-nCoV spike protein to HIV-1 gp120 and Gag

Posted on March 26, 2020 by Mimi Castellanos

bioRxiv preprint first posted online Jan. 31, 2020; doi: http://dx.doi.org/10.1101/2020.01.30.927871. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.

Uncanny similarity of unique inserts in the 2019-nCoV spike protein to HIV-1 gp120 and Gag

Prashant Pradhan$1,2, Ashutosh Kumar Pandey$1, Akhilesh Mishra$1, Parul Gupta1, Praveen Kumar Tripathi1, Manoj Balakrishnan Menon1, James Gomes1, Perumal Vivekanandan*1and Bishwajit Kundu*1

1Kusuma School of biological sciences, Indian institute of technology, New Delhi-110016, India. 2Acharya Narendra Dev College, University of Delhi, New Delhi-110019, India
$Equal contribution

* Corresponding authors- email: bkundu@bioschool.iitd.ac.in vperumal@bioschool.iitd.ac.in

Abstract:

We are currently witnessing a major epidemic caused by the 2019 novel coronavirus (2019- nCoV). The evolution of 2019-nCoV remains elusive. We found 4 insertions in the spike glycoprotein (S) which are unique to the 2019-nCoV and are not present in other coronaviruses. Importantly, amino acid residues in all the 4 inserts have identity or similarity to those in the HIV- 1 gp120 or HIV-1 Gag. Interestingly, despite the inserts being discontinuous on the primary amino acid sequence, 3D-modelling of the 2019-nCoV suggests that they converge to constitute the receptor binding site. The finding of 4 unique inserts in the 2019-nCoV, all of which have identity /similarity to amino acid residues in key structural proteins of HIV-1 is unlikely to be fortuitous in nature. This work provides yet unknown insights on 2019-nCoV and sheds light on the evolution and pathogenicity of this virus with important implications for diagnosis of this virus.

Introduction

Coronaviruses (CoV) are single-stranded positive-sense RNA viruses that infect animals and humans. These are classified into 4 genera based on their host specificity: Alphacoronavirus, Betacoronavirus, Deltacoronavirus and Gammacoronavirus (Snijder et al., 2006). There are seven known types of CoVs that includes 229E and NL63 (Genus Alphacoronavirus), OC43, HKU1, MERS and SARS (Genus Betacoronavirus). While 229E, NL63, OC43, and HKU1 commonly infect humans, the SARS and MERS outbreak in 2002 and 2012 respectively occurred when the virus crossed-over from animals to humans causing significant mortality (J. Chan et al., n.d.; J. F. W. Chan et al., 2015). In December 2019, another outbreak of coronavirus was reported from Wuhan, China that also transmitted from animals to humans. This new virus has been temporarily termed as 2019-novel Coronavirus (2019-nCoV) by the World Health Organization (WHO) (J. F.- W. Chan et al., 2020; Zhu et al., 2020). While there are several hypotheses about the origin of 2019-nCoV, the source of this ongoing outbreak remains elusive.

The transmission patterns of 2019-nCoV is similar to patterns of transmission documented in the previous outbreaks including by bodily or aerosol contact with persons infected with the virus.

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Casesofmildtosevereillness,anddeathfromtheinfectionhavebeenreportedfromWuhan. This outbreak has spread rapidly distant nations including France, Australia and USA among others. The number of cases within and outside China are increasing steeply. Our current understanding is limited to the virus genome sequences and modest epidemiological and clinical data. Comprehensive analysis of the available 2019- nCoV sequences may provide important clues that may help advance our current understanding to manage the ongoing outbreak.

The spike glycoprotein (S) of cornonavirus is cleaved into two subunits (S1 and S2). The S1 subunit helps in receptor binding and the S2 subunit facilitates membrane fusion (Bosch et al., 2003; Li, 2016). The spike glycoproteins of coronoviruses are important determinants of tissue tropism and host range. In addition the spike glycoproteins are critical targets for vaccine development (Du et al., 2013). For this reason, the spike proteins represent the most extensively studied among coronaviruses. We therefore sought to investigate the spike glycoprotein of the 2019-nCoV to understand its evolution, novel features sequence and structural features using computational tools.

Methodology
Retrieval and alignment of nucleic acid and protein sequences

We retrieved all the available coronavirus sequences (n=55) from NCBI viral genome database (https://www.ncbi.nlm.nih.gov/) and we used the GISAID (Elbe & Buckland-Merrett, 2017)[https://www.gisaid.org/] to retrieve all available full-length sequences (n=28) of 2019- nCoV as on 27 Jan 2020. Multiple sequence alignment of all coronavirus genomes was performed by using MUSCLE software (Edgar, 2004) based on neighbour joining method. Out of 55 coronavirus genome 32 representative genomes of all category were used for phylogenetic tree development using MEGAX software (Kumar et al., 2018). The closest relative was found to be SARS CoV. The glycoprotein region of SARS CoV and 2019-nCoV were aligned and visualized using Multalin software (Corpet, 1988). The identified amino acid and nucleotide sequence were aligned with whole viral genome database using BLASTp and BLASTn. The conservation of the nucleotide and amino acid motifs in 28 clinical variants of 2019-nCoV genome were presented by performing multiple sequence alignment using MEGAX software. The three dimensional structure of 2019-nCoV glycoprotein was generated by using SWISS-MODEL online server (Biasini et al., 2014) and the structure was marked and visualized by using PyMol (DeLano, 2002).

Results

Uncanny similarity of novel inserts in the 2019-nCoV spike protein to HIV-1 gp120 and Gag

Our phylogentic tree of full-length coronaviruses suggests that 2019-nCoV is closely related to SARS CoV [Fig1]. In addition, other recent studies have linked the 2019-nCoV to SARS CoV. We therefore compared the spike glycoprotein sequences of the 2019-nCoV to that of the SARS CoV (NCBI Accession number: AY390556.1). On careful examination of the sequence alignment we found that the 2019- nCoV spike glycoprotein contains 4 insertions [Fig.2]. To further investigate if these inserts are present in any other corona virus, we performed a multiple

sequence alignment of the spike glycoprotein amino acid sequences of all available coronaviruses (n=55) [refer Table S.File1] in NCBI refseq (ncbi.nlm.nih.gov) this includes one sequence of 2019-nCoV[Fig.S1]. We found that these 4 insertions [inserts 1, 2, 3 and 4] are unique to 2019-nCoV and are not present in other coronaviruses analyzed. Another group from China had documented three insertions comparing fewer spike glycoprotein sequences of coronaviruses . Another group from China had documented three insertions comparing fewer spike glycoprotein sequences of coronaviruses (Zhou et al., 2020).

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Figure 1: Maximum likelihood genealogy show the evolution of 2019- nCoV: The evolutionary history was inferred by using the Maximum Likelihood method and JTT matrix-based model. The tree with the highest log likelihood (12458.88) is shown. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using a JTT model, and then selecting the topology with superior log likelihoodbioRxiv preprint first posted online Jan. 31, 2020; doi: http://dx.doi.org/10.1101/2020.01.30.927871. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.

value. This analysis involved 5 amino acid sequences. There were a total of 1387 positions in the final dataset. Evolutionary analyses were conducted in MEGA X.

Figure 2: Multiple sequence alignment between spike proteins of 2019-nCoV and SARS. The sequences of spike proteins of 2019-nCoV (Wuhan-HU-1, Accession NC_045512) and of SARS CoV (GZ02, Accession AY390556) were aligned using MultiAlin software. The sites of difference are highlighted in boxes.

We then analyzed all available full-length sequences (n=28) of 2019-nCoV in GISAID (Elbe & Buckland-Merrett, 2017) as on January 27, 2020 for the presence of these inserts. As most of these sequences are not annotated, we compared the nucleotide sequences of the spike glycoprotein of all available 2019-nCoV sequences using BLASTp. Interestingly, all the 4 insertions were absolutely (100%) conserved in all the available 2019- nCoV sequences analyzed [Fig.S2, Fig.S3].

We then translated the aligned genome and found that these inserts are present in all Wuhan 2019- nCoV viruses except the 2019-nCoV virus of Bat as a host [Fig.S4]. Intrigued by the 4 highly conserved inserts unique to 2019-nCoV we wanted to understand their origin. For this purpose, we used the 2019-nCoV local alignment with each insert as query against all virus genomes and considered hits with 100% sequence coverage. Surprisingly, each of the four inserts aligned with short segments of the Human immunodeficiency Virus-1 (HIV-1) proteins. The amino acid positions of the inserts in 2019-nCoV and the corresponding residues in HIV-1 gp120 and HIV-1 Gag are shown in Table 1. The first 3 inserts (insert 1,2 and 3) aligned to short segments of amino acid residues in HIV-1 gp120. The insert 4 aligned to HIV-1 Gag. The insert 1 (6 amino acid residues) and insert 2 (6 amino acid residues) in the spike glycoprotein of 2019-nCoV are 100% identical to the residues mapped to HIV-1 gp120. The insert 3 (12 amino acid residues) in 2019- nCoV maps to HIV-1 gp120 with gaps [see Table 1]. The insert 4 (8 amino acid residues) maps to HIV-1 Gag with gaps.

Although, the 4 inserts represent discontiguous short stretches of amino acids in spike glycoprotein of 2019-nCoV, the fact that all three of them share amino acid identity or similarity with HIV-1 gp120 and HIV-1 Gag (among all annotated virus proteins) suggests that this is not a random fortuitous finding. In other words, one may sporadically expect a fortuitous match for a stretch of 6-12 contiguous amino acid residues in an unrelated protein. However, it is unlikely that all 4 inserts in the 2019-nCoV spike glycoprotein fortuitously match with 2 key structural proteins of an unrelated virus (HIV-1).

The amino acid residues of inserts 1, 2 and 3 of 2019-nCoV spike glycoprotein that mapped to HIV-1 were a part of the V4, V5 and V1 domains respectively in gp120 [Table 1]. Since the 2019- nCoV inserts mapped to variable regions of HIV-1, they were not ubiquitous in HIV-1 gp120, but were limited to selected sequences of HIV-1 [ refer S.File1] primarily from Asia and Africa.

The HIV-1 Gag protein enables interaction of virus with negatively charged host surface (Murakami, 2008) and a high positive charge on the Gag protein is a key feature for the host-virus interaction. On analyzing the pI values for each of the 4 inserts in 2019-nCoV and the corresponding stretches of amino acid residues from HIV-1 proteins we found that a) the pI values were very similar for each pair analyzed b) most of these pI values were 10±2 [Refer Table 1] . Of note, despite the gaps in inserts 3 and 4 the pI values were comparable. This uniformity in the pI values for all the 4 inserts merits further investigation.

As none of these 4 inserts are present in any other coronavirus, the genomic region encoding these inserts represent ideal candidates for designing primers that can distinguish 2019-nCoV from other coronaviruses.

Motifs	Virus Glycoprotein	Motif Alignment	HIV protein and Variable region	HIV Genome Source Country/ subtype	Number of Polar Residues	Total Char ge	pI Valu e
Insert 1	2019- nCoV (GP) HIV1(GP120)	71 76 TNGTKR TNGTKR 404 409	gp120- V4	Thailand */ CRF01_ AE	5 5	2 2	11 11
Insert 2	2019- nCoV (GP) HIV1(GP120)	145 150 HKNNKS HKNNKS 462 467	gp120- V5	Kenya*/ G	6 6	2 2	10 10
Insert 3	2019- nCoV (GP) HIV1(GP120)	245 256 RSYL- – – -TPGDSSSG RTYLFNETRGNSSSG 136 150	gp120- V1	India*/C	8 10	2 1	10.84 8.75
Insert 4	2019- nCoV (Poly P) HIV1(gag)	676 684 QTNS———————–PRRA QTNSSILMQRSNFKG PRRA 366 384	Gag	India*/C	6 12	2 4	12.00 12.30

Table 1: Aligned sequences of 2019-nCoV and gp120 protein of HIV-1 with their positions in primary sequence of protein. All the inserts have a high density of positively charged residues. The deleted fragments in insert 3 and 4 increase the positive charge to surface area ratio. *please see Supp. Table 1 for accession numbers

The novel inserts are part of the receptor binding site of 2019-nCoV

To get structural insights and to understand the role of these insertions in 2019-nCoV glycoprotein, we modelled its structure based on available structure of SARS spike glycoprotein (PDB: 6ACD.1.A). The comparison of the modelled structure reveals that although inserts 1,2 and 3 are at non-contiguous locations in the protein primary sequence, they fold to constitute the part of glycoprotein binding site that recognizes the host receptor (Kirchdoerfer et al., 2016) (Figure 4). The insert 1 corresponds to the NTD (N-terminal domain) and the inserts 2 and 3 correspond to the CTD (C-terminal domain) of the S1 subunit in the 2019-nCoV spike glycoprotein. The insert 4 is at the junction of the SD1 (sub domain 1) and SD2 (sub domain 2) of the S1 subunit (Ou et al., 2017). We speculate, that these insertions provide additional flexibility to the glycoprotein binding site by forming a hydrophilic loop in the protein structure that may facilitate or enhance virus-host interactions.

Figure 3. Modelled homo-trimer spike glycoprotein of 2019-nCoV virus. The inserts from HIV envelop protein are shown with colored beads, present at the binding site of the protein.

Evolutionary Analysis of 2019-nCoV

It has been speculated that 2019-nCoV is a variant of Coronavirus derived from an animal source which got transmitted to humans. Considering the change of specificity for host, we decided to study the sequences of spike glycoprotein (S protein) of the virus. S proteins are surface proteins that help the virus in host recognition and attachment. Thus, a change in these proteins can be reflected as a change of host specificity of the virus. To know the alterations in S protein gene of 2019-nCoV and its consequences in structural re-arrangements we performed in-sillico analysis of 2019-nCoV with respect to all other viruses. A multiple sequence alignment between the S protein amino acid sequences of 2019-nCoV, Bat-SARS-Like, SARS-GZ02 and MERS revealed that S protein has evolved with closest significant diversity from the SARS-GZ02 (Figure 1).

Insertions in Spike protein region of 2019-nCoV

Since the S protein of 2019-nCoV shares closest ancestry with SARS GZ02, the sequence coding for spike proteins of these two viruses were compared using MultiAlin software. We found four new insertions in the protein of 2019-nCoV- “GTNGTKR” (IS1), “HKNNKS” (IS2), “GDSSSG” (IS3) and “QTNSPRRA” (IS4) (Figure 2). To our surprise, these sequence insertions were not only absent in S protein of SARS but were also not observed in any other member of the Coronaviridae family (Supplementary figure). This is startling as it is quite unlikely for a virus to have acquired such unique insertions naturally in a short duration of time

Insertions share similarity to HIV

The insertions were observed to be present in all the genomic sequences of 2019-nCoV virus available from the recent clinical isolates (Supplementary Figure 1). To know the source of these insertions in 2019-nCoV a local alignment was done with BLASTp using these insertions as query with all virus genome. Unexpectedly, all the insertions got aligned with Human immunodeficiency Virus-1 (HIV-1). Further analysis revealed that aligned sequences of HIV-1 with 2019-nCoV were derived from surface glycoprotein gp120 (amino acid sequence positions: 404-409, 462-467, 136- 150) and from Gag protein (366-384 amino acid) (Table 1). Gag protein of HIV is involved in host membrane binding, packaging of the virus and for the formation of virus-like particles. Gp120 plays crucial role in recognizing the host cell by binding to the primary receptor CD4.This binding induces structural rearrangements in GP120, creating a high affinity binding site for a chemokine co-receptor like CXCR4 and/or CCR5.

Discussion

The current outbreak of 2019-nCoV warrants a thorough investigation and understanding of its ability to infect human beings. Keeping in mind that there has been a clear change in the preference of host from previous coronaviruses to this virus, we studied the change in spike protein between 2019-nCoV and other viruses. We found four new insertions in the S protein of 2019-nCoV when compared to its nearest relative, SARS CoV. The genome sequence from the recent 28 clinical isolates showed that the sequence coding for these insertions are conserved amongst all these isolates. This indicates that these insertions have been preferably acquired by the 2019-nCoV, providing it with additional survival and infectivity advantage. Delving deeper we found that these insertions were similar to HIV-1. Our results highlight an astonishing relation between the gp120 and Gag protein of HIV, with 2019-nCoV spike glycoprotein. These proteins are critical for the viruses to identify and latch on to their host cells and for viral assembly (Beniac et al., 2006). Since surface proteins are responsible for host tropism, changes in these proteins imply a change in host specificity of the virus. According to reports from China, there has been a gain of host specificity in case 2019-nCoV as the virus was originally known to infect animals and not humans but after the mutations, it has gained tropism to humans as well.

Moving ahead, 3D modelling of the protein structure displayed that these insertions are present at the binding site of 2019-nCoV. Due to the presence of gp120 motifs in 2019-nCoV spike glycoprotein at its binding domain, we propose that these motif insertions could have provided an enhanced affinity towards host cell receptors. Further, this structural change might have also increased the range of host cells that 2019-nCoV can infect. To the best of our knowledge, the function of these motifs is still not clear in HIV and need to be explored. The exchange of genetic material among the viruses is well known and such critical exchange highlights the risk and the need to investigate the relations between seemingly unrelated virus families.

Conclusions

Our analysis of the spike glycoprotein of 2019-nCoV revealed several interesting findings: First, we identified 4 unique inserts in the 2019-nCoV spike glycoprotein that are not present in any other coronavirus reported till date. To our surprise, all the 4 inserts in the 2019-nCoV mapped to

short segments of amino acids in the HIV-1 gp120 and Gag among all annotated virus proteins in the NCBI database. This uncanny similarity of novel inserts in the 2019- nCoV spike protein to HIV-1 gp120 and Gag is unlikely to be fortuitous. Further, 3D modelling suggests that atleast 3 of the unique inserts which are non-contiguous in the primary protein sequence of the 2019-nCoV spike glycoprotein converge to constitute the key components of the receptor binding site. Of note, all the 4 inserts have pI values of around 10 that may facilitate virus-host interactions. Taken together, our findings suggest unconventional evolution of 2019-nCoV that warrants further investigation. Our work highlights novel evolutionary aspects of the 2019-nCoV and has implications on the pathogenesis and diagnosis of this virus.

References

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Fig.S1 Multiple sequence alignment of glycoprotein of coronaviridae family, representing all the four inserts.

Fig.S2: All four inserts are present in the aligned 28 Wuhan 2019-nCoV virus genomes obtained from GISAID. The gap in the Bat-SARS Like CoV in the last row shows that insert 1 and 4 is very unique to Wuhan 2019-nCoV.

Fig.S3 Phylogenetic tree of 28 clinical isolates genome of 2019-nCoV including one from bat as a host.

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Supplementary Fig 4. Genome alingment of Coronaviridae family. Highlighted black sequences are the inserts represented here.

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Decoding the evolution and transmissions of the novel pneumonia coronavirus (SARS-CoV-2) using whole genomic data

Posted on March 26, 2020 by Mimi Castellanos

Title page

Decoding the evolution and transmissions of the novel pneumonia coronavirus (SARS-CoV-2) using whole genomic data

Wen-Bin Yu1,2 * Ph.D., Guang-Da Tang3,4 Ph.D., Li Zhang5 Ph.D., Richard T. Corlett1,2 Ph.D.

1Center for Integrative Conservation, Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, Mengla, Yunnan 666303, China
2Center of Conservation Biology, Core Botanical Gardens, Chinese Academy of Sciences, Mengla, Yunnan 666303, China

3Henry Fok College of Biology and Agriculture, Shaoguan University, Shaoguan 512005, China 4College of Forestry and Landscape Architecture, South China Agricultural University, Guangzhou 510642, China
5Chinese Institute for Brain Research, Beijing 102206, China

* Author for corresponding: yuwenbin@xtbg.ac.cn

chinaXiv:202002.00033v2

Summary
Background The outbreak of COVID-19 started in mid-December 2019 in Wuhan, Central China. Up to February 19, 2020, SARS-CoV-2 has infected more than 75,000 people in China, and another 25 countries across five continents. In this study, we used 93 complete genomes of SARS-CoV-2 from the GISAID EpiFluTM database to investigate the evolution and human-to-human transmissions of SARS-CoV-2 in the recent two months.

Methods Haplotype analyses were conducted on the alignment of coding regions using DnaSP. Evolutionary analysis of haplotypes used NETWORK. Population size changes were estimated using both DnaSP and Arlequin. Expansion date of population size was calculated based on the expansion parameter tau (τ) using the formula t=τ/2u.

Findings Eight coding-regions have 120 substitution sites, including 79 non-synonymous and 40 synonymous substitutions. Forty-two non-synonymous substitutions changed the biochemical property of amino acids. No combinations were detected. Fifty-eight haplotypes were classified into five groups: 31 haplotypes were found in samples from China and 31 in samples from other countries. The rooted network suggested that H13 and H35 were ancestral haplotypes, and H1 (which with its descendants included all samples from the Hua Nan market) was derived from the H3 haplotype. Population size of SARS-CoV-2 was estimated to have had a recent expansion on 6 January 2020, and an early expansion on 8 December 2019.

Interpretation Genomic variations of SARS-CoV-2 are still low in comparisons with published genomes of SARS-CoV and MERS-CoV. Phyloepidemiologic analyses suggested that the SARS-CoV-2 source at the Hua Nan market was imported from elsewhere. The crowded market then boosted SARS-CoV-2 circulation and spread it to the whole city in early December 2019. Furthermore, phyloepidemiologic approaches have recovered specific directions of human-to-human transmissions and the sources for international infected cases.

Funding Ten Thousand Talents Program of Yunnan, and Chinese Academy of Sciences.

chinaXiv:202002.00033v2

Evidence before this study

Up to February 19, 2020, SARS-CoV-2 had infected more than 75,000 people worldwide. Tracing back to the first identified COVID-19 patient on 1 December, SARS-CoV-2 has been circulated in humans for more than two months. However, it is still unclear whether the Hua Nan market was the birthplace of the virus, and how it has been transmitted and spread subsequently. We searched PubMed, preprint archives, and Google Scholar for articles published up to February 18, 2020, that contained information about the COVID-19 outbreak using the terms “2019-nCoV” or “SARS-CoV-2”, “coronavirus”, “evolution” or “phylogeny”, “GISAID”, “seafood market”, “transmission”, and “Wuhan”. We found six studies using fewer than 55 genomes of SARS-CoV-2 for phylogenetic analyses and molecular dating using BEAST.

Added value of this study

We found that 120 substitution sites were evenly distributed in eight coding regions, without evident recombination events. An earlier expansion date could be traced back to 8 December 2019. Genomic evidence did not support the Hua Nan market as the birthplace of SARS-CoV-2. In the first two months, most infected people were linked to Wuhan, but some infected patients outside China may link to Guangdong or other places.

Implications of all the available evidence

We suggest that SARS-CoV-2 may have already circulated widely among humans in Wuhan before December 2019, probably beginning in mid to late November. Some infected patients may have been overlooked because they had mild symptoms. We have demonstrated that a phylogenetic approach can be incorporated into epidemiological studies to search for the original source of SARS-CoV-2 and identify the direction of human-to-human transmissions.

chinaXiv:202002.00033v2

Introduction

Betacoronaviruses are characterized by enveloped, positive-sense, single-stranded RNA, and hosted in animals, particularly in mammals.1 Before December 2019, four species/strains of Betacoronavirus, HKU1, MERS-CoV, OC43 and SARS-CoV, had been reported to cause severe human diseases.1 The fifth species/strain, a novel betacoronavirus SARS-CoV-22 causing human pneumonia (i.e. COVID-19), was first reported in Wuhan, Hubei, Central China.3,4 Up to February 19, 2020, SARS-CoV-2 has infected more than 75,000 people in all provinces/regions of China, and another 25 countries across Africa, Asia, Europe, North America, and Oceania.5 Because SARS-CoV-2 can transmit from human to human,6 the massive exodus of people before the Chinese Spring Festival boosted the infection frequencies, as predicted.7 Daily confirmed infection cases were more than 2,000 between January 30 and 16 February 16, 2020, and the highest was more than 15,100,5 almost twice the total number of infection cases of SARS-CoV.8

As a member of subgenus Sarbecovirus, SARS-CoV-2 has been suggested to be of bat origin,3,9 and may have been transmitted to humans through non-bat intermediate mammals (e.g. pangolins10). Medical information for the first 41 infected patients in Wuhan showed that 27 patients were linked to the Hua Nan seafood market,6,11 which sold living wild mammals. This suggests a high possibility that SARS-CoV-2 originated in the market, then the infected people transmitted it to other people out of the market. However, this conclusion has been debated because the first identified infected person and 12 others had no link to the Hua Nan market. Some researchers have therefore argued that the Hua Nan market was not the original and/or only source of SARS-CoV-2 transmission to humans.12 The market was closed on 1 January 2020, making it very difficult to identify the intermediate animal vectors of SARS-CoV-2. In the absence of information on potential intermediary reservoirs, the origin and transmission pattern of SARS-CoV-2 are still unresolved.

Since the outbreak of COVID-19 was first identified in Wuhan in mid-December 2019, the first infected individuals identified in other provinces and regions of China, and other countries, during January 2020, have been assumed to have been infected in Wuhan or through contact with people from Wuhan.13-16 For example, the first identified infected patient and his four family members from Shenzhen visited Wuhan between Dec 29, 2019 and Jan 4, 2020,13 and the first identified infected patient in the United States visited family in Wuhan and returned to Washington State on January 15, 2020.14 SARS-CoV-2 can transmit from human to human,6 so Wuhan has been assumed to be the birthplace of SARS-CoV-2. However, this assumption has not been fully validated because the Hua Nan market has not been confirmed as the single source of SARS-CoV-2 transmission to humans and other possible original sources of SARS-CoV-2 have not been identified in Wuhan yet. It is evident, however, that the Hua Nan market boosted SARS-CoV-2 transmission to humans at an early stage of the pneumonia outbreak in Wuhan, after

which it spread rapidly with infected travelers to the whole of China and to other countries. 4

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In this study, we used 93 genomes of SARS-CoV-2 from the GISAID EpiFluTM database17 (access date 12 February 2020) to decode the evolution and transmissions of SARS-CoV-2 in the recent two months. Our aims were to: 1) characterize genomic variations of SARS-CoV-2; 2) infer the evolutionary relationships of the worldwide samples; and 3) deduce the transmission history of SARS-CoV-2 in Wuhan and out of Wuhan to the world.

Methods

To decode the evolutionary history of SARS-CoV-2, we retrieved 96 complete genomes from GISAID (Table S1, access by 12 February, 2020).17 The genome EPI_ISL_402131 (bat-RaTG13-CoV, hereafter) from GISAID was also included as the outgroup, because it is the closest sister betacoronavirus to SARS-CoV-2.3 The 97 genome sequences were aligned using MAFFT,18 then the alignment was manually checked using Geneious (Biomatters, New Zealand). In the alignment, we found that the genome EPI_ISL_404253 contains six ambiguous sites at variable positions and EPI_ISL_407079 and EPI_ISL_408978 have 175 “N” and 1,476 “N” bases, respectively, so these three genomes were excluded in this study. In addition, four genomes (EPI_ISL_407071, EPI_ISL_407894, EPI_ISL_407896, and EPI_ISL_409067) have their own private ambiguous sites, which were conservatively replaced by the common nucleotide at that position in the alignment. In the alignment, the 5’UTR and 3’UTR contain missing and ambiguous sites, so both regions were excluded in the following analyses.

The alignment was then imported into DnaSP19 for haplotype analyses. Population size changes were estimated based on a constant population size hypothesis using DnaSP, in combination with neutrality tests (Tajima’s D and Fu’s Fs). We also used Arlequin20 to test the sudden population expansion hypothesis and to calculate the expansion parameter tau (τ) if sudden population expansion is not rejected. We used the formula t=τ/2u to estimate the time since expansion (in days). In the formula, u is the cumulative substitution rate per year (for the genome sequence, so we used the formula u = μk to calculate it, where μ is the substitution rate per site per year, and k is the genome sequence length (29,358 bp for the CDS matrix). In this study, the substitution rate was set as 0·92×10-3 (95% CI, 0·33×10-3-1·46×10-3) substitution/site/year based on the most recent estimation for SARS-CoV-2.21 A median-joining network of haplotypes was generated by the NETWORK program22 with bat-RaTG13-CoV as the outgroup. Phylogenomic analyses of haplotypes were performed using IQ-TREE23. We conducted likelihood mapping and SH-like approximate likelihood ratio tests to assess the phylogenetic information and branch supports, respectively.

Results and discussion
Genomic variations of SARS-CoV-2
Genome size of SARS-CoV-2 varied from 29,782 bp to 29,903 bp. The aligned matrix was 29,910

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bp in length including 140 variable sites. The coding regions contained 120 substitution sites (Figure S1), which were classified as 58 haplotypes (Table S2). Nucleotide diversity (Pi) was 0·15×10-3±0·02×10-3 (Standard Deviation, SD, hereafter). Haplotype diversity (Hd) was 0·953±0·016 (SD) and variance of Hd was 0·26×10-3.

There are 120 substitution sites found in eight coding sequence (CDS) regions, i.e. Replicase polyprotein CDS (75 sites, 0·35% of the whole sequence), Spike glycoprotein CDS (16 sites, 0·42%), ORF3 (7 sites, 0·75%), Membrane glycoprotein CDS (4 sites, 0·60%), ORF7 (3 sites, 0·82%), ORF8 (3 sites, 0·82%), Nucleocapsid protein CDS (11 sites, 0·77%), and ORF10 (1 site, 0.86%), including 79 transitions (65·83%) and 41 transversions (34·17%). A chi-squared test showed that the distribution of substitution sites across ten CDS regions in the genome was even (χ2=1·958, df=9, P=0·99). Substitution sites at the 1st to 3rd frame positions are 27 (25·55%), 44 (40·0%), and 49 (44·55%), respectively. The 120 mutation sites are associated with 119 codons, including 79 non-synonymous (65·83%) and 40 synonymous (33·61%) substitutions. There were 42 non-synonymous substitutions (53·17%) which changed the biochemical properties of the amino acid (AA). The details for each CDS gene are shown in Figure 1 and Table S3. It is not clear whether non-synonymous substitutions and the biochemical property changes of amino acids might change the infectious activities of SARS-CoV-2. The current samplings showed that H1 haplotype has been found in 19 patients, but most haplotypes were just sequenced once. One possible explanation is that a common haplotype from the Hua Nan market (Figure 2, Table S1) was rapidly circulated at an early stage of human-to-human transmissions.

In comparisons with published genomes of SARS-CoV24 and MERS-CoV,25 genomic variations of SARS-CoV-2 are still low, without evident recombination sites/regions (Rm=2, P=1·0) at this time. According to the collection dates of the sequenced samples, haplotypes H1 and H3 were found in two samples at intervals of more than 30 days, and multiple samples over 20 days (Figure 2, Table S1). Although the incubation period can be over 24 days, there was only one case of this out of 1099 observations.26 Estimation of DNA substitution rates using 90 genomes of SARS-CoV-2 showed that the rate for SARS-CoV-2 (0·92×10-3, 95% CI: 0·33-1·46×10-3 substitution/site/year21) was lower than the rates for SARS-CoV (95% CI: 0·80-2·38×10-3 substitution/site/year27) and MERS-CoV (1·12×10-3, 95% CI: 0· ×10-3 substitution/site/year28; ×10-3, 95% CI: 0· ×10-3 substitution/site/year29). It therefore looks as if SARS-CoV-2 is still undergoing stable evolution. Due to mild symptoms and low mortality30,31, the immune system of the infected humans may provide a suitable environment for propagation of SARS-CoV-2. SARS-CoV-2 is highly infectious31 and is able to infect humans not only through the mucous membranes of the nose and mouth, but also use mucous membranes in the eyes,32 which may boost regional circulation and large-scale spread. Some large mutations may have occurred in Wuhan or other regions, but the strict quarantine policy over China since 23 January 2020 may have reduced the circulation and spreading of some mutants.

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88-1·

or 0·

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83-1·

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Of 93 genomes of SARS-CoV-2, 39 (41·93%) were from infected patients in 11 countries on four continents and encoded 31 haplotypes (Hd=0·987±0·009 (SD), Pi=0·16×10-3±0·01×10-3), with 27 nationally/regionally private haplotypes. The 54 genomes (58.07%) from China also encoded 31 haplotypes (Hd=0·906±0·001 (SD), Pi=0·14×10-3±0·03×10-3). A proportion Z-test showed significant differences in haplotype diversity of samples between China and other countries (χ2=4·024, df=1, P<0·05). The high haplotype diversity found in samples from other countries may be because the sampling dates were mostly after 22 January 2020, while those in China were before this date (Table S1 and Figure S2). In addition, the low level of radiation exposure on long-distance international flights33 may have accelerated mutation rates of SARS-CoV-2.34

Population size expansion of SARS-CoV-2

Constant population size of SARS-CoV-2 was rejected (Ramos-Onsins and Rozas’s R2=0·025, P<0·001; Raggedness r=0·011, P<0·05) using DnaSP (also see Figure S3), while both Fu’s test (Fs=-67.681.964, P<0·001) and Tajima’s D test (D=-2.701, P<0.001) indicated that the population size of SARS-CoV-2 was rapidly increasing. Mismatch distribution analysis using Arlequin strongly supported that the population of SARS-CoV-2 underwent sudden expansion (τ=2·887, Sum of Squared deviation=0·541×10-3, P=0·88, Harpending’s Raggedness index=0·010, P=0·88). The calculated expansion was 28·72 days (95% CI: 12·29-54·36 days) ago. Of the 93 genomes, the latest one was sampled on 3 February 2020, so the estimated expansion date was on 6 January 2020 (95% CI: 11 December 2019-22 January 2020), when China CDC started to activate a Level-2 emergency response.6 Until 6 January 2020, 129 patients were identified as SARS-CoV-2 infected through field investigations.6 Of 22 genomes (17·05 % of 129 patients) sequenced before 6 January 2020, 13 haplotypes (22.41% of 58 haplotypes) were recovered, which were H1 and its derived descendant haplotypes, and H3 (Figures 2 and 3). The CDS’s emergency response greatly reduced public activities and travels, and may have reduced the local circulation and large-scale spread in the following weeks of January.

Furthermore, mismatch distribution analysis of the 22 genomes before 6 January 2020 also showed a sudden population expansion of SARS-CoV-2 at an earlier stage of transmission (τ=2·818, Sum of Squared deviation=0·010, P=0·41, Harpending’s Raggedness index=0·046, P=0·57, Tajima’s D=-2·241, P<0·001; Fu’s Fs=-7·834, P<0·001). This earlier population expansion time was estimated at 28·38 days (95% CI: 12·00-54·36 days) before 5 January 2020, which was the latest sampling date of the 22 genomes. This earlier expansion was thus estimated to have occurred on 8 December 2019 (95% CI: 13 November 2019-26 December 2019), when there was only one infected patient officially reported.6,11 Therefore, SARS-CoV-2 had already circulated widely among humans in Wuhan before December 2019, probably beginning in mid to late November.21

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Evolutionary relationships of SARS-CoV-2 haplotypes

The evolutionary network of 58 haplotypes of SARS-CoV-2, with bat-RaTG13-CoV as the outgroup, is shown in Figure 3A. Five main groups can be recognized. In the network, H1, H3, and H13 were three core haplotypes, so Groups A-C were recognized using them as the central (i.e., ancestral super-spreader) haplotypes. Groups D and E were recognized based on two new super-spreader haplotypes, H56 and a medium vector mv2, which was a hypothesized (often ancestral) haplotype not sampled in the current samples. These two groups can be also treated as subgroups of Group C.

In the network, four satellite haplotypes and H35 connected to the H13 haplotype (Group A), and nine satellite haplotypes and H38+H45 and H50 haplotypes connected to H3 (Group B). The evolutionary network showed that bat-RaTG13-CoV connected through a hypothesized haplotype (mv1) to the H13 and H38 haplotypes by single mutations at positions 18,067 (S, synonymous substitution) and 29,102 (S), referring to the alignment length 29,910 bp. The connections between the H3 and H1 haplotypes are two mutations at positions 8,789 (S) and 28,151 (Ns, non-synonymous substitution). The H1 haplotype, the most abundant, included 19 samples, while 26 satellite haplotypes and H40+(H43 and H47) haplotypes are directly derived from the H1 haplotype (Group C). Moreover, five haplotypes of Group D and four haplotypes of Group E should be also derived from the H1 haplotype.

Where are the original sources from?

The evolutionary network suggests that the hypothesized haplotype mv1 may be from an intermediate host or the first infected humans. From those connections, both H13 and H38 would be suggested as ancestral haplotypes. The SH-like approximate likelihood ratio test showed both haplotype H13’s group and H38 (with H45) could be the most basal clades in phylogenies of the 58 haplotypes (Figure S4), but phylogenetic information of the alignment was informative (Figure S5). Two main evolutionary paths of available haplotypes can be from H13 through H3 to H1, or from H38 through H3 to H1 (Figure 3C). Both scenarios demonstrate H3 was the key connection from an ancestral haplotype to H1. Neither H13 nor H38 has samples from Wuhan (Hubei) (Figure 3). H13 was only recovered from five Shenzhen (Guangdong) samples, including the father (patient 2) of the familial cluster, who was one of the first identified infected patients in Guangdong.13 Two derived haplotypes were also only found in Shenzhen (H14 from the grandson of patient 2), and the other three haplotypes were found in three samples from Japan and one sample from Arizona in the United States (Figure 3). According to an epidemiological study, the Shenzhen family traveled to Wuhan after the outbreak was announced, and they could have been infected during their visit in Wuhan from a hospital or an unknown common source13. This suggests that H13 should have originated from Wuhan, but none of the available samples from Wuhan encode haplotypes in Group A. Genetically, haplotypes of Group A have links to only

Wuhan haplotype H3 (only one sample EPI_ISL_406801, with no link to the Hua Nan market). It 8

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is possible that H13 was newly derived from H3 in the family from Shenzhen (Figure 3C) and did not spread in Wuhan, or that no samples have been sequenced yet. However, this scenario is not supported by the evolutionary network. H38 has three genomes from the same patient (Table S1), who was the first identified infected patient in the United States.14 He should have been infected while visiting his family in China. The original source of H38 can be explained as that of H13, which is also derived from H3, and the derived H45 was from a Chongqing patient who was working in Wuhan.

The H3 haplotype has only one sample from Wuhan, which was not linked to the Hua Nan market,9 and the other samples in this group were from other countries and regions (Figure 3). Noteworthily, all the samples from the Hua Nan market had the H1 haplotype or its derived haplotypes (H2, H8-H12, see Figure 2 and Table S1), indicating that there were circulated infections within the market in the short term. It is possible that SARS-CoV-2 in the Hua Nan market had been transmitted from other places (Figure 3D), or at least, that Hua Nan market did not host the original source of SARS-CoV-2. As the first identified infected patients had no link to the market,11 it is possible that infected humans transmitted SARS-CoV-2 to workers or sellers in the market, after which it rapidly circulated there. The crowded market boosted SARS-CoV-2 transmissions to buyers and spread it to the whole city in early December 2019, corresponding to the estimated population expansion time.

Regional and worldwide circulation and spread

Of the 54 genomes from patients in China, Chongqing (3 samples), Guangdong (18), Hubei (22), Taiwan (2), and Zhejiang (4) have more than two samples, and the other five provinces sequenced one sample. Hubei (Wuhan) samples from 24 December 2019 to 5 January 2020 encoded 13 haplotypes, belonging to Groups C (H1 and 11 satellite haplotypes) and B (only H3). These relationships indicate a rapid transmission and circulations of SARS-CoV-2 in Wuhan at an early stage of transmission. H1 (no satellite haplotypes) and H3 haplotypes are the ancestors of haplotypes out of Wuhan/Hubei. Eighteen Guangdong samples, collected from 10 to 23 January 2020, encoded 15 haplotypes, belonging to Groups A, C and E, showing that there were multiple sources imported into Guangdong. Three haplotypes (H14, H15 and H17) may have evolved locally, indicating that human-to-human transmissions happened when SARS-CoV-2 initially spread to Shenzhen of Guangdong.13 Two samples from Taiwan encoded H3 and H24 in Groups B and D, respectively, and three samples from Chongqing encoded H1, H40, and H45 in Groups B and C, respectively. There were two sources imported into these two provinces/regions. Four Zhejiang samples encoded H1 and H24 in Group C, which was only imported from the source of the H1 haplotype.

The samples outside of China encoded 31 haplotypes belonging to Groups A-E. Of these, 27

haplotypes are private by regional samplings, only two Thailand samples were the H1 haplotype, 9

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one each from Australia and Belgium were the H3 haplotype, one sample from the United States was the H19 haplotype, and one sample from Singapore was the H40 haplotype. Twelve samples, encoding 10 haplotypes, were from patients in five countries in Asia. Seven haplotypes linked to Wuhan and three haplotypes linked to Guangdong (Shenzhen). Human-to-human transmission may have happened from patients with H53 to H52 haplotypes in Tokyo, Japan, who were repatriated Japanese from Wuhan.35 Five Oceanian samples, encoding six haplotypes in Groups B, C, and D, were from patients from three states in Australia, all with links to Wuhan. Patients with H3, with H25 and H26, and with H55 (linked to H1) were directly from Wuhan, and human-to-human transmission was from patients of H25 to H26, who were in a same tour group in Queensland.36 The connection between patients with H56 and H27 is not clear, because the patient with H56 flew to Sydney from Wuhan on 25 January 2020, and the patient with H27 flew to Melbourne from Wuhan on 15 January 2020. One possibility is that there was an intermediary spreader, who also transmitted SARS-CoV-2 to other patients in France, the United States, and Taiwan. Eight European samples, encoding seven haplotypes, were from patients in four countries. Patients in Belgium37 and Germany16 traveled to or stayed in Wuhan. Patients in England did not report a link to Wuhan,38 but a familial transmission was recovered from H28 to H29. Patients in France may have been infected by three different sources, i.e. H44 linked to Wuhan, H43 may link to Chongqing/Singapore, and H30 may link to an intermediary spreader.

Of the 13 genomes from the United States, three were from the same patient in Washington encoding the same haplotype H38, while the other three samples encoded eight haplotypes, covering all five groups (Figure 3A), so the sources of imported infections are complicated. Three haplotypes (H1 (California), H19 (Wisconsin), and H38 (Washington)) were linked to Wuhan, and three (H19 (Wisconsin), H35 (Arizona), H42 (California)) to five (H41 (California) and H58 (Illinois)) haplotypes may link to Guangdong. The remaining haplotypes (H36 (California), H37 (California), and H57 (Massachusetts)) linked to patients out of China (H54 (Vietnam) and H56 (Australia)), who were from Wuhan.15,39 It is not clear where they were infected. There is no human-to-human transmission evidence in the United States from the 11 cases.

Phylogenetic approaches provide insight into the epidemiology of SARS-CoV-2

Epidemiological study of SARS-CoV-2 using traditional approaches is very difficult, because it was not identified as a new coronavirus until 29 December, and some infected people with mild symptoms or without symptoms16,40 may have been overlooked in late November and early December. Moreover, the Hua Nan market, which was considered as the birthplace of SARS-CoV-2, has been closed since 1 January 2020. In this study, we have used genomic data for SARS-CoV-2 to infer evolutionary relationships of the 58 haplotypes, and to suggest that H1 and its descendant haplotypes from the Hua Nan market should be derived from the H3 haplotype, which was not linked to the market (Figure 3C). This suggests that the source of the coronavirus in

the Hua Nan market was imported from elsewhere, as also suggested by other researchers.12 The 10

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phylogenetic network indicated that H13 and H38 should be ancestral haplotypes that connected to the outgroup bat-RaTG13-CoV through a hypothesized intermediate haplotype. Because the currently available samples do not include the first identified infected patient and other patients from early December, the most common ancestral haplotype might be missed. If there are any frozen samples from those patients, it would be worth doing genomic sequencing for phyloepidemiologic study to help to locate the birthplace of SARS-CoV-2 in Wuhan. Meanwhile, we expect that the H13 and H38 haplotypes will be found in some samples of infected patients in Wuhan if more samples are sequenced in future. This will be very important in the search for the original sources of SARS-CoV-2, because both H13 and H38 tend to be ancestral haplotypes.

The evolutionary network of haplotypes can be used to recover the directions of human-to-human transmissions at the local scale and spread at the larger scale. The central haplotype can be considered as the super-spreader haplotype, and the tip haplotype is the most recent descendant haplotype. The transmission direction can be identified using the connection information of tips and branches. For example, the confirmed patients from the Hua Nan market shared the common ancestral haplotype H1, indicating they were infected from a common source, who may have been a super-spreader in the market. This transmission phenomenon may also have happened in Shenzhen with the patients of Group A. This approach has recovered specific directions of human-to-human transmission in the Shenzhen family (H13 → H14), the Queensland tour group (H25 → H26), the England family (H28 → H29), and the repatriated Japanese from Wuhan (H53 → H52). Most international infections link to Wuhan directly or indirectly, but for some of them it is not clear exactly where they were infected. As discussed above, we have found that some patients in Japan and United States might have been infected in Guangzhou, and one patient in France might have been infected in Chongqing or Singapore. We suspect that super-spreaders mediate the spreading from China to worldwide. At least, the infected people with H56 and mv2, as well as H54, contributed at least three haplotypes (Figure 3A).

Contributors

W-BY conceived the research, analyzed the data, interpreted the results, and wrote the draft manuscript; WBY and GDT collected data; all authors reviewed and approved the final version of the manuscript.

Declaration of interests

We declare no competing interests.

Acknowledgements

We are grateful to scientists and researchers for depositing whole genomic sequences of Novel Pneumonia Coronavirus (SARS-CoV-2) at the Global Initiative on Sharing All Influenza Data (GISAID) EpiFluTM; to GISAID database for allowing us to access the sequences for

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non-commercial scientific research; and to Dr. Jin Chen and Dr. De-Zhu Li for their valuable comments and suggestions on conceiving the research and an early version of the manuscript. WBY also thanks his wife Dr. Nan Jiang for supporting him in daily life during the quarantine of the Novel Pneumonia outbreak, so he can focus on this research. This study was supported by grants from the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB31000000), Ten Thousand Talents Program of Yunnan for Top‐notch Young Talents, and the open research project of “Cross-Cooperative Team” of the Germplasm Bank of Wild Species, Kunming Institute of Botany, Chinese Academy of Sciences.

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35. Japan tightens immigration as 3 more infected by coronavirus. 2020. http://www.asahi.com/ajw/articles/AJ202002020013.html (accessed 2 February 2020).

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37. Belgium: First case of coronavirus confirmed in Belgium February 3. 2020. https://www.garda.com/fr/crisis24/alertes-de-securite/311116/belgium-first-case-of-coronavirus-co nfirmed-in-belgium-february-3 (accessed 4 February 2020).

38. UK confirms first new coronavirus case; rises risk level. 2020. https://www.pharmaceutical-technology.com/news/uk-coronavirus-case/ (accessed 31 January 2020).
39. Fifth Australian coronavirus case confirmed as 21-year-old UNSW student. 2020. https://www.sbs.com.au/news/fifth-australian-coronavirus-case-confirmed-as-21-year-old-unsw-st udent (accessed 27 January 2020).

40. Heymann DL, Shindo N. COVID-19: what is next for public health? Lancet 2020: 10.1016/S0140-6736(20)30374-3.

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Iodine: the Forgotten Weapon

Posted on March 9, 2020 by Mimi Castellanos

https://realrawfood.com/sites/default/files/article/Iodine-Weapon%20Against%20Viruses.pdf

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Dr Ruggiero gives a talk on Coronavirus COVID-19

Posted on March 9, 2020 by Mimi Castellanos

Dr Ruggiero told us that he has worked for many years with things like the HIV virus. (Late 80’s)

He told us of some Chinese research which was published on a public research domain that shows the Coronavirus Kovid19, has spikes that are the same as HIV. He said that the part about the HIV type spikes has been proven to be true. However, the conclusions about how this happened are still in debate.

Therefore, theoretically, the drugs that work for HIV should work for Coronavirus Kovid19. No tests have been done at this point, but Dr. Ruggiero says that theoretically that makes sense.

Dr Ruggiero said that the HIV virus spikes and the Coronavirus spikes are very strongly polarized. He states that the things that he has “built into” Bravo Yogurt and imuno have the polarity components to neutralize this aspect of the Coronavirus. Therefore anyone who has a deficiency in these areas can increase their ability to have the necessary environment in their body to neutralize it.

These are the same things that we have been talking about at www.bravocoop.com and www.imunothesolution.com. Further, our shopping cart has specific info about each product. Please login to see all products available.

Bravo Yogurt

Imuno

This is Dr. Ruggiero’s recording. I have asked him for the TCM paper and his slides or reference section and I will post it here as soon as I get it.

Please direct your questions to Mimi Castellanos 760-815-8830

We will have a Q&A with Dr. Ruggiero as soon as we can get on the schedule.

https://www.healthyenergetics.com/collections/accessories-for-bravo-and-rerum/products/sign-up-for-the-next-q-a-with-dr-ruggiero

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Someone asked “How many NGs are in imuno?”

Posted on February 25, 2020 by Mimi Castellanos

Let us consider first, what is a NG?

A NG abbreviates a nanogram. It is basically a measurement which is one thousand-millionth of a gram. This measurement used to measure the weight of the Gc Protein molecules contained in a solution.

imuno contains different compounds which are measured as indicated on the label. Gc Protein has not been specifically identified.

Find more info and purchase at www.healthyenergetics.com

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imuno® is over 100 times more effective than pure GcMAF

Posted on February 25, 2020 by Mimi Castellanos

imuno® is over 100 times more effective than pure GcMAF

19th February 2020: Dr Marco Ruggiero, MD. PhD.

Elevated serum alpha-N-acetylgalactosaminidase (nagalase) is associated with a number of life threatening and/or debilitating conditions ranging from cancer (Korbelik et al., 1998; Greco et al., 2009; Thyer et al., 2013) to viral infections caused by influenza virus and HIV (Yamamoto and Urade, 2005; Yamamoto, 2006), bacterial infections (Caines et al., 2008), alcoholism (Zoga et al., 2017), and autism (Bradstreet et al., 2012). Nagalase, an enzyme, was first proposed as a marker for cancer and viral infections and its increased serum activity in these conditions was associated with immune system deficiency because nagalase prevents formation of the Gc protein derived Macrophage Activating Factor (GcMAF), an immune stimulant cytokine (Yamamoto and Naraparaju, 1997; Yamamoto and Urade, 2005; Yamamoto, 2006). According to the original hypothesis proposed by Yamamoto and Colleagues, cancer cells and cells infected by viruses produced and released nagalase that caused immune deficiency that, in turn, favored the progression of cancer or viral infections. Therefore, elevated serum nagalase activity in cancer and viral infections was considered a consequence of cancer or viral-infected cells releasing nagalase. In apparent agreement with this hypothesis, successful immunotherapy of cancer with GcMAF was associated with a decrease of serum nagalase activity (Thyer et al., 2013; Schwalb et al., 2016) that was attributed to the effects of GcMAF on macrophages that, once activated, attacked cancer cells with consequent decrease of the number of cancer cells producing nagalase.

This interpretation, however, was contradicted by the observation that in autism, just like in cancer, successful immunotherapy with GcMAF was associated with significant decrease of serum nagalase activity (Bradstreet et al., 2012). Since the autistic subjects successfully treated with GcMAF had no signs of cancer cells producing nagalase, nor of any other concurrent disease or infections (Bradstreet et al., 2012), a novel hypothesis concerning the role of nagalase was proposed. According to this novel hypothesis, elevated nagalase activity has to be considered more a pathogenetic factor for cancer and other conditions rather than a simple marker. In simpler words, elevated nagalase activity may be one of the causative factors leading to cancer, autism or other diseases because elevated nagalase impairs the function of the immune system and immune deficiency plays a pivotal role in the onset and development of cancer and other diseases. Such a role for nagalase is now well established as exemplified by the title of a recent review that reads ” Is α-N-acetylgalactosaminidase the key to curing cancer?” (Saburi et al., 2017a). Based on these considerations, strategies aiming at reducing serum nagalase activity may prove useful in the fight against cancer, autism, viral infections and all other conditions associated with elevated serum nagalase activity.

Here we demonstrated for the first time that GcMAF directly binds to, and inhibits, human nagalase in vitro, thus elucidating the mechanism of action of GcMAF and explaining its effectiveness in a variety of conditions ranging from cancer to autism (Saburi et al., 2017a; Saburi et al., 2017b; Bradstreet et al., 2012; Greilberger and Herwig, 2020). In addition, we demonstrate that a novel supplement containing low-molecular-weight microbial chondroitin sulfate, ultrapure phosphatidylcholine, and vitamin D3, (imuno®, imuno Corporation, Vanuatu) shows more than 100 fold higher activity than purified GcMAF.

Human nagalase and GcMAF were purified at R.E.D. Laboratories (Zellik, Belgium). The
experiment was performed using microtiter plates coated with a specific antibody able to capture human nagalase. Samples were incubated with a standardized dilution of a pool of 300 human sera from healthy subjects and, after 1 h incubation and exhaustive washing, nagalase-GcMAF or nagalase-imuno® complexes were detected with a horse radish peroxidase conjugate of a rabbit antibody. In order to establish the kinetics of interaction between nagalase and GcMAF, or imuno®, the serum pool was mixed either with 200 ng of purified Gc-MAF, or with a 1:100 dilution of imuno® in phosphate buffered saline (PBS). Since preliminary experiments indicated that undiluted imuno® exceeded the binding capacity of nagalase in the test, imuno® was diluted 1:100 in PBS. The mixture was then incubated at room temperature for 4, 24, 48, 72 and 120 h. Values for nagalase binding activity in the absence of GcMAF or imuno®, with only PBS in the reaction mixture, were taken as

1.00. The experiment was repeated twice and the results reported in Fig. 1 are the means of the two experiments.

As shown in Fig. 1, purified GcMAF bound human nagalase only after 4 h incubation, reached a peak at 48 h, and returned below baseline values at 120 h. imuno®, on the other hand, had an initial value (at 0 h) higher than PBS, thus demonstrating immediate, intrinsic GcMAF activity against human nagalase. At every time point, imuno® showed significantly higher activity in comparison with purified GcMAF and, at 120 h, imuno® activity was still well above baseline, thus indicating a stronger, more prolonged activity. It is worth noticing that imuno® was diluted 100 fold and, therefore, it may be argued that its activity against human nagalase is more than 100 fold higher than that of purified GcMAF.

It is also important to consider that these results were obtained in vitro, that is in the absence of any variable or confounding factor that may hamper interpretation of results observed in clinical settings. Direct interaction between imuno® and nagalase may help explaining the effectiveness of imuno® recently observed by Antonucci and Colleagues in those conditions where GcMAF had proven effective in the past (Antonucci et al., 2019a; Antonucci et al., 2019b) and lead to propose imuno® as a more potent and intrinsically safer substitute for human-blood-derived GcMAF. The significantly higher potency of imuno® is to be ascribed to its peculiar molecular design that was described in detail in two recent papers (Ruggiero and Pacini, 2018a; Ruggiero and Pacini, 2018b); in brief, imuno® reproduces that physical-chemical features of GcMAF at a much higher molecular efficiency and density. The N- acetylgalactosamine active site of GcMAF is present in much higher concentration in imuno® thanks to the presence of low-molecular-weight chondroitin sulfate, a sulfated polysaccharide that is composed by an alternating chain of Nacetylgalactosamine and glucuronic acid. The hydrophobic moieties of GcMAF, that are the regions binding vitamin D and fatty acids, are present in imuno® thanks to the binding of phosphatidylcholine to chondroitin sulfate. Vitamin D3, essential for increasing the potency of GcMAF as demonstrated by Greilberger and Herwig in 2020, is intercalated in the proto-cellular structure formed by the core of chondroitin sulfate surrounded by phosphatidylcholine (Ruggiero and Pacini, 2018a). In fact, imuno® was designed taking into account the physical-chemical features of GcMAF that were published 2013 when a molecular model of GcMAF interaction with the cell membrane was described (Thyer et al., 2013). In this model, optimal interaction was achieved when GcMAF was non-covalently bound to a fatty acid – in that example, oleic acid – and vitamin D3 that are the conditions reproduced, at a much higher molecular efficiency and density, in imuno®.

Buy it here: www.healthyenergetics.com

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Dr Ruggiero on the protective elements of Bravo & imuno on pandemic viruses such as Coronavirus

Posted on February 19, 2020 by Mimi Castellanos

Explore Bravo & imuno’s unique properties

to protect you from viral pandemic

Dr Marco Ruggiero has recently communicated that imuno and Bravo are ideally suited to help with the coronavirus.

Bravo, thanks to its unique composition in phages (friendly viruses that fight pathogenic viruses) and its proven efficacy on empowering the immune system may be used to help prevent infection by the coronavirus that is causing the current epidemic.

As you know, the phages in Bravo are known to fight a number of pathogenic viruses that include HIV, and it was recently demonstrated that the coronavirus responsible for the current epidemic, contains HIV sequences that are responsible for its infectivity and are the target of Bravo’s phages https://www.biorxiv.org/content/10.1101/714154v2.

Dr. Ruggiero says, “I take the liberty to write that I am 99% (100% would be unrealistic) confident that imuno will help with the coronavirus for two reasons. The first and most obvious is its general effect on the immune system as described in all the papers where it has been mentioned.”

“The second is related to the specific anti-viral properties of chondroitin sulfate that are further amplified by the molecular arrangement of imuno. As you may know by now, the coronavirus responsible for the current epidemic attacks human cells because it has spikes from HIV that no one knows, at the present, whether they were intentionally inserted or are result of a casual viral recombination. (highly improbable but not impossible)”

Needless to say, the chondroitin sulfate in imuno is much more potent/bioavailable that the molecules used in those studies for at least two reasons: it is homogeneous and low-molecular-weight; it is arranged in a supramolecular structure that makes it more bioavailable and efficient in binding to the HIV spikes.

Because of these evidences, I am 99% sure that imuno will work against coronavirus.

Combination with Bravo may prove synergistic as we have observed in the case of myeloma and in the case of Dr. Carter that I have just submitted to the Am J Immunol.

Bravo will work through the phages and the natural GcMAF and it will complement the effects of imuno. Most likely, Bravo and imuno will work even if administered alone, but the combination may prove synergistic.

More on Coronavirus including studies- http://simplymimi.net/archives/1629

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More on Bravo & imuno with the Coronavirus

Posted on February 19, 2020 by Mimi Castellanos

Whatever the case, it is thanks to the HIV spikes that the coronavirus attacks human cells and, therefore, anti-HIV/anti-retroviral drugs – that should not work on coronavirus since it is not a retrovirus – appear to be effective in fighting the current disease. Please take a look at the news pasted below from an Italian Government-run media outlet and hospital clearly describing the effectiveness of anti-HIV drugs in two cases currently treated in Italy. By the way, according to this outlet, WHO (OMS in Italian) recommended the use of anti-HIV drugs, a very strange occurrence since they were not recommended with SARS (another type of coronavirus) and, most important, coronavirus is not a retrovirus and anti-HIV drugs were supposed to be specific for HIV and not for other viruses. Obviously there is something wrong, but we leave speculations and conspiracy theories to others.

http://www.rainews.it/dl/rainews/articoli/Coronavirus-protesta-della-Cina-verso-gli-altri-paesi-per-lo-stop-dei-voli-Piu-di-28mila-contagi-565-morti-14fbd0fd-410a-494e-b6c9-9b55abdb46ad.html

“I coniugi “ricevono dal 4 febbraio terapia antivirale sperimentale. Tali farmaci sono indicati dall’Organizzazione Mondiale della Sanità come i più promettenti sulla base dei dati disponibili”. E’ quanto si legge nel bollettino. Il lopinavir/ritonavir, spiegano i medici, è un antivirale comunemente utilizzato per la infezione da HIV che mostra attività antivirale anche sui coronavirus. – See more at: http://www.rainews.it/dl/rainews/articoli/Coronavirus-protesta-della-Cina-verso-gli-altri-paesi-per-lo-stop-dei-voli-Piu-di-28mila-contagi-565-morti-14fbd0fd-410a-494e-b6c9-9b55abdb46ad.html”

All this is to say that chondroitin sulfate is known to have anti-HIV/anti-retroviral properties as evidenced by two patents

https://patents.google.com/patent/EP0240098A2/en

https://patents.google.com/patent/EP0319676A2/en

and peer-reviewed scientific articles

https://www.ncbi.nlm.nih.gov/pubmed/26650729

https://www.ncbi.nlm.nih.gov/pubmed/23962762

https://www.ncbi.nlm.nih.gov/pubmed/11366557

https://www.ncbi.nlm.nih.gov/pubmed/11366556

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Dark Green Leafies – How to prepare them for a delicious dish

Posted on February 17, 2020 by Mimi Castellanos

“If you eat dark green, you WILL become lean!”

1) Kale
Possibly the most common of the bitter greens, kale is fairly mild in flavor, and it’s delicious both raw or cooked.

Just make sure to remove the fibrous stems before cooking!

Recipe: Slice kale leaves thinly and add to a bowl. Add a generous pour of extra virgin olive oil and a pinch of sea salt, and rub the salt/oil mixture into the leaves, massaging thoroughly. Finish the salad with a splash of lemon juice, red wine vinegar and, chopped avocado.

TIP: Greens grow in sandy soil, so be sure to double-rinse your greens in cool, flowing water to get that grit out.

2) Swiss Chard
Chard tastes like a cross between kale and cabbage. It can be eaten raw, but it’s best when cooked.

Eat the stems for extra fiber and crunch — they’re delicious!

Recipe: Thinly slice leaves and stems and set aside. Heat olive oil and garlic in a large saucepan. Cook until garlic is fragrant. Add chopped pecans and toast, then add chard and cook until wilted. Finish with a splash of balsamic vinegar.

Tip: If these greens are too bitter for you, make sure to add plenty of healthy fat, like extra virgin olive oil. It really cuts the bitterness… and tastes great!

3) Mustard Greens
This southern staple is curly leafed and has a peppery bite. Eat them cooked, and make sure to remove the stems first.

Recipe: Tear leaves into bite-sized pieces, removing stems. Add to a large pot with sliced onions, red wine vinegar, chicken stock, and sliced garlic. Cook covered for 1-2 hours over low heat until tender.

TIP: Another great way to add flavor and cut bitterness is with something acidic, like vinegar or even a squeeze of lemon juice!

4) Collard Greens
If you’ve ever spent time in the South, you’ve had collard greens… but you can actually eat them raw, and they’re a lot more nutritious this way.

Since the leaves are so broad, these relatively mild greens actually make great wraps — instead of tortillas. Just make sure to remove the tough stems if you’re eating them raw.

Recipe: Trim stem off leaves, and place in bowl of warm water with lemon juice. Let soak for 10 minutes, then dry and slice down the center stem to make leaves easy to wrap.

Fill with avocado, the protein of your choice, pickled onions, and a drizzle of lime juice, and wrap like a burrito.

TIP: Raw greens are delicious — and SO full of nutrients. Consider shredding greens like a salad, using them as a wrap, or subbing them into your favorite slaw.

So if you haven’t been a huge fan of bitter greens in the past, I strongly encourage you to try one of the four recipes above… even better, try all of them – you’re sure to find something you love.

Whatever you do, don’t skip your bitter greens! They’re just too important. And always remember my two favorite sayings:

“More bitter, more better.”

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Freeze Dried Free Range Milk

Posted on December 24, 2019 by Mimi Castellanos

Freeze Dried Free Range Milk

in 1 liter or 1/2 liter sizes

Why use this milk?

It doesn’t go bad
It don’t use freezer space
It doesn’t taste like powdered instant from childhood. (Shutter)
This milk has no additives, Vit A additive is a bad one for kids
It has all it’s fresh cream
It is dried in it’s peak of freshness
It is packed in mylar with oxygen removers
If unopened, it is rated to remain fresh and usable for 24 years
It has it’s casein removed
It has been gently home pasteurized

Your choice of Goat or Cow – 1/2 liter – $10.

ALSO sheep, camel, OR water buffalo – 1/2 liter – market price

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Simply Mimi

Uncanny similarity of unique inserts in the 2019-nCoV spike protein to HIV-1 gp120 and Gag

Decoding the evolution and transmissions of the novel pneumonia coronavirus (SARS-CoV-2) using whole genomic data

Iodine: the Forgotten Weapon

Dr Ruggiero gives a talk on Coronavirus COVID-19

Someone asked “How many NGs are in imuno?”

imuno® is over 100 times more effective than pure GcMAF

Dr Ruggiero on the protective elements of Bravo & imuno on pandemic viruses such as Coronavirus

More on Bravo & imuno with the Coronavirus

Dark Green Leafies – How to prepare them for a delicious dish

Freeze Dried Free Range Milk

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